US009943609B2 (12 ) United States Patent ( 10 ) Patent No. : US 9 , 943 ,609 B2 Yurkovetskiy et al. ( 45 ) Date of Patent: * Apr. 17, 2018 ( 54 ) PROTEIN -POLYMER -DRUG CONJUGATES ( 52 ) U . S. CI. CPC . .. . A61K 47/ 48692 (2013 .01 ) ; A61K 31/ 4745 (71 ) Applicant: Mersana Therapeutics, Inc. , ( 2013 .01 ) ; A61K 47/ 59 ( 2017 .08 ) ; A61K 47/ 64 Cambridge, MA (US ) ( 2017 . 08 ) ; A61K 47/ 6803 ( 2017 .08 ) ; A61K 47/ 6849 ( 2017 .08 ) ; A61K 47 /6883 ( 2017 .08 ) ; (72 ) Inventors : Aleksandr V. Yurkovetskiy , Littleton , CO7K 7 / 02 ( 2013 . 01 ) ; COOK 16 /32 ( 2013 .01 ) ; MA (US ) ; Mao Yin , Needham , MA CONG 65/ 334 ( 2013. 01 ); C08G 65/ 33396 (US ); Timothy B . Lowinger , Carlisle , (2013 .01 ) MA (US ) ; Joshua D . Thomas , Natick , (58 ) Field of Classification Search MA (US ) ; Charles E . Hammond , None Billerica , MA (US ) ; Cheri A . See application file for complete search history . Stevenson , Haverhill , MA (US ) ; Natalya D . Bodyak , Brookline , MA (56 ) References Cited (US ) ; Patrick R . Conlon , Wakefield , MA (US ) ; Dimitry R . Gumerov , U . S . PATENT DOCUMENTS Waltham , MA (US ) 4 ,046 ,722 A 9 /1977 Rowland 4 ,460 ,560 A 7 / 1984 Tokes et al. (73 ) Assignee : Mersana Therapeutics, Inc ., 5 , 410 ,024 A 4 / 1995 Pettit et al. Cambridge , MA (US ) 6 ,080 ,751 A * 6 / 2000 Stehlin A61K 31 /44 514 / 283 ( * ) Notice : Subject to any disclaimer, the term of this 6 ,602 ,498 B28 / 2003 Shen patent is extended or adjusted under 35 6 ,822 ,086 B1 11/ 2004 Papisov U .S . C . 154 (b ) by 0 days. (Continued ) This patent is subject to a terminal dis FOREIGN PATENT DOCUMENTS claimer . CN 1997401 A 7 / 2007 (21 ) Appl. No. : 15 / 001, 119 EP 112720 A2 7 / 1984 ( Continued ) ( 22 ) Filed : Jan . 19 , 2016 (65 ) Prior Publication Data OTHER PUBLICATIONS Yurkovetskiy et al. (Mol Pharm . 2004 ; 1( 5 ): 375 -382 ). * US 2016 / 0220696 A1 Aug. 4 , 2016 PubChem CID 104842 ( downloaded on May 22 , 2017 from URL : < https: // pubchem .ncbi .nlm .nih . gov / compound / 7 - Ethyl - 10 hydroxycamptothecin # section = Top > ) . * Related U .S . Application Data Burke et al. , “ Design , Synthesis , and Biological Evaluation of Antibody -Drug Conjugates Comprised of Potent Camptothecin (60 ) Continuation of application No . 14 /457 ,955 , filed on Analogues .” Bioconj. Chem . 20 . 6 ( 2009 ): 1242 - 1250 . Aug . 12 , 2014 , now Pat . No . 9 , 254 ,339 , which is a Doronina , S . et al. “ Novel Peptide Linkers for Highly Potent continuation of application No. 13 / 944 , 561, filed on Antibody -Auristatin Conjugate ” , Bioconjugate Chemistry, 2008 , Jul. 17 , 2013 , now Pat. No. 8 ,808 ,679 , which is a vol. 19 , No. 10 , p . 1960 - 1963 . division of application No . 13 /493 , 899, filed on Jun . 11 , 2012 , now Pat . No . 8 ,685 , 383 . (Continued ) Primary Examiner — Jennifer Pitrak McDonald ( 60 ) Provisional application No . 61/ 495 ,771 , filed on Jun . Assistant Examiner — Sergio Coffa 10 , 2011 , provisional application No . 61 /501 , 000 , (74 ) Attorney, Agent, or Firm — Cooley LLP ; Heidi A . filed on Jun . 24 , 2011, provisional application No . Erlacher; Xixi Sun 61/ 513 , 234 , filed on Jul . 29, 2011 , provisional application No . 61 / 566 , 935 , filed on Dec . 5 , 2011 , (57 ) ABSTRACT provisional application No . 61 /605 ,618 , filed on Mar. Auristatin compounds and conjugates thereof are provided 1 , 2012 , provisional application No. 61 /618 , 499, filed herein . The conjugate comprises a protein based recogni on Mar. 30 , 2012 . tion -molecule (PBRM ) and a polymeric carrier substituted with one or more - LP - D , the protein based recognition (51 ) Int. Ci. molecule being connected to the polymeric carrier by L ” . A61K 31 /74 ( 2006 .01 ) Each occurrence of D is independently an Auristatin com AIK 47/ 48 ( 2006 . 01 ) pound . LP and L are linkers connecting the therapeutic COZK 7 /02 ( 2006 .01 ) agent and PBRM to the polymeric carrier respectively . Also A61K 31/ 4745 ( 2006 .01 ) disclosed are polymeric scaffolds useful for conjugating CO7K 16 /32 ( 2006 . 01 ) with a PBRM to form a polymer - drug- PBRM conjugate A61K 47 /59 ( 2017 .01 ) described herein , compositions comprising the conjugates , A6IK 47 /64 ( 2017 .01 ) methods of their preparation , and methods of treating vari A61K 47/ 68 ( 2017 .01 ) ous disorders with the conjugates or their compositions . C08G 65 / 333 ( 2006 .01 ) C08G 65 / 334 ( 2006 .01 ) 20 Claims, 5 Drawing Sheets US 9, 943 , 609 B2 Page 2

(56 ) References Cited WO WO 2000064486 A2 11 / 2000 WO WO 2001010468 A2 2 /2001 U . S . PATENT DOCUMENTS WO WO 2004009082 A1 1 / 2004 wo WO 2004009774 A2 1 / 2004 6 ,911 , 197 B2 6 / 2005 Shen WO WO 2004010957 A2 2 / 2004 7 , 160, 924 B2 1 / 2007 Kinstler et al . WO WO 2004073656 A2 9 / 2004 7 , 276 , 497 B2 10 / 2007 Chari et al. Wo WO 2005023294 A2 3 / 2005 7 , 329 ,721 B2 2 /2008 Kozlowski et al. wo WO 2005081711 A2 9 / 2005 7 ,432 ,330 B2 10 /2008 Kozlowski et al. wo WO 2005123140 A2 12 / 2005 7 , 432 , 331 B2 10 / 2008 Kozlowski et al. WO WO 2006044986 Al 4 / 2006 7 , 553 , 816 B2 6 / 2009 Senter et al. WO WO 2007008848 A2 1 / 2007 7 , 659 , 361 B2 2 /2010 Kozlowski et al. WO WO 2007103288 A2 9 / 2007 7 , 790 , 150 B2 9 / 2010 Papisov et al . WO WO 2007109567 AL 9 / 2007 7 , 872 , 082 B2 1 /2011 Kozlowski et al . WO WO 2007140371 A2 12 / 2007 7 , 910, 661 B2 3 /2011 Kozlowski et al . WO WO 2008052187 A2 5 / 2008 7 ,977 , 465 B2 7 / 2011 Ng et al. WO WO 2008076333 A2 6 / 2008 7 , 994 ,272 B2 8 / 2011 Kozlowski et al. WO WO 2008119036 10 / 2008 8 , 030 , 459 B2 10 /2011 Papisov et al . WO WO 2009052249 Al 4 / 2009 8 , 034 , 959 B2 10 / 2011 Ng et al. WO WO 2009117531 A1 9 / 2009 8 , 058, 385 B2 11 /2011 Kozlowski et al. WO WO 2010114940 A 10 / 2010 8 . 106 . 131 B2 1 / 2012 Kozlowski et al. WO WO 2010126552 AL 11 / 2010 8 ,227 ,555 B2 7 /2012 Kozlowski et al. WO WO 2010138719 A1 12 / 2010 8 , 227 ,558 B2 7/ 2012 Kozlowski et al . WO WO 2011120053 AL 9 / 2011 8 ,304 , 511 B2 11/ 2012 Kozlowski et al. WO WO 2011130598 A1 10 / 2011 8 , 454 , 946 B2 6 / 2013 Shen WO WO 2012066581 AL 5 / 2012 8 , 562, 965 B2 10 / 2013 McManus et al. WO WO 2013173337 A2 11/ 2013 8 , 685, 383 B2 4 /2014 Yurkovetskiy et al . 8 , 765, 111 B2 7 /2014 Shen 8 ,808 , 679 B2 8 / 2014 Yurkovetskiy et al. OTHER PUBLICATIONS 8 , 815 , 226 B2 8 / 2014 Yurkovetskiy et al . 8 , 821 , 850 B2 9 / 2014 Yurkovetskiy et al . Hamblett et al. , “ Effects of Drug Loading on the Antitumor Activity 8 , 835 ,556 B2 9 / 2014 Kozlowski et al . of a Monoclonal Antibody Drug Conjugate . " Clin . Cancer Res. 9 , 144 , 615 B2 9 /2015 Yurkovetskiy et al. 10 ( 2004 ): 7063 - 7070 . 9 , 254 , 339 B2 2 / 2016 Yurkovetskiy et al . Herceptin , Mar . 2015 , CAS 180288 -69 - 1 . 2002 /0082362 Al 6 / 2002 Brocchini et al. Lambert et al . “ Drug - Conjugated Monoclonal Antibodies for the 2004 / 0105840 A1 6 / 2004 Kinstler et al. Treatment of Cancer .” Curr. Opin . Pharmacal. 5 ( 2005 ) :543 - 549 . 2004 /0166089 Al 8 / 2004 Yu et al. Lash , “ Making the Case for Antibody -Drug Conjugates. ” In Vivo . 2005 / 0049387 Al 3 / 2005 Van et al . 28 . 11 ( 2010 ) : 32 - 38 . (Article # 2010800200 ) . 2005 /0169933 A1 8 / 2005 Steeves et al . Noguchi et al. , “ Preparation and Properties of the Immunoconjugate 2005 /0169968 A1 8 / 2005 Elmaleh et al. Composed of Anti - Human Colon Cancer Monoclonal Antibody and 2005 /0186174 A1 8 / 2005 Bossard Mitomycin C -Dextran Conjugate . " Bioconj. Chem . 3 . 2 ( 1992 ) : 132 2007/ 0190018 A1 8 /2007 Papisov et al. 137 . 2008 /0176958 Al 7 / 2008 Davis et al. 2009 /0148396 A1 6 / 2009 Akullian et al . Senter , “ Potent Antibody Drug Conjugates for Cancer Therapy. " 2010 /0129314 Al 5 / 2010 Singh et al. Curr . Opin . Chem . Biol. 13 ( 2009 ) : 1 - 10 . 2010 /0203007 AL 8 / 2010 Li et al. Tumey et al . , “ Mild Method for Succinimide Hydrolysis on ADCs : 2010 /0305149 Al 12 /2010 Yurkovetskiy et al . Impact on ADC Potency , Stability , Exposure , and Efficacy . " 2011 / 0020343 AL 1 / 2011 Senter et al. Bioconjug Chem . , 2014 : 25 ( 10 ) : 1871- 80 . 2011/ 0044967 A1 2 /2011 Elmaleh et al. Ulbrich et al. “ HPMA Copolymers With pH - Controlled Release of 2011/ 0070248 AL 3 / 2011 Ichikawa et al . Doxorubicin . ” J . Control. Release . 87 . 1 - 3 ( 2003 ) : 33 - 47 . 2011/ 0243880 A1 10 /2011 Yurkovetskiy et al. Zhao et al. , “ Synthesis and Evaluation of Hydrophilic Linkers for 2014 / 0017265 A1 1/ 2014 Yurkovetskiy et al. Antibody - Maytansinoid Conjugates .” J. Med . Chem . 2011, 54 , 2014 / 0193437 A1 7 / 2014 Lin et al . 3606 - 3623 . 2014 / 0256957 AL 9 / 2014 Shen Yurkovetskiy et al . , “ Fully Degradable Hydrophilic Polyals for 2016 /0067353 A1 3/ 2016 Yurkovetskiy et al . Protein Modification . ” Biomacromol. 6 . 5 ( 2005 ) :2648 - 2658 . Yurkovetskiy et al. , “ Synthesis of a Macromolecular Camptothecin FOREIGN PATENT DOCUMENTS Conjugate with Dual Phase Drug Release. ” Molecular Pharmaceutics, 2004 , 1( 5 ): 375 -382 . 2006 - 507232 AL 3 /2006 53 2007 -504253 AL 3 / 2007 * cited by examiner atent Apr . 17 , 2018 Sheet 1 of 5 US 9 ,943 , 609 B2

700 . Endpoint. + Vehicle + Example 8 , 15 .6 mg/ kg Example 8 , 5 .2 mg/ kg TumorVolume(mm3) e Example 8 , 1 .6 mg/ kg e?isuseš Example 8 , 0 . 5 mg/ kg + Example 6 o * * Non 22mi 20 me 36 gG *A * So* *go TN 83 DitaDays Post Dose Figure 1

1000 Endpoint Vehicle a + Trastuzumab , 15 mg/ kg Example 7 , 7 .5 mg/ kg TumorVolume(mm3) + Example 6 + Trastuzumab , 15 mg/ kg + Example 54 , 20 mg/ kg

A 8 M Days Post Dose Figure 2 atent Apr . 17 , 2018 Sheet 2 of 5 | US 9, 943, 609 B2

Endpoint Vehicle + Trastuzumab, 15 mg / kg - Example 51 / TumorVolume(mm3) + Trastuzumab, 15 mg / kg Example 52, 7 . 5 mg / kg

wwww? ??????? | 6 ppp Days Post Dose Figure 3

- Vehicle Endpoint Example 7, 3. 5mg /kg awkx3 Example 7 , 10 mg / kg , TumorVolume(mm) ??p? qdx1 Example 7, 10 mg /kg , If qWkx3

190?????? Days Post Dose

Figure 4 atent Apr . 17 , 2018 Sheet 3 of 5 US 9 ,943 , 609 B2

Vehicle + Example 60, 7 mg/ kg TumorVolume(mm3)

of H + + + + + + + + + + ? ? 4 ???? ?? ?? ? $ 4 ?$ ? ? 9 ? ? ? Days Post Dose Figure 5

1 , 000 , 000 Conjugated mL) 100 ,000 HPV - - - - 10 ,000 - - - 1 - HPV Plasmaconcentration(ng/ Trastuzumab conjugate 1, 000 ( estimate ) Total 100 Trastuzumab 10 + 0 50 100 150 200 Time (hr )

Figure 6 atent Apr . 17 , 2018 Sheet 4 of 5 US 9 ,943 , 609 B2

go

Š Tumor + Liver Š TotalDrugConcetration(ng/mL) Kidney

öngyŠ Muscle

ð

q

oot * 50 100 150 200 Time (hr )

Figure 7 U . S . Patent Apr. 17, 2018 Sheet 5 of5 ? US 9, 943, 609 B2

- - Vehicle Endpoint . - Example 70, 2mg / kg - Example 52, 2mg/kg TumorVolume(mm) ?T1 ?T1 T21 + Example 70, 4mg / kg ?Y T1 * Example 52 , 4mg/ kg ? ?IP ?T4 g3 ? ? 33338?| | | | ???????? | \ Sep???psees Days Post Dose

Figure 8 US 9 ,943 ,609 B2 PROTEIN -POLYMER -DRUG CONJUGATES tration of a drug to the target such that maximum cytotox icity for the drug is achieved . RELATED APPLICATIONS This application is a continuation of U . S . patent applica - 5 SUMMARY OF THE INVENTION tion Ser. No. 14 /457 ,955 , filed on Aug. 12 , 2014 , now The present invention relates to a protein -polymer - drug allowed , which is a continuation of U . S . patent application conjugate that is biodegradable , biocompatible and exhibits Ser. No . 13 / 944 ,561 , filed on Jul. 17 , 2013 , issued as U . S . high drug load as well as strong binding to target antigen . Pat. No . 8 , 808 ,679 on Aug . 19 , 2014 , which is a division of The present invention also relates to a polymeric scaffold U .S . patent application Ser. No . 13 /493 , 899 , filed Jun . 11, 10 useful to conjugate with a protein based recognition -mol 2012 , issued as U . S . Pat. No . 8 ,685 ,383 on Apr. 1 , 2014 , ecule (PBRM ) so as to obtain the protein - polymer- drug which claims the benefit of and priority under 35 USC § conjugate . 119 ( e ) to U . S . Provisional Patent Application Nos . 61/ 495 , In one aspect, the invention features a polymeric scaffold 771, filed Jun . 10 , 2011 ; 61/ 501 ,000 , filed Jun . 24 , 2011 ; useful to conjugate with a PBRM . The scaffold comprises a 61/ 513 , 234 , filed Jul. 29 , 2011 ; 61 /566 , 935 , filed Dec . 5 , 15 polymeric carrier, one or more - LP - D connected to the 2011 ; 61/ 605 ,618 , filed Mar. 1 , 2012 ; and 61/ 618 ,499 , filed polymeric carrier , and one or more LP connected to the Mar. 30 , 2012 . The contents of each of these applications are polymeric carrier which is suitable for connecting a PBRM hereby incorporated by reference in their entireties. to the polymeric carrier , wherein : each occurrence of D is independently a therapeutic agent BACKGROUND OF THE INVENTION 20 having a molecular weight 55 kDa ; Traditionally , pharmaceuticals have primarily consisted the polymeric carrier is a polyacetal or polyketal, of small molecules that are dispensed orally ( as solid pills LP is a linker having the structure : and liquids ) or as injectables . Over the past three decades, formulations ( i . e . , compositions that control the route and / or 25 rate of drug delivery and allow delivery of the therapeutic agent at the site where it is needed ) have become increas — RLI - C = 0 ) — DI — ingly common and complex . Nevertheless , many questions and challenges regarding the development ofnew treatments *- -~ = = + as well as the mechanisms with which to administer them 30 remain to be addressed . For example , many drugs exhibit with R 1 connected to an oxygen atom of the polymeric limited or otherwise reduced potencies and therapeutic carrier and LP connected to D , and effects because they are either generally subject to partial degradation before they reach a desired target in the body , or accumulate in tissues other than the target , or both . 35 One objective in the field of drug delivery systems, therefore , is to deliver medications intact to specifically targeted areas of the body through a system that can stabilize the drug and control the in vivo transfer of the therapeutic agent utilizing either physiological or chemical mechanisms, 40 denotes direct or indirect attachment of D to LDI, and LD or both . contains a biodegradable bond so that when the bond is Antibody - drug conjugates have been developed as target - broken , D is released from the polymeric carrier in an active specific therapeutic agents . Antibodies against various can - form for its intended therapeutic effect ; cer cell - surface antigens have been conjugated with different LD is a carbonyl- containing moiety ; cytotoxic agents that inhibit various essential cellular targets 45 LP is a linker different from LP and having the structure: such as microtubules (maytansinoids , auristatins , taxanes : - RL2 _ C ( O ) - L P1 with RL2 connected to an oxygen atom U . S . Pat. Nos. 5 , 208 ,020 ; 5 , 416 , 064 ; 6 ,333 ,410 ; 6 , 441, 163 ; of the polymeric carrier and LP suitable for connecting 6 ,340 ,701 ; 6 ,372 ,738 ; 6 ,436 ,931 ; 6 ,596 ,757 ; and 7 ,276 , 497 ) ; DNA (calicheamicin , doxorubicin , CC - 1065 analogs ; directly or indirectly to a PBRM ; U . S . Pat. Nos . 5 , 475 , 092 ; 5 , 585 ,499 ; 5 , 846 ,545 ; 6 , 534 ,660 ; 50 each of R 1 and R22 independently is absent, alkyl, het 6 ,756 ,397 ; and 6 ,630 ,579 ) . Antibody conjugates with some eroalkyl, cycloalkyl, or heterocycloalkyl; and of these cytotoxic drugs are actively being investigated in L ' is a moiety containing a functional group that is the clinic for cancer therapy (Ricart , A . D . , and Tolcher, A . capable of forming a covalent bond with a functional group W . , 2007 , Nature Clinical Practice , 4 , 245 - 255 ; Krop et al . , of a PBRM . 2010 , J . Clin . Oncol. , 28 , 2698 -2704 ) . However, existing 55 The polymeric scaffold can include one or more of the antibody - drug conjugates have exhibited a few limitations. following features , A major limitation is their inability to deliver a sufficient L ' is a linker having the structure : concentration of drug to the target site because of the limited number of targeted antigens and the relatively moderate cytotoxicity of cancer drugs like methotrexate , daunorubi- 60 cin , maytansinoids, taxanes, and vincristine . One approach - RLI - C = 0 ) — LDI? LP2 to achieving significant cytotoxicity is by linkage of a large man number of drug molecules either directly or indirectly to the antibody. However such heavily modified antibodies often display impaired binding to the target antigen and fast in 65 in which LP2 is a moiety containing a functional group that vivo clearance from the blood stream . Therefore , there is a is capable of forming a covalent bond with a functional need to improve the ability to deliver a sufficient concen group of a PBRM , and US 9 , 943 ,609 B2

is an integer from 1 to about 40 , mz is an integer from 1 to about 18 , and /or m , is an integer from 1 to about 140 ( e . g , m , being about 1 - 90 ) . When the PHF in Formula ( la ) has a molecular weight 5 ranging from about 6 kDa to about 20 kDa ( i. e . , the sum of denotes direct or indirect attachment of LP2 to LDI. m , m?, m2, and mz ranging from about 45 to about 150 ) , m2 The functional group of LP1 or LP2 is selected from is an integer from 2 to about 20 , mz is an integer from 1 to - SRP, - S — S -LG , maleimido , and halo , in which LG is a about 9 , and /or m , is an integer from 1 to about 75 ( e . g , m? leaving group and RP is H or a sulfur protecting group . 10 beingmg about 4* -45 * ). LDI comprises X - (CH ) C ( o ) with X directly When the PHF in Formula (la ) has a molecular weight connected to the carbonyl group ofR1 _ C ( = O ) , in which ranging from about 8 kDa to about 15 kDa ( i. e ., the sum of X is CH ,, O , or NH , and v is an integer from 1 to 6 . m , m?, m2 , and mz ranging from about 60 to about 110 ), m2 LP1 or LP2 contains a biodegradable bond . is an integer from 2 to about 15 , mz is an integer from 1 to Each of R21 and R22 is absent. 15 about 7 , and /or m , is an integer from 1 to about 55 ( e . g , m , The polymeric carrier of the scaffold of the invention is a being about 4 -30 ). polyacetal, e .g ., a PHF having a molecular weight (i . e. , MW When the PHF in Formula ( la ) has a molecular weight of the unmodified PHF ) ranging from about 2 kDa to about ranging from 20 kDa to 300 kDa ( i . e ., the sum of m , m , m2, 300 kDa. 20 and mz ranging from about 150 to about 2200 ) , m , is an For conjugating a PBRM having a molecular weight of 40 integer from 3 to about 300 , mz is an integer from 1 to about kDa or greater ( e . g . , 80 kDa or greater ) , the polymeric 110 , and /or m , is an integer from 1 to about 660 ( e . g , mi carrier of the scaffold of the invention is a polyacetal , e .g ., being about 10 - 250 ). a PHF having a molecular weight ( i. e ., MW of the unmodi- When the PHF in Formula (la ) has a molecular weight fied PHF) ranging from about 2 kDa to about 40 kDa ( e . g ., 25 man about 6 - 20 kDa or about 8 - 15 kDa ) . 19. 8 , 25 ranging from 40 kDa to 150 kDa ( i. e ., the sum of m , m?, m2 , For conjugating a PBRM having a molecular weight of and mz ranging from about 300 to about 1100 ) , m , is an 200 kDa or less ( e. g ., 80 kDa or less ), the polymeric carrier integer from 4 to about 150 , mz is an integer from 1 to about of the scaffold of the invention is a polyacetal, e. g. , a PHF 75, and /or m , is an integer from 1 to about 330 ( e. g, m , being having a molecular weight ( i. e ., MW of the unmodified 30 about 15 - 100 ) . PHF) ranging from about 20 kDa to about 300 kDa ( e . g . , When the PHF in Formula (la ) has a molecular weight ranging from about 50 kDa to about 100 kDa ( i . e ., the sum about 40 - 150 kDa or about 50 - 100 kDa ) . of m , m?, m2, and mz ranging from about 370 to about 740 ) , The scaffold is of Formula ( la ): m , is an integer from 5 to about 100 , mz is an integer from 35 1 to about 40 , and/ or m , is an integer from 1 to about 220 ( la) ( e .g , m , being about 15 - 80) . The scaffold further comprises a PBRM connected to the wa polymeric carrier via L . 40 One or more PBRMs are connected to one drug -carrying ??OH OH polymeric carrier . The scaffold ( e . g . , a PBRM - polymer - drug conjugate ) is of - mi1111 Formula ( Ib ) :

45 et ( Ib )

OH 50 n2 + 13mz OH OH ] ,17 OH

Jn 1

D LP2 55 wherein : m is an integer from 1 to about 2200 , B ?? m , is an integer from 1 to about 660 , m , is an integer from 1 to about 300 , 02 m2 Jm3 mz is an integer from 1 to about 110 , and the sum of m , m , , m , and mz ranges from about 15 to DI TDI about 2200 . When the PHF in Formula ( la ) has a molecular weight 65 ni mi ranging from about 2 kDa to about 40 kDa ( i. e . , the sum of m , m?, m2 , and mz ranging from about 15 to about 300 ) ,m2 US 9 ,943 ,609 B2

- continued 0 . (Ic )

OH OH ?? an4 OH ni

ni

IP2

min ?? OH PBRM , 0 12m2 wherein : DI 1 D1 mi ni | P2

OH

between LP2 and PBRM denotes direct or indirect attach M4 ment of PBRM to LP ? , each occurrence of PBRM indepen ?? [ dently has a molecular weight of less than 200 kDa , 30 m is an integer from 1 to about 2200 , wie m , is an integer from 1 to about 660 , P2 m , is an integer from 3 to about 300, nan mz is an integer from 0 to about 110 , 35 m4 is an integer from 1 to about 60 ; and wherein : the sum of m , m?, m2, mz and m4 ranges from about 150 terminal to about 2200 . 40 In Formula ( Ib ), m , is an integer from about 10 to about 660 ( e . g , about 10 - 250 ) . When the PHF in Formula ( Ib ) has a molecular weight ranging from 40 kDa to 150 kDa (i . e ., the sum of m ,m7 , m2 , as mz, and my ranging from about 300 to about 1100 ) , m , is an attached to LP2 denotes direct or indirect attachment of LP2 integer from 4 to about 150 , mz is an integer from 1 to about to PBRM such that the D -carrying polymeric carrier is 75 , m , is an integer from 1 to about 30 , and /or my is an connected to the PBRM , integer from 1 to about 330 ( e . g , m , being about 10 -330 or m is an integer from 1 to 300 , 50 m , is an integer from 1 to 140 , about 15 - 100 ) . m , is an integer from 1 to 40 , When the PHF in Formula (Ib ) has a molecular weight mz is an integer from 0 to 18 , ranging from about 50 kDa to about 100 kDa (i . e ., the sum m , is an integer from 1 to 10 ; and of m , m?, m2, mz, and m , ranging from about 370 to about the sum of m , m?, my, mz, and m , ranges from 15 to 300 ; 740 ) , m , is an integer from 5 to about 100 , m , is an integer 55 provided that the total number of L " - attached to the PBRM is 10 or less . from 1 to about 40 , m4 is an integer from 1 to about 20 , In Formula (Ic ) , m , is an integer from 1 to about 120 ( e . g , and/ or m , is an integer from 1 to about 220 ( e . g , m , being about 1- 90 ) and /or mz is an integer from 1 to about 10 (e . g , about 15 - 80 ) . about 1 - 8 ). Alternatively or additionally , one or more drug -carrying 60 When the PHF in Formula (Ic ) has a molecular weight polymeric carriers are connected to one PBRM . The scaffold ranging from about 6 kDa to about 20 kDa ( i . e ., the sum of m , m?, m2, m3, and m4 ranging from about 45 to about 150 ) , ( e . g . , a PBRM -polymer -drug conjugate ) comprises a PBRM 1m ,, is an integer from 2 to about 20 , mz is an integer from 1 with a molecular weight of greater than 40 kDa and one or to about 9 , and / or m , is an integer from 1 to about 75 ( e . g , more D -carrying polymeric carriers connected too the PBRM , 65 m , being about 4 -45 ) . in which each of the D -carrying polymeric carrier indepen When the PHF in Formula ( Ic ) has a molecular weight dently is of Formula ( Ic ): ranging from about 8 kDa to about 15 kDa (i .e ., the sum of US 9 ,943 ,609 B2 m , m?, m2, mz , and m4 ranging from about 60 to about 110 ) , -continued m , is an integer from 2 to about 15 , m , is an integer from 1 @ to about 7 , and /or m , is an integer from 1 to about 55 ( e . g , w m , being about 4 - 30 ). Each occurrence of D independently is selected from 5 - SHIA . vinca alkaloids, auristatins, tubulysins, duocarmycins , kinase inhibitors , MEK inhibitors , KSP inhibitors , and ana logs thereof. ? @ IP is R21 _ C10XD -MPI - YD -MP2 - ZP -MP3 - OP MP4 - with Mp4 directly connected to D , in which N3 XD is 04 - S — , - N (R ) — , or absent, in which R I is hydrogen , an aliphatic , heteroaliphatic , carbocyclic , or @ heterocycloalkyl moiety , C = O )RB , - C ( O )ORIB , or minm - SO2R1B , or — N (R ') — is a heterocycloalkyl moiety , - NH2; wherein RIB is hydrogen , an aliphatic , heteroaliphatic , car- 15 4 CÁNH, bocyclic , or heterocycloalkylmoiety ; each of Yº , Z , and Q ” , independently , is absent or a @ biodegradable linker moiety selected from the group con sisting of — S S - , C O ) O - , - C ( O )NR ? — , - OC (= O ) - , - NR - C ( O ) - , - OCE010 , OC 20 ( O )NR2 , NR ? C = O ) O - , - NR CEO) NR3 — , - C ( OR ) O - , - C (OR )S — , - C (ORP ) NR3 — , C (SR² ) 0 , C (SR2 ) , C (SR2 ) NR3— , C ( NRPR ) 0 , - C (NRPR3 ) S — , - C (NR²R3 ) NR * - , - C ( O ) - , - SC OAc; ( O ) , SC ( = O ) S - , OC (= O ) S , SCE= 0 ) 0 , 25 @ CES) S , SCE= S ) , OCCES) , CES) O - , - SC (= S ) O - , - OCES) S — , - OCESSO , SC ym ES) S — , - CENR2) 0 — , - CENR2) S , CENR2) NR3 — , - OCENR ?) — , - SCENR ? ) — , NR3C EE ENR ? ) — , NR 80 , - , - NR²NR , CEO) 30 NR2NRJ, NR ? NR°C ~ O ) , OC ( — O0NR2NRºº, - NR²NRC = 00 — , - C = S )NR ?NR3 — , - NR²NRC i ( S ) , C (= NR4) NR²NR3 — , NR ?NRCE = NR4) , NHNH ) ; O ( N - CR3 ) , ( CR = NO , CFO) NR2 — mi (N CR°) , ( CR > > N ) NR°C ( O ) - , SO , - , 5 . - NR SO NRG , SO NR2 — , and polyamide, wherein each occurrence of R², Rº, and R4 independently is hydro gen or an aliphatic , heteroaliphatic , carbocyclic , or hetero cyclic moiety , or each occurrence of — NR ? — or - NR²NR3 — is a heterocycloalkyl moiety ; and 4 each of MPI, MP2, MD3, and MP4 independently , is absent or a non - biodegradable linker moiety selected from the group consisting of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, a carbocyclic moiety , a het erocyclic moiety , and a combination thereof, and each of 45 w MPI, MP2, and MP3 optionally contains one or more - C = O ) — but does not contain any said biodegradable ( 10 ) linker moiety ; provided that for each LDI at least one of XD, YP , ZP , and OMe; QP is not absent. 50 Each Ph

min Ph ( 11 ) 55 - m SS mm when not connected to PBRM , independently comprises a terminal group WP, in which each WP independently is : 60 ( 12 ) S - S !wo - SH ; ON US 9, 943, 609 B2 10 - continued fiachEach R14- independency independently isto a( 13 ) RS a RS2

T COOR 3 , RS2 ? N - RSI, (14 ) 5 RSI COOR3 RSRslo mi RS2 (15 ) .oso per 1 R $ COOR $3 , HO 15 wi et in which ris 1 or 2 and each of RSI , Rs2 , and R $3 is hydrogen , ( 16) an aliphatic , heteroaliphatic , carbocyclic , or heterocy 20 cloalkyl moiety . Each Ps25 - ni IP2 when connected to PBRM , independently is - XP -MP1 - YP ( 18 ) 30 MP2 -ZP -MP3 - OP -MP4 -, with XP directly connected to the carbonyl group ofRI _ C ( O ) and MP4 directly connected to PBRM , in which XP is 04 , - S — , - N (R !) , or absent, in which R1 is hydrogen , an aliphatic , heteroaliphatic , carbocyclic , or xo 35 heterocycloalkyl moiety , CEO )RIB , CEO ) ORIB , or ( 19 ) - SO2R1B , or — N ( R ' ) — is a heterocycloalkyl moiety , wherein R1B is hydrogen , an aliphatic , heteroaliphatic , car bocyclic , or heterocycloalkyl moiety ; each of Y ? , z® , and Q , independently , is absent or a 40 biodegradable linker moiety selected from the group con NO2; sisting of S - S — , C ( O ) O - , C ( O )NR2 — , ( 20 ) OC ( = O ) — , - NR²C ( O ) - , - OCEO ) O - , - OC N ( O ) NR2 , NR²C40) 0 — , - NR²C ( O )NR3 _ , RIR - C (OR ) O - , - C (OR )S — , - C (OR )NR3 — , - C ( SR2) 450 , C (SR2 ) S — , C ( SR2) NR3 , C ( NR²R3) 0 C (NR²Rº ) , - C (NR ? R * )NR4 , - C40) S , SC ( O ) - , - S ( O ) - , - OC ( O ) - , - SC0 0 — , ( 21) CCS) S — , - SCES) - , - OCCS) , CES) O - , — SC ( S ) 0 — OCESS — , - OCS ) 0 — — SC 50 GS) S — , - CENR20 , - C NR ) — , - C NR ) or NR3 — , OC NR ?) — , - SCENR2) , NRC ( NR ?) — , - NR S02 — , - NR²NR3 — , CEO ) NR NR3 , NR NRC = 0 ) , OCEO) NR ?NR3 , - NR²NRC ( O ) O - , - C ( = S )NR²NR3 — , - NR²NRC on 55 ( S ) , CENR4) NR ?NR3 - , - NR²NRC = NR4) - O ( N = CR ) , (CR = N ) O , CEO )NR2 — ( N = CR3) , (CR = N ) NR²C40 , 50 - , - NR ’ SO NR3 — , - SO2NR2 — , and polyamide , wherein each occurrence of R², R3, and R4 independently is hydro 60 gen or an aliphatic , heteroaliphatic , carbocyclic , or hetero cyclic moiety , or each occurrence of — NR2– or in which R ' s is a leaving group ( e . g . , halide or RC ( O ) O - NRPNR3 _ is a heterocycloalkyl moiety ; and in which R is hydrogen , an aliphatic , heteroaliphatic , car each ofMP1 , MP2, MP3, and MP4 independently , is absent bocyclic , or heterocycloalkyl moiety ), RIA is a sulfur pro - or a non -biodegradable linker moiety selected from the tecting group , and ring A is cycloalkyl or heterocycloalkyl, 65 group consisting of alkyl, alkenyl, alkynyl, heteroalkyl, and R ) is hydrogen , an aliphatic , heteroaliphatic , carbocy heteroalkenyl, heteroalkynyl , a carbocyclic moiety , a het clic , or heterocycloalkyl moiety . erocyclic moiety , and a combination thereof, and each of US 9 ,943 ,609 B2 11 12 MPI , MP2 , and MP3 optionally contains one or more -continued ( C = 0 ) — but does not contain any said biodegradable linker moiety ; provided that for each manXP -$

_ _ _ 10 mi connected to PBRM , at least one of XP , YP , ZP , and is CH3 not absent. Each of Mºl and MP1 independently is C1- 6 alkuloralkyl or C1C .- 6 15 La heteroalkyl. ; and Each of MP2, MP3, MP4, MP² , MP3, and MP4 , indepen www mm dently is absent, C1- 6 alkyl , cycloalkyl, heteroalkyl , hetero cycloalkyl, or a combination thereof. 20 H2 In each 1p 25 wat NH IP2 mm - in which q is an integer from 0 to 12 and each of p and t independently is an integer from 0 to 3 . at most one of MP2 and MP3 has one of the following 30 Also within the scope of the invention is a method of structures : preparing a scaffold described above . The method comprises providing a polymeric carrier that is substituted with one or more - LP - D and one or more - R C O )- LDI, and reacting the polymeric carrier with a compound containing mm 35 an LP2 moiety to produce the scaffold comprising a poly meric carrier substituted both with one or more - LP -D and winner po with3 one or more 40 — RII - C ( = O ) — 1D -man 5 1 P2 tu hart Alternatively , the method comprises providing a polymeric carrier that is substituted with one or more IS from hot 50 - R - C ( = O ) — DI -min 5

m and one or more — RI — CEO ) -LDI , and reacting the mi polymeric carrier with D containing a functional group that ( CH3) 1- 2 is capable of forming a covalent bond with — R _ C ( O ) Ll to produce the scaffold comprising a polymeric carrier substituted both with one or more -LP - D and with one or M 60 more (CH3 ) 1 - 2 mi

- R - C ( = O ) — IDISvin mm wi 65 c = o= * US 9 ,943 ,609 B2 13 14 The invention investiga also ata features kata1 . a3emprant compound of ParadaFormula (XIIen ) 1414 or (XIIa ) :

H3C CH3 H3C . ( XII)

or NY IT: CH3 0 CH3 CH3 0 OCH3 0 CH3 NHN - - R40 (XIIa ) H3C , CH3 H3C CH3 CH3 LA

CH3 0 CH3 CH3 OCH3 0 HzC CH3 0 NHN — - K40R40 or a pharmaceutically acceptable salt thereof, - continued wherein CH , continued0 R40 is selected from the group consisting of w Rio de elected from the group comising or s NH2; @ w i w OH ; ( 10 ) w C NH2;

Xh ( 11 ) mi CH3 @ wi OH : mm beforeyou ( 12 ) JOH ; NH2;

XIL ( 13 ) CH3 Ã @ m NH2; NH2; mm Xe ( 14 ) CH3 @ mu NH2; NH2; CH3 ý ( 15 ) CH3 CH ; E mm NH2; ti g ( 16 ) NH2; so ymin hten to11- 12 NH2 XL o US 9 ,943 ,609 B2 15 16 - continued Lºl is a moiety containing a functional group that is (17 ) capable of forming a covalent bond with a functional group of a PBRM . (CH2 ) a - NH2; The D - free scaffold useful to conjugate with a PBRM and atsuit 5 a D can have one or more of the following features . ( 18 ) L ’ is a linker having the structure : wamt – C( H ( CH3 ) = ( CH2 )- NH2 ; 10 frastructures (19 ) — REI - C ( == 0 ) — 1Di ni

mu ; and in which LP2 is a moiety containing a functional group that ( 20 ) is capable of forming a covalent bond with a functional CH3 group of a PBRM , and

Y CH3CHz:; 20 w NH2 nen a is an integer from 1 to 6 ; and c is an integer from 0 to 3 . 25 R40 can be denotes direct or indirect attachment of LP2 to LDI. The functional group of Lºl or LP2 is selected from - SRP , - S - S -LG , maleimido , and halo , in which LG is a leaving group and Rp is H or a sulfur protecting group . OH Di comprises — X — (CH2 ) . C40 ) with X directly connected to the carbonyl group of R4 _ CCO ) , in which

w X is CH , O , or NH , and v is an integer from 1 to 6 . NH2 , or LP1 or LP2 contains a biodegradable bond . 35 Each of R21 and R22 is absent. CH3 The polymeric carrier of the D - free scaffold is a polyac etal, e . g . , a PHF having a molecular weight ( i. e ., MW of the unmodified PHF ) ranging from about 2 kDa to about 300 mwin 40 kDa. For conjugating a PBRM having a molecular weight of 40 In another aspect , the invention features a polymeric kDa or greater ( e. g ., 80 kDa or greater ), the polymeric scaffold useful to conjugate with both a protein based carrier of the D - free scaffold is a polyacetal , e . g ., a PHF recognition -molecule (PBRM ) and a therapeutic agent (D ). 45 having a molecular weight (i .e . , MW of the unmodified The scaffold ( i . e . , the one free of any D ) comprises a PHF ) ranging from about 2 kDa to about 40 kDa ( e . g . , about polymeric carrier, one or more L connected to the poly - 6 -20 kDa or about 8 - 15 kDa ). meric carrier which is suitable for connecting a PBRM to the For conjugating a PBRM having a molecular weight of polymeric carrier , and one or more — R1_ C ( O ) - LDI 200 kDa or less ( e . g ., 80 kDa or less ), the polymeric carrier connected to the polymeric carrier via R " , wherein : of the D - free scaffold of the invention is a polyacetal, e . g . , the polymeric carrier is a polyacetal or polyketal , a PHF having a molecular weight ( i . e . ,MW of the unmodi RZI is connected to an oxygen atom of the polymeric fied PHF ) ranging from about 20 kDa to about 300 kDa ( e . g ., carrier , about 40 - 150 kDa or about 50 - 100 kDa ). IDI is a linker suitable for connecting a D molecule to the polymeric carrier, in which each occurrence of D is inde - 55» The D - free scaffold is of Formula ( Id ): pendently a therapeutic agent having a molecular weight 55 kDa; LP is a linker different from Rii _ C ( = O ) - LDI, and ( Id ) having the structure : R22 _ C ( = O )- LP1 with RL2 con nected to an oxygen atom of the polymeric carrier and L ? 00 suitable for connecting to a PBRM ; each of R21 and RL2 independently is absent, alkyl, het I OH OH OH eroalkyl, cycloalkyl, or heterocycloalkyl; LP is a moiety containing a functional group that is 65 m1 capable of forming a covalent bond with a functional group LDI of D , and US 9 ,943 ,609 B2 17 18 - continued -continued

? OH

? m3 m3 TDI

mi 10 wi TP2 LP2 wherein : m is an integer from 1 to about 2200 , m , is an integer from 1 to about 660 , ?? mz is an integer from 1 to about 110 , and the sum of m ,m? , and mz ranges from about 15 to about m4 2200 . IDI When the PHF in Formula ( Id ) has a molecular weight 20 ranging from about 2 kDa to about 40 kDa (i .e ., the sum of m , m?, and mz ranging from about 15 to about 300 ) , mz is P2 an integer from 1 to about 18 , and/ or m , is an integer from 1 to about 140 ( e . g , m , being about 2 - 120 ) . min When the PHF in Formula ( Id ) has a molecular weight 25 PBRM ranging from about 6 kDa to about 20 kDa ( i. e ., the sum of m , m?, and mz ranging from about 45 to about 150 ), mz is wherein : an integer from 1 to about 9 , and / or m , is an integer from 1 to about 75 ( e . g , m? being about 6 -60 ) . When the PHF in Formula ( Id ) has a molecular weight 30 ranging from about 8 kDa to about 15 kDa ( i. e . , the sum of m , m , , and m , ranging from about 60 to about 110 ) , mz is an integer from 1 to about 7 , and / or m , is an integer from 1 to mm about 55 ( e . g , m , being about 6 -45 ) . When the PHF in Formula (Id ) has a molecular weight 35 ranging from 20 kDa to 300 kDa ( i . e . , the sum of m , m?, and between LP2 and PBRM denotes direct or indirect attach mz ranging from about 150 to about 2200 ) , mz is an integer ment of PBRM to LP ?, PBRM has a molecular weight of less from 1 to about 110 , and / or m , is an integer from 1 to about than 200 kDa, 660 ( e . g , m , being about 13 - 550 ) . 40 m is an integer from 1 to 2200 , When the PHF in Formula (Id ) has a molecular weight m , is an integer from 1 to 660 , ranging from 40 kDa to 150 kDa ( i. e ., the sum of m , m ], and m , ranging from about 300 to about 1100 ) , m , is an integer mz is an integer from 0 to 110 , from 1 to about 75 , and /or m , is an integer from 1 to about m4 is an integer from 1 to about 60; and 330 ( e . g , m , being about 20 - 250 ) . 45 the sum of m , m?, m2, mz and m4ranges from about 150 When the PHF in Formula (Id ) has a molecular weight to theabout 2200 . ranging from about 50 kDa to about 100 kDa ( i. e ., the sum in F of m , m , , and mz ranging from about 370 to about 740e ) ,sum mz In Formula ( le ) , m , is an integer from about 10 to about is an integer from 1 to about 40 , and / or m , is an integer from 660 ( e . g , about 14 - 550 ). 1 to about 220 ( e . g , m , being about 20 - 180 ) . When the PHF in Formula ( le ) has a molecular weight The D - free scaffold further comprises a PBRM connected ranging from 40 kDa to 150 kDa ( i . e . , the sum of m , m , , mz, to the polymeric carrier via L . and m4 ranging from about 300 to about 1100 ) , mz is an One or more PBRMs are connected to one D - free poly integer from 1 to about 75 , m , is an integer from 1 to about meric carrier. 30 , and /or m , is an integer from 1 to about 330 ( e . g , m , being The D - free scaffold is of Formula ( le ) : about 20 -250 ) . When the PHF in Formula (le ) has a molecular weight ranging from about 50 kDa to about 100 kDa ( i. e . , the sum ( le ) of m , m?, m3, and m4 ranging from about 370 to about 740 ), mz is an integer from 1 to about 40 , m4 is an integer from 1 60 to about 20 , and / or m , is an integer from 1 to about 220 ( e . g , m , being about 20 - 180 ) . Alternatively or additionally , one or more D - free poly OH OH ] OH meric carriers are connected to one PBRM . The scaffold comprises a PBRM with a molecular weight of greater than 11 65 40 kDa and one or more polymeric carriers connected to the IDI PBRM , in which each of the polymeric carrier indepen to tastedently is of Formula ( Ih ) : US 9 , 943 ,609 B2 20 As used herein , the terms " polymeric scaffold ” or simply (Ih ) " scaffold ” and “ conjugate ” are used interchangeably when the scaffold comprises one or more PBRM and one or more D molecules . In yet another aspect , the invention encompasses a con jugate comprising a polymeric carrier, one or more - LP -D OH connected to the polymeric carrier, and a protein based OH OH?? recognition -molecule (PBRM ) connected to the polymeric tapos07 - 1 carrier via L ” , wherein : LDI 10 each occurrence of D is independently a therapeutic agent ( e .g ., a drug ) having a molecular weight 55 kDa ; the polymeric carrier is a polyacetal or polyketal, LP is a linker having the structure : - R21 — C =0 XD MP YP -MP2 - ZP -MP3 - QP -MP4 - , with R 1 connected to an ??? 15 oxygen atom of the polymeric carrier and MP4 connected to D ; 03 -m3 L is a linker having the structure : R2_ C ( O ) XP D1 MP1 - YP -MP2 -ZP -MP3 - QP -MP4 -, with R12 connected to an min oxygen atom of the polymeric carrier and MP4 connected to 20 the protein based recognition -molecule ; LP2 each of Rl and RL2 independently is absent, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl; each of XD and XP , independently is 0 % , - S — , - N ( R ) — , or absent, in which R ' is hydrogen , an aliphatic , 25 heteroaliphatic , carbocyclic , or heterocycloalkyl moiety, C ORB, - C ( O )ORIB , SO2R1B or — N ( R ' ) — is m4 a heterocycloalkyl moiety , wherein RIB is hydrogen , an IDI aliphatic , heteroaliphatic , carbocyclic , or heterocycloalkyl moiety ; 30 each of YD , YP , ZP , ZP , PP , and QP , independently , is miw absent or a biodegradable linker moiety selected from the min group consisting of S S - , - C ( O )O - , - C ( O ) NR2 — , - OC30 - , - NR²C ( O ) - , - OCE00 — , - OC (= O )NR2 NR2C400 — , - NR2C = O ) wherein : 35 NR3 , C (ORP ) 0 — , C (OR ) , C (OR )NR3 _ , terminal C ( SR2) , C (SR )S — - C (SR2 ) NR3 — , C ( NRPR3 ) 0 , C (NR²R3 ) S — , - C (NR²R3 ) NR4 - C ( O ) S , - SCO ) - , - SCO ) S — , - OC O ) S - , - S ( O )O - , - CES) S — , - SCES) - , - OC 40 (ES ) , C (= S )O , SCES) O , OCCS) S , OCUS) 0 , SCES) S — , CNRP) 0 inn CNRS , CENR3) NR3 _ , OCENR2) , SC (= NR ) — , NRC = NR2) , NR 80 , — , attached to LP2 denotes direct or indirect attachment of LP2 NR ?NR , CEO) NR ?NR3 — NR NR3C ( O ) - , to PBRM such that the D - carrying polymeric carrier is 45 OC ( = O )NR2NR3 _ , — NR NRC ( = O ) O - , - C ( S ) connected to the PBRM , NR ?NR3 — , - NR²NR3C1= S ) , C ( = NR4 )NR ? NR3 — , m is an integer from 1 to 300 , - NR²NR3C ( = NR4) , O (N = CR3) , (CR = N ) m , is an integer from 1 to 140 , 0 , C ( O )NR2 (N - CR3 ) , (CR = N ) - NR²C ( 0 ) — , - S03 — , - NR SO NR3 - , SO NR2 — , and mz is an integer from 0 to 18 , 50 polyamide , wherein each occurrence of R2, R }, and R4 m4 is an integer from 1 to 10 ; and independently is hydrogen or an aliphatic , heteroaliphatic , the sum of m , m , , mz, and m , ranges from 15 to 300 ; carbocyclic , or heterocyclic moiety , or each occurrence of provided that the total number of LP2 attached to the PBRM - NR2 — or - NR²NR3 — is a heterocycloalkyl moiety ; and is 10 or less . each of MPI, MP2, MP3, MP4, MP1, MP2 , MP3 and MP4 , In Formula ( 1h ), m , is an integer from 2 to about 130 ( e .g , 55 independently , is absent or a non - biodegradable linker moi about 3 - 120 ) and / or mz is an integer from 1 to about 10 (g e . e ty selected from the group consisting of alkyl. alkenvl. about 1 - 8 ) . alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, a carbo When the PHF in Formula ( Ih ) has a molecular weight cyclic moiety , a heterocyclic moiety , and a combination ranging from about 6 kDa to about 20 kDa ( i . e ., the sum of thereof, and each of MPI, MP2 , MP3 , MP1, MP2, and MP3 m , m , mz, and m , ranging from about 45 to about 150 ) , mz 60 optionally contains one or more ( C = 0 ) — but does not is an integer from 1 to about 9 , and /or my is an integer from contain any said biodegradable linker moiety ; 6 to about 75 ( e . g , m , being about 7 -60 ) . provided that for each LP , at least one of XD , YP , ZP , and When the PHF in Formula (Ih ) has a molecular weight PP is not absent, and for each L , at least one of XP , YP , ZP , ranging from about 8 kDa to about 15 kDa ( i . e . , the sum of and QP is not absent. m , m?, m3, and m4 ranging from about 60 to about 110 ) , mz 65 The conjugate can include one or more of the following is an integer from 1 to about 7 , and / or my is an integer from features. 6 to about 55 ( e . g , m , being about 7 -45 ) . The polymeric carrier can be a polyacetal, e .g ., PHF . ? US 9, 943 , 609 B2 ? 21 ? 22 For each LP , MPI is not absent when XD is absent. - continued For each LP , MPl is not absent when XP is absent. (11 ) The polymeric carrier can be further substituted with one or more - RE _ C ( O ) - XP- MP1 - P - MP2 - w ", in which , each Wº independently is : NNNNNN

? ( 12 )

- SH; nnnnn ONY (13 )

N ; ( 14 )

NH) ; ( 15 ) ?? | NNNNNN OAc; OHH : NH ?323458893 NNNN RV . O ww NANANA NHNH ;

non RIK - 0 6 - NNNNN mmm ?OH: NNNNN NHNH2;

? OMe ; wmn Ph ?? ? ?OH: ??? Ph 33333388 US 9 ,943 ,609 B2 23 24 - continued aliphatic , heteroaliphatic , carbocyclic , or heterocycloalkyl (23 ) moiety ; and R1K is a leaving group ( e . g ., halide or RC ( O ) O in which R is hydrogen , an aliphatic , heteroaliphatic , m carbocyclic , or heterocycloalkyl moiety ) . 5 The polymeric carrier can be further substituted with one or more R22 - C O ) XP- MP1 - YP- MP2 - WP , in which each WP independently is : ?

O - SH ; dhan SR1A . -NO2 ; minmunminn RIK TO inn — N3 ; 07 - NH2; RIR N min RIJ (28 ) 30 s

RIK N

in 35 RII OAc; RIJ (29 )

Z

Vio Z 40 o8-27

(30 ) 45 NHNH ; in mm RIR

(31 ) 50 mm ni 55 (32 ) win EL ( 10 )

COOH (33 ) 60 OMe; in which R14 is a sulfur protecting group , each of ring A and Ph B , independently , is cycloalkyl or heterocycloalkyl, R " is an 65 aliphatic , heteroaliphatic , carbocyclic or heterocycloalkyl min Ph moiety ; ring D is heterocycloalkyl ; R is hydrogen , an US 9, 943, 609 B2 25 26 - continued -continued ( 11 ) ( 22 ) mm min in which R1K is a leaving group ( e . g ., halide or RC ( 0 ) 0 — (12 ) in which R is hydrogen , an aliphatic , heteroaliphatic , car 10 bocyclic , or heterocycloalkyl moiety ) , R14 is a sulfur pro tecting group , and ring A is cycloalkyl or heterocycloalkyl, ON and Rl' is hydrogen , an aliphatic , heteroaliphatic , carbocy ( 13 ) clic , or heterocycloalkyl moiety . For example , R14 is 15 m RSR $

COOR $3 no N — RSI, ( 14 ) 20 mi

?? Rs1 - COOR3COOR $3 25 RSS RS2 my RS2 OSOzR 3 , RS COORs3 , ( 15) w

HO . 30 in which ris 1 or 2 and each of RSI, R $2 and R $ 3 is hydrogen , an aliphatic , heteroaliphatic , carbocyclic , or heterocy (16 ) cloalkyl moiety . Ring A can be C3- cycloalkyl or 5 -19 membered hetero 35 cycloalkyl. Ring A can be ma $ mm

#

Ring B can be C3- cycloalkyl or 3 - 12 membered hetero cycloalkyl. (19 ) 50 Ring D can be piperazinyl or piperidinyl. Each of RsI, Rs2 , and R $3 can be hydrogen or C1- 6 alkyl. Each PBRM independently can be a peptide , a peptide mimetic , an antibody, or an antibody fragment. NO2; 55 Each of Mºl and MPl independently can be C1- 6 alkyl or (20 ) C1- 6 heteroalkyl . RIR Each of MP2, MP3, MP4, MP2 , MP3 , and MP4, indepen mu dently can be absent, C1-6 alkyl, cycloalkyl, heteroalkyl, 60 heterocycloalkyl, or a combination thereof. (21 ) For each LP , at most two of MP2, MP3 , and MP4 can be absent. For each LP , at most two of MP2, MP3, and MP4 can be mi 65 absent. For each Lº , at most one of MP2 and MP3 can have one of the following structures: US 9 ,943 ,609 B2 27 US9 , 943 ,609 B2 28 " w mm N tylnatw , wityfountant

10 OE ?? ??? ? ???ME ????? mi ? ??

$1 tytoot 20 takrat S win 25 nim m (CH3 ) 1 -2 mi ( CH3) 1 - 2

30 tykt( CH3) 1- 2 tomt( CH3) 1- 2 mm 35 win U mm

40 mm mu 45 Ips mm - CH ;

4 -50 Yo1 - , and - , and Ips ??3 CH3 Xehet . 55 Xoat

- mi S- thlytuartP .60 toni bontert in which q is an integer from 0 to 12 and each of p and t in which q is an integer from 0 to 12 and each of p and t independently is an integer from 0 to 3 . independently is an integer from 0 to 3 . 65 For each LP , each of -MP2 -Z ” , “ ZP -MP3 -, _ ZP For each L , at most one of MP2 and MP3 can have one MP2 -, and -MP3 - ZP , independently can have one of the of the following structures: following structures : US 9 ,943 ,609 B2 30 - continued ( 10 ) mm man ww min Ó Ph wi ( 11 ) pornmiwatan www . texHot ( 12 ) mm mam ? NH mm toy 09 ( 13 )

w

" toy ( 14 )

u PI min wi ( 15 ) 5

Z n w Z m amor mm th ( 16 ) mi NHN quemu Roll x ( 17 ) mm

NHN = . mi mine m thing ( 18 ) ?

RW wwowe titmuha Z mm US 9 ,943 ,609 B2 31 32 - continued -continued ( 19 ) @ 5 mm mi ?

minn @ (20 ) 10 w RIJ min E Howy mi — NH mo mm toyo HindsRIJ min mm win0=

- ; and mm

RIJ i mim " tit (24 ) theyNHN

ma nh in which ring A or B independently is cycloalkyl or hetero + cycloalkyl; R " is an aliphatic , heteroaliphatic , carbocyclic , or heterocycloalkyl moiety ; Rl is hydrogen , an aliphatic , heteroaliphatic , carbocyclic , or heterocycloalkyl moiety ; 55 and ring D is heterocycloalkyl. For each LP, each of -MP2 -ZP , ZP- MP3 -, ZP -MP2 , HHwww RIJ and -MP3 -Z - , independently , can have one of the follow ing structures: ( 10 ) 8 mm mi mim mu ? Ph US 9 ,943 ,609 B2 33 34 -continued - continued 0 ( 11 ) RIJ

wr ; and ??

W myfarersmi (12 ) wa in which ring A is cycloalkyl or heterocycloalkyl and R ) is hydrogen , an aliphatic , heteroaliphatic , carbocyclic , or het oy erocycloalkyl moiety .

Each of X and XP, independently can be absent. LTH Each of X and XP, independently can be O or NH . 07 Each of X and XP , independently can be 25 MPI YP MP2 or O = C WP m mm tot . wa

Each of YD and YP independently can be — S - S , OCO , C00 — , CONH — , or NHCO — . Each of Q and independently can be absent, S - 40 S OCO , COO , CONH , NHCO , - OCONHNH — or - NHNHCOO — . MP2 In particular, this invention features a conjugate of For mula (I ) : MP3 45

MP4 PBRM . 50 wherein each of n , n?, n2, nz, and n4, is the molar fraction of OH OH ] the corresponding polymer unit ranging between 0 and 1 ; n + ni + n2+ nz + 14 = 1; provided that none of n , n2, and n4 is 0 . In Formula ( I ) above, the disconnection or gap between 55 the polyacetal units indicates that the units can be connected to each other in any order . In other words , the appending groups that contain D , PBRM , WD , and WP, can be ran OH domly distributed along the polymer backbone . In the protein -polymer - drug conjugate of Formula ( I) , 60 each D can be the same or differentmoiety and each PBRM can be the same or different moiety . ni The ratio between n , and n , can be greater than 1 : 1 , and MD1 up to 200 : 1 ( e . g . , up to 100 : 1 ), e. g . , between 2 : 1 and 40 : 1 ; VD between 5 : 1 and 20 : 1 ; between 10 : 1 and 50 : 1 , between 25 : 1 MD2 65 and 50 : 1 , or between 30 : 1 and 50 : 1 . WD The ratio between n , and n , can be about 50 : 1 , 40 : 1 , 25 : 1 , 20 : 1 , 10 : 1 , 5 : 1 or 2 : 1 . US 9 ,943 ,609 B2 35 36 In another aspect, the invention provides compositions each group ) after IV administration of vehicle , PBRM - drug comprising the conjugates , methods for their preparation , polymer conjugate PHF -GA - (HPV - Alanine ) - ( Trastuzumab and methods of use thereof in the treatment of various M - ( PEG ) , - ) , ( Example 8 , HPV :trastuzumab about 16 : 1 to disorders, including, but not limited to cancer. 18 : 1 ) at 15 . 6 mg/ kg , 5 . 2 mg/ kg , 1 . 6 mg/kg and 0 . 5 mg/ kg The invention also features a drug - polymer conjugate 5 respectively and drug polymer conjugate PHF -GA - (HPV ( e . g . , therapeutic agent - polymer conjugate ) that is similar to Alanine ) -SH (Example 6 (dosed at a Vinca dose that was the protein - polymer - drug conjugate described above except equivalent to that present in Example 8 at 15 . 6 mg/ kg ) dosed that drug -polymer conjugate does not contain a PBRM . In once every week for 3 weeks on day 1 , day 8 and day 15 this embodiment the polymer - drug conjugate may comprise respectively . a plurality of drug moieties in which each D can be the same or different. In this embodiment, n , is 0 in the conjugate of FIG . 2 is a graph showing the tumor response in mice Formula (I ). The methods of producing the drug -polymer inoculated subcutaneously with BT474 tumors ( n = 12 for conjugates and methods of treating various disorders ( e . g . , each group ) after IV administration of vehicle ; PBRM cancer ) are also contemplated and described herein . ( trastuzumab ) at 15 mg/ kg ; PBRM - drug polymer conjugates The invention also features a protein -polymer conjugate PHF -GA - (HPV - Alanine ) -( trastuzumab -MCC ) ( Example 7 , ( e . g ., PBRM -polymer conjugate ) that is similar to the pro - 15 HPV : trastuzumab about 19 : 1 to 22 : 1 ) at 7 . 5 mg/ kg and tein -polymer - drug conjugate described above except that PHF -GA -( HPV -Alanine )- (Rituximab -MCC ) (Example 54 , protein - polymer conjugate does not contain a drug. In this HPV : Rituximab about 12 : 1 to 15 : 1 ) at 20 mg/ kg ; drug embodiment the protein - polymer conjugate may comprise a polymer conjugate PHF -GA - (HPV - Alanine ) -SH (Example plurality of protein moieties in which each PBRM can be the 6 ) (dosed at a Vinca dose that was equivalent to that present same or different. In this embodiment, n , is 0 in the conju - 20 in Example 7 at 15 mg /kg ) in combination with trastuzumab gate of Formula ( I ) . The methods of producing the drug at 15 mg/ kg dosed once every week for 3 weeks on day 1 , polymer conjugates or polymeric scaffolds and methods of day 8 and day 15 respectively . treating various disorders ( e . g ., cancer ) are also contem - FIG . 3 is a graph showing the tumor response in mice plated and described herein . The target cancer can be anal, inoculated subcutaneously with BT474 tumors ( n = 12 for astrocytoma, leukemia , lymphoma, head and neck , liver, 25 each group ) after IV administration of vehicle ; PBRM testicular, cervical, sarcoma, hemangioma, esophageal, eye , laryngeal ,mouth , mesothelioma, skin ,myeloma , oral, rectal, ( trastuzumab ) at 15 mg/ kg ; PBRM -drug polymer conjugates throat, bladder, breast , uterus, ovary , prostate , lung , colon , PHF -GA - ( Auristatin F -hydroxypropylamide - L - Alanine ) pancreas , renal, or gastric cancer . ( Trastuzumab -MCC ) (Example 52, Auristatin F : Trastu The invention further relates to a pharmaceutical compo zumab about 20 : 1 to 22 : 1 ) at 7 . 5 mg/ kg ; drug polymer sition comprising a polymeric scaffold or conjugate 30 conjugate PHF -GA - SH -( Auristatin F - propylamide- L - Ala described herein and a pharmaceutically acceptable carrier. nine ) (Example 51 ) ( dosed at an auristatin dose that was In yet another aspect , the invention relates to a method of equivalent to that present in Example 52 at 15 mg/ kg ) in diagnosing a disorder in a subject suspected of having the combination with trastuzumab at 15 mg/ kg dosed once every disorder . The method comprises administering an effective week for 3 weeks on day 1 , day 8 and day 15 respectively . amount of the conjugate described herein to the subject 35 FIG . 4 is a graph showing the tumor response in mice suspected of having the disorder or performing an assay to inoculated subcutaneously with BT474 tumors ( n = 10 for detect a target antigen /receptor in a sample from the subject each group ) after IV administration of vehicle ; PBRM - drug so as to determine whether the subject expresses target polymer conjugates PHF -GA - (HPV - Alanine ) - ( Trastu antigen or receptor. zumab -MCC ) (Example 7 , HPV : trastuzumab about 19 : 1 to Unless otherwise defined , all technical and scientific 40 22 : 1 ) at 3 . 5 mg/ kg dosed once every week for 3 weeks on terms used herein have the same meaning as commonly day 1 , day 8 and day 15 respectively ; PBRM -drug polymer understood by one of ordinary skill in the art to which this conjugates PHF -GA - (HPV - Alanine ) - ( Trastuzumab -MCC ) invention belongs . In the specification , the singular forms (Example 7 , HPV : trastuzumab about 19 : 1 to 22 : 1 ) at 10 also include the plural unless the context clearly dictates mg/ kg dosed as a single dose on day 1 ; PBRM - drug polymer otherwise . Although methods and materials similar or 45 conjugates PHF -GA - (HPV - Alanine ) - ( Trastuzumab -MCC ) equivalent to those described herein can be used in the (Example 7 , HPV : trastuzumab about 19 : 1 to 22 : 1 ) at 10 practice or testing of the present invention , suitable methods mg/ kg dosed once every week for 3 weeks on day 17 , day and materials are described below . All publications, patent 2 4 and day 31 respectively. applications , patents and other references mentioned herein FIG . 5 is a graph showing the tumor response in mice are incorporated by reference . The references cited herein 50 inoculated subcutaneously with BT474 tumors ( n = 10 for are not admitted to be prior art to the claimed invention . In each group ) after IV administration of vehicle or 30 kDa the case of conflict, the present specification , including PHF -GA - (HPV - Alanine ) - ( Trastuzumab -Fab ) ( Example 60 , definitions, will control. In addition , the materials , methods HPV : trastuzumab - Fab about 10 : 1 to 14 : 1 ) at 7 mg/ kg dosed and examples are illustrative only and are not intended to be once every week for 3 weeks on day 1 , day 8 and day 15 limiting . 55 respectively . One of the advantages of the present invention is that the FIG . 6 is a graph showing the plasma PK for the conju protein - polymer -drug conjugates or the polymeric scaffolds gated HPV and trastuzumab after IV bolus administration of described herein greatly enhances the bioavailability of the PBRM - drug -conjugate PHF -GA -( HPV - Alanine ) - ( Trastu drugs to be delivered and /or enhances the bioavailability of zumab - M - ( PEG ) 2 ) as in Example 8 (HPV :trastuzumab the protein attached to the polymeric carrier . Other features 60 about 16 : 1 to 18 : 1) at 15 mg/kg (based on trastuzumab ). and advantages of the invention will be apparent from the FIG . 7 is a graph showing the accumulation of HPV in following detailed description and claims. various organs of the mice after IV bolus administration of PBRM -drug -conjugate PHF -GA - (HPV -Alanine ) -( Trastu BRIEF DESCRIPTION OF FIGURES zumab - M - ( PEG ) ) as in Example 8 (HPV : trastuzumab 65 about 16 : 1 to 18: 1 ) at 15 mg/ kg (based on trastuzumab ) . FIG . 1 is a graph showing the tumor response in mice FIG . 8 is a graph showing the tumor response in mice inoculated subcutaneously with NCI- N87 cells (n = 10 for inoculated subcutaneously with BT474 tumors (n = 10 for US 9 ,943 ,609 B2 37 38 each group ) after IV administration of vehicle ; PBRM - drug “ Protecting group” : as used herein , the term protecting polymer conjugates PHF -GA - (Auristatin F -hydroxypropyl - group means that a particular functional moiety , e . g . , O , S , amide - L - Alanine ) - ( Trastuzumab -MCC ) (Example 52 , or N , is temporarily blocked so that a reaction can be carried Auristatin F : Trastuzumab about 24 : 1 to 28 : 1 ) and drug out selectively at another reactive site in a multifunctional polymer conjugate PHF -GA -SS -Dimethyl - NO , -( Auristatin 5 compound . In preferred embodiments , a protecting group F -hydroxypropylamide - L -Alanine ) - ( S - S - Trastuzumab ) reacts selectively in good yield to give a protected substrate that is stable to the projected reactions; the protecting group ( Example 70 , Auristatin F : Trastuzumab about 9 : 1 to 13 : 1 ) at must be selectively removed in good yield by readily 2 mg/ kg and 4 mg/ kg dosed once every week for 3 weeks on available, preferably nontoxic reagents that do not attack the day 1 , day 8 and day 15 respectively . 10 other functional groups ; the protecting group forms an easily separable derivative (more preferably without the generation DETAILED DESCRIPTION OF CERTAIN of new stereogenic centers ) ; and the protecting group has a PREFERRED EMBODIMENTS OF THE minimum of additional functionality to avoid further sites of INVENTION reaction . As detailed herein , oxygen , sulfur, nitrogen and 15 carbon protecting groups may be utilized . For example , in The present invention provides novel protein -polymer certain embodiments , certain exemplary oxygen protecting drug conjugates, polymeric scaffolds for making the conju groups may be utilized . These oxygen protecting groups gates , synthetic methods for making the conjugates or poly include , but are not limited to methyl ethers, substituted meric scaffolds , pharmaceutical compositions containing methyl ethers ( e . g ., MOM (methoxymethyl ether ) , MTM them and various uses of the conjugates . 20 (methylthiomethyl ether ), BOM (benzyloxymethyl ether ), The present invention also provides novel polymer -drug and PMBM ( p -methoxybenzyloxymethyl ether ) ) , substi conjugates, synthetic methods for making the conjugates, tuted ethyl ethers , substituted benzyl ethers , silyl ethers pharmaceutical compositions containing them and various ( e .g ., TMS ( trimethylsilyl ether ), TES (triethylsilylether ), uses of the conjugates . TIPS ( triisopropylsilyl ether ), TBDMS (t - butyldimethylsilyl The present invention further provides novel drug deriva - 25 ether ), tribenzyl silyl ether, and TBDPS (t -butyldiphenyl tives, synthetic methods for making the derivatives, phar - silyl ether ), esters ( e . g . , formate , acetate , benzoate ( Bz ) , maceutical compositions containing them and various uses trifluoroacetate , and dichloroacetate ), carbonates, cyclic of the drug derivatives . acetals and ketals . In certain other exemplary embodiments , nitrogen protecting groups are utilized . Nitrogen protecting Definition / Terminology 30 groups , as well as protection and deprotection methods are known in the art . Nitrogen protecting groups include , but are Certain compounds of the present invention , and defini - not limited to , carbamates ( including methyl, ethyl and tions of specific functional groups are also described in more substituted ethyl carbamates ( e . g . , Troc ) , amides , cyclic detail herein . For purposes of this invention , the chemical imide derivatives , N - Alkyl and N - Aryl amines, imine elements are identified in accordance with the Periodic Table 35 derivatives, and enamine derivatives. In yet other embodi of the Elements , CAS version , Handbook of Chemistry and ments , certain exemplary sulphur protecting groups may be Physics , 75th Ed . , inside cover, and specific functional utilized . The sulfur protecting groups include, but are not groups are generally defined as described therein . Addition - limited to those oxygen protecting group describe above as ally , general principles of organic chemistry, as well as well as aliphatic carboxylic acid ( e . g ., acrylic acid ) , maleim specific functional moieties and reactivity , are described in 40 ide , vinyl sulfonyl, and optionally substituted maleic acid . " Organic Chemistry ” , Thomas Sorrell , University Science Certain other exemplary protecting groups are detailed Books, Sausalito : 1999 , the entire contents of which are herein , however, it will be appreciated that the present incorporated herein by reference. Furthermore, it will be invention is not intended to be limited to these protecting appreciated by one of ordinary skill in the art that the groups ; rather, a variety of additional equivalent protecting synthetic methods , as described herein , utilize a variety of 45 groups can be readily identified using the above criteria and protecting groups. utilized in the present invention . Additionally , a variety of The use of the articles “ a ” , “ an ” , and “ the ” in both the protecting groups are described in " Protective Groups in following description and claims are to be construed to Organic Synthesis ” Third Ed . Greene , T . W . and Wuts , P . G . , cover both the singular and the plural, unless otherwise Eds. , John Wiley & Sons , New York : 1999 , the entire indicated herein or clearly contradicted by context . The 50 contents of which are hereby incorporated by reference . terms “ comprising ” , “ having” , “ including" , and " contain - “ Leaving group ” refers to a molecular fragment that ing" are to be construed as open terms ( i .e ., meaning departs with a pair of electrons in heterolytic bond cleavage . “ including but not limited to " ) unless otherwise noted . Leaving groups can be anions or neutral molecules . Leaving Additionally whenever “ comprising ” or another open - ended groups include, but are not limited to halides such as Cl- , term is used in an embodiment, it is to be understood that the 55 Br , and I" , sulfonate esters , such as para - toluenesulfonate same embodiment can be more narrowly claimed using the (“ ftosylate” , TSO - ) , and RC ( 0 ) 0 in which R is hydrogen , intermediate term “ consisting essentially of” or the closed an aliphatic , heteroaliphatic , carbocyclic , or heterocy term " consisting of. ” cloalkyl moiety . Recitation of ranges of values are merely intended to All methods described herein can be performed in any serve as a shorthand method of referring individually to each 60 suitable order unless otherwise indicated herein or otherwise separate value falling within the range , unless otherwise clearly contradicted by context. The use of any and all indicated herein , and each separate value is incorporated into examples, or exemplary language ( e . g . , " such as ” ) provided the specification as if it were individually recited herein . A herein , is intended merely to better illustrate the invention range used herein , unless otherwise specified , includes the and is not to be construed as a limitation on the scope of the two limits of the range . For example , the expressions “ X 65 claims unless explicitly otherwise claimed . No language in being an integer between 1 and 6 ” and “ x being an integer the specification is to be construed as indicating that any of 1 to 6 " both mean “ x being 1 , 2 , 3 , 4 , 5 , or 6 ” . non - claimed element is essential to what is claimed . US 9 ,943 ,609 B2 39 40 “ Antibody ” refers to an immunoglobulin molecule of the such as , e . g. , antineoplastic agents ), compounds are “ bio class IgG including but not limited to IgG subclasses ( IgG1, compatible” if their addition to normal cells in vitro , at 2 , 3 and 4 ) and class IgM which is able to specifically bind concentrations similar to the intended systemic in vivo to a specific epitope on an antigen . Antibodies can be intact concentrations, results in less than or equal to 1 % cell death immunoglobulins derived from natural sources or from 5 during the time equivalent to the half - life of the compound recombinant sources and can be immunoreactive portions of in vivo ( e . g . , the period of time required for 50 % of the intact immunoglobulins. Antibodies may exist in a variety of compound administered in vivo to be eliminated / cleared ) , forms including, for example , polyclonal antibodies , mono - and their administration in vivo induces minimal and medi clonal antibodies, camelized single domain antibodies, intra cally acceptable inflammation , foreign body reaction , immu cellular antibodies ( “ intrabodies” ), recombinant antibodies , 10 notoxicity , chemical toxicity and / or other such adverse anti- idiotypic antibodies , domain antibodies, linear anti - effects . In the above sentence, the term “ normal cells ” refers body , multispecific antibody, antibody fragments, such as, to cells that are not intended to be destroyed or otherwise Fv, Fab , Fab ', Fab '- SH , F (ab ') 2 , single chain variable frag - significantly affected by the compound being tested . ment antibodies ( scFv ) , Fc , pFc ' , scFvFc , disulfide Fv ( dsfv ) , “ Biodegradable ” : As used herein , " biodegradable ” poly bispecific antibodies (bc - scFv ) such as BiTE antibodies ; 15 mers are polymers that are susceptible to biological process camelid antibodies , resurfaced antibodies, humanized anti - ing in vivo . As used herein , “ biodegradable ” compounds or bodies , fully human antibodies, single - domain antibody moieties are those that, when taken up by cells , can be ( sdAb , also known as NANOBODY® ) , chimeric antibodies, broken down by the lysosomal or other chemical machinery chimeric antibodies comprising at least one human constant or by hydrolysis into components that the cells can either region , dual -affinity antibodies such as , dual -affinity retar - 20 reuse or dispose of without significant toxic effect on the geting proteins (DARTTM ), divalent (or bivalent) single - cells . The term “ biocleavable ” as used herein has the same chain variable fragments (di - scFvs , bi- scFvs ) including but meaning of " biodegradable ” . The degradation fragments not limited to minibodies, diabodies, triabodies or tribodies, preferably induce little or no organ or cell overload or tetrabodies , and the like , and multivalent antibodies . “ Anti - pathological processes caused by such overload or other body fragment” refers to at least a portion of the variable 25 adverse effects in vivo . Examples of biodegradation pro region of the immunoglobulin molecule that binds to its cesses include enzymatic and non - enzymatic hydrolysis , target , i. e ., the antigen -binding region . As used herein , the oxidation and reduction . Suitable conditions for non - enzy term “ antibody ” refers to both the full - length antibody and matic hydrolysis of the biodegradable protein -polymer - drug antibody fragments unless otherwise specified . conjugates (or their components, e . g ., the biodegradable " Protein based recognition -molecule ” or “ PBRM ” refers 30 polymeric carrier and the linkers between the carrier and the to a molecule that recognizes and binds to a cell surface antibody or the drug molecule ) described herein , for marker or receptor such as, a transmembrane protein , sur example , include exposure of the biodegradable conjugates face immobilized protein , or protoglycan . Examples of to water at a temperature and a pH of lysosomal intracellular PBRMs include but are not limited to , antibodies ( e . g ., compartment. Biodegradation of some protein - polymer Trastuzumab , Cetuximab , Rituximab , Bevacizumab , 35 drug conjugates (or their components , e . g . , the biodegrad Epratuzumab , Veltuzumab , Labetuzumab ) or peptides able polymeric carrier and the linkers between the carrier (LHRH receptor targeting peptides , EC - 1 peptide) , lipoca - and the antibody or the drug molecule ), can also be lins , such as , for example , anticalins , proteins such as , for enhanced extracellularly , e . g . in low pH regions of the example, interferons, lymphokines , growth factors, colony animal body , e . g . an inflamed area , in the close vicinity of stimulating factors , and the like , peptides or peptide mimics, 40 activated macrophages or other cells releasing degradation and the like . The protein based recognition molecule, in facilitating factors . In certain preferred embodiments , the addition to targeting the modified polymer conjugate to a effective size of the polymer carrier at pH - 7 . 5 does not specific cell , tissue or location , may also have certain detectably change over 1 to 7 days , and remains within 50 % therapeutic effect such as antiproliferative ( cytostatic and /or of the original polymer size for at least several weeks . At cytotoxic ) activity against a target cell or pathway . The 45 pH - 5 , on the other hand , the polymer carrier preferably protein based recognition molecule comprises or may be detectably degrades over 1 to 5 days , and is completely engineered to comprise at least one chemically reactive transformed into low molecular weight fragments within a group such as , - COOH , primary amine , secondary amine two - week to several -month time frame . Polymer integrity in — NHR , — SH , or a chemically reactive amino acid moiety such tests can be measured , for example , by size exclusion or side chains such as , for example , tyrosine, histidine , 50 HPLC . Although faster degradation may be in some cases cysteine, or lysine . preferable , in general it may be more desirable that the “ Biocompatible” as used herein is intended to describe polymer degrades in cells with the rate that does not exceed compounds that exert minimal destructive or host response the rate ofmetabolization or excretion of polymer fragments effects while in contact with body fluids or living cells or by the cells . In preferred embodiments, the polymers and tissues . Thus a biocompatible group , as used herein , refers to 55 polymer biodegradation byproducts are biocompatible . an aliphatic , cycloalkyl, heteroaliphatic , heterocycloalkyl, “ Bioavailability " : The term “ bioavailability ” refers to the aryl, or heteroaryl moiety , which falls within the definition systemic availability (i . e ., blood /plasma levels ) of a given of the term biocompatible , as defined above and herein . The amount of drug or compound administered to a subject . term " Biocompatibility ” as used herein , is also taken to Bioavailability is an absolute term that indicates measure mean that the compounds exhibit minimal interactions with 60 ment of both the time (rate ) and total amount (extent ) of drug recognition proteins, e . g ., naturally occurring antibodies, or compound that reaches the general circulation from an cell proteins , cells and other components of biological administered dosage form . systems, unless such interactions are specifically desirable. “Hydrophilic ” : The term “ hydrophilic ” as it relates to Thus , substances and functional groups specifically intended substituents on the polymer monomeric units does not to cause the above minimal interactions, e . g . , drugs and 65 essentially differ from the common meaning of this term in prodrugs, are considered to be biocompatible . Preferably the art, and denotes chemical moieties which contain ion (with exception of compounds intended to be cytotoxic , izable , polar, or polarizable atoms, or which otherwise may US 9 , 943, 609 B2 41 42 be solvated by water molecules . Thus a hydrophilic group , artificially created ( e . g ., via chemical synthesis ) that have a as used herein , refers to an aliphatic , cycloalkyl, heteroali relatively low molecular weight. Preferred small molecules phatic , heterocycloalkyl, aryl or heteroaryl moiety , which are biologically active in that they produce a local or falls within the definition of the term hydrophilic , as defined??? systemic effect in animals , preferably mammals , more pref above . Examples of particular hydrophilic organic moieties 5 erably humans . In certain preferred embodiments , the small which are suitable include , without limitation , aliphatic or molecule is a drug and the small molecule is referred to as heteroaliphatic groups comprising a chain of atoms in a “ drug molecule ” or “ drug ” or “ therapeutic agent” . In certain range of between about one and twelve atoms, hydroxyl, embodiments , the drug molecule has MW less than or equal hydroxyalkyl , amine, carboxyl, amide , carboxylic ester, to about 5 kDa . In other embodiments , the drug molecule has thioester , aldehyde, nitryl, isonitryl, nitroso , hydroxylamine , 10 MW less than or equal to about 1 . 5 kDa . In embodiments , mercaptoalkyl, heterocycle , carbamates , carboxylic acids the drug molecule is selected from vinca alkaloids , aurista and their salts , sulfonic acids and their salts , sulfonic acid tins, tubulysins, duocarmycins, kinase inhibitors, MEK esters , phosphoric acids and their salts , phosphate esters , inhibitors , KSP inhibitors , and analogs thereof. Preferably , polyglycol ethers , polyamines , polycarboxylates , polyesters though not necessarily , the drug is one that has already been and polythioesters . In preferred embodiments of the present 15 deemed safe and effective for use by an appropriate gov invention , at least one of the polymer monomeric units ernmental agency or body , e . g . , the FDA . For example , include a carboxyl group (COOH ), an aldehyde group drugs for human use listed by the FDA under 21 C . F . R . (CHO ) , a methylol (CH2OH ) or a glycol ( for example , $ $ 330 . 5 , 331 through 361, and 440 through 460 ; drugs for CHOH - CH2OH or CH — (CH2OH )2 ) . veterinary use listed by the FDA under 21 C . F . R . $ 8500 The term “ hydrophilic ” as it relates to the polymers of the 20 through 589 , incorporated herein by reference , are all con invention generally does not differ from usage of this term sidered suitable for use with the present hydrophilic poly in the art , and denotes polymers comprising hydrophilic mers . functional groups as defined above . In a preferred embodi Classes of drug molecules that can be used in the practice ment, hydrophilic polymer is a water -soluble polymer . of the present invention include , but are not limited to , Hydrophilicity of the polymer can be directly measured 25 anti - cancer substances , radionuclides , vitamins , anti - AIDS through determination of hydration energy, or determined substances, antibiotics , immunosuppressants , anti - viral sub through investigation between two liquid phases, or by stances, enzyme inhibitors , neurotoxins , opioids, hypnotics , chromatography on solid phases with known hydrophobic anti - histamines , lubricants , tranquilizers , anti - convulsants , ity, such as , for example , C4 or C18 . muscle relaxants and anti -Parkinson substances , anti -spas " Polymeric Carrier " : The term polymeric carrier , as used 30 modics and muscle contractants including channel blockers , herein , refers to a polymer or a modified polymer, which is miotics and anti- cholinergics , anti - glaucoma compounds , suitable for covalently attaching to or can be covalently anti- parasite and /or anti -protozoal compounds, modulators attached to one or more drug molecules with a designated of cell - extracellular matrix interactions including cell linker and / or one or more PBRMs with a designated linker. growth inhibitors and anti - adhesion molecules , vasodilating “ Physiological conditions " : The phrase " physiological 35 agents , inhibitors of DNA , RNA or protein synthesis , anti conditions ” , as used herein , relates to the range of chemical hypertensives , analgesics, anti -pyretics , steroidal and non ( e . g . , pH , ionic strength ) and biochemical (e . g ., enzyme steroidal anti - inflammatory agents, anti - angiogenic factors , concentrations ) conditions likely to be encountered in the anti- secretory factors , anticoagulants and / or antithrombotic extracellular fluids of living tissues . For most normal tissues , agents, local anesthetics, ophthalmics, prostaglandins , anti the physiological pH ranges from about 7 . 0 to 7 . 4 . Circu - 40 depressants , anti- psychotic substances , anti - emetics , imag lating blood plasma and normal interstitial liquid represent ing agents. Many large molecules are also drugs. typical examples of normal physiological conditions. A more complete , although not exhaustive , listing of “ Polysaccharide” , " carbohydrate ” or “ oligosaccharide” : classes and specific drugs suitable for use in the present The terms " polysaccharide” , " carbohydrate " , or " oligosac - invention may be found in “ Pharmaceutical Substances : charide ” are known in the art and refer, generally , to sub - 45 Syntheses , Patents , Applications ” by Axel Kleemann and stances having chemical formula (CH2O ) n , where generally Jurgen Engel, Thieme Medical Publishing , 1999 and the n > 2 , and their derivatives. Carbohydrates are polyhydroxy - “Merck Index : An Encyclopedia of Chemicals , Drugs, and aldehydes or polyhydroxyketones , or change to such sub Biologicals ” , Edited by Susan Budavari et al. , CRC Press , stances on simple chemical transformations , such as hydro - 1996 , both of which are incorporated herein by reference . In lysis , oxidation or reduction . Typically , carbohydrates are 50 preferred embodiments , the drug used in this invention is a present in the form of cyclic acetals or ketals (such as, therapeutic agent that has antiproliferative (cytostatic and / or glucose or fructose ). These cyclic units (monosaccharides ) cytotoxic ) activity against a target cell or pathway . The drug may be connected to each other to form molecules with few may have a chemically reactive group such as , for example , ( oligosaccharides ) or several (polysaccharides ) monosac - COOH , primary amine , secondary amine — NHR , OH , charide units . Often , carbohydrates with well defined num - 55 SH , - C ( O ) H , C (O ) R , C ( O ) NHR25 , C ( S )OH , ber, types and positioning of monosaccharide units are - S ( O ) ,OR25 , — P (0 ) OR25 , CN , NC or ONO , in called oligosaccharides , whereas carbohydrates consisting which R is an aliphatic , heteroaliphatic , carbocyclic or of mixtures of molecules of variable numbers and /or posi heterocycloalkyl moiety and R25 is a hydrogen , an aliphatic , tioning ofmonosaccharide units are called polysaccharides . heteroaliphatic , carbocyclic , or heterocyclic moiety . The terms “ polysaccharide ” , " carbohydrate ” , and “ oligosac - 60 “ Drug derivative ” or “ modified drug ” or the like as used charide ” , are used herein interchangeably . A polysaccharide herein , refers to a compound that comprises the drug mol may include natural sugars ( e . g . , glucose , fructose , galac - ecule intended to be delivered by the conjugate of the tose , mannose , arabinose , ribose , and xylose ) and /or deriva invention and a functional group capable of attaching the tives of naturally occurring sugars ( e . g ., 2 ' - fluororibose , drug molecule to the polymeric carrier. 2 ' - deoxyribose , and hexose ) . 65 “ Active form " as used herein refers to a form of a " Small molecule ” : As used herein , the term " small mol - compound that exhibits intended pharmaceutical efficacy in ecule ” refers to molecules, whether naturally -occurring or vivo or in vitro . In particular , when a drugmolecule intended US 9 ,943 ,609 B2 43 44 to be delivered by the conjugate of the invention is released ing in the formation of a stable compound . Representative from the conjugate , the active form can be the drug itself or alkynyl groups include ethynyl, 2 -propynyl (propargyl ) , its derivatives , which exhibit the intended therapeutic prop - 1 -propynyl , and the like . erties. The release of the drug from the conjugate can be In certain embodiments , the alkyl, alkenyl and alkynyl achieved by cleavage of a biodegradable bond of the linker 5 groups employed in the invention contain about 1- 20 ali which attaches the drug to the polymeric carrier. The active phatic carbon atoms. In certain other embodiments , the drug derivatives accordingly can comprise a portion of the alkyl , alkenyl, and alkynyl groups employed in the invention contain about 1 - 10 aliphatic carbon atoms. In yet other linker. embodiments , the alkyl, alkenyl, and alkynyl groups “ Diagnostic label” : As used herein , the term diagnostic 10 employed in the invention contain about 1 -8 aliphatic carbon label refers to an atom , group of atoms, moiety or functional atoms. In still other embodiments , the alkyl, alkenyl, and group , a nanocrystal , or other discrete element of a compo alkynyl groups employed in the invention contain about 1 - 6 sition ofmatter , that can be detected in vivo or ex vivo using aliphatic carbon atoms. In yet other embodiments , the alkyl, analytical methods known in the art . When associated with alkenyl, and alkynyl groups employed in the invention a conjugate of the present invention , such diagnostic labels 15 contain about 1 - 4 carbon atoms. Illustrative aliphatic groups permit the monitoring of the conjugate in vivo . Alternatively thus include , but are not limited to , for example, methyl. or additionally, constructs and compositions that include ethyl, n -propyl , isopropyl, allyl, n -butyl , sec - butyl, isobutyl, diagnostic labels can be used to monitor biological functions tert - butyl, n -pentyl , sec -pentyl , isopentyl, tert -pentyl , or structures . Examples of diagnostic labels include , without n -hexyl , sec -hexyl , moieties and the like , which again , may limitation , labels that can be used in medical diagnostic 20 bear one or more substituents . Alkenyl groups include , but procedures, such as, radioactive isotopes (radionuclides ) for are not limited to , for example , ethenyl, propenyl, butenyl, gamma scintigraphy and Positron Emission Tomography 1 -methyl - 2 -buten - 1 - yl, and the like . Representative alkynyl (PET ) , contrast agents for Magnetic Resonance Imaging groups include , but are not limited to , ethynyl, 2 - propynyl (MRI ) ( for example paramagnetic atoms and superparamag - (propargyl ) , 1 - propynyl and the like . netic nanocrystals ) , contrast agents for computed tomogra - 25 “ Alkylene ” as used herein , the term alkylene by itself or phy and other X - ray - based imaging methods, agents for part of another term refers to a saturated , branched or ultrasound - based diagnostic methods ( sonography ) , agents straight chain having two monovalent radical centers for neutron activation ( e. g ., boron , gadolinium ), fluoro derived by the removal of two hydrogen atoms from the phores for various optical procedures, and , in general moi same or two different carbon atoms of a parent alkane . 30 Alkylene radicals include , but are not limited to , methylene , eties which can emit, reflect , absorb , scatter or otherwise 1 , 2, ethylene, 1, 3 -propyl , and the like . Suitable alkylenes affect electromagnetic fields or waves ( e .g . gamma- rays, include, but are not limited to methylene , ethylene, propyl X -rays , radiowaves, microwaves , light) , particles (e . g. alpha ene , butylene , pentylene , hexylene , heptylene , ocytylene , particles , electrons , positrons, neutrons , protons ) or other nonylene , decalene , and the like . The term “ cycloalkylene ” forms of radiation , e . g . ultrasound . 35 similarly refers to bivalent cycloalkyl. Cycloalkylene radi “ Aliphatic ” : In general, the term aliphatic , as used herein , cals include, but are not limited to , 1 , 1 - cyclopentylene , includes both saturated and unsaturated , straight chain (1 . e ., 1 . 2 - cyclopentylene , 1 , 1 -cyclobutylene , 1 , 3 - cyclobutylene , unbranched ) or branched aliphatic hydrocarbons , which are etc optionally substituted with one or more functional groups . “ Heteroaliphatic ” : as used herein , the term heteroaliphatic As will be appreciated by one of ordinary skill in the art , 40 refers to aliphatic moieties in which one or more carbon " aliphatic ” is intended herein to include , but is not limited to , atoms in the main chain have been substituted with a alkyl, alkenyl, alkynyl moieties . Thus, as used herein , the heteroatom . Thus, a heteroaliphatic group refers to an ali term “ alkyl” includes straight and branched alkyl groups. An phatic chain which contains one or more oxygen , sulfur, analogous convention applies to other generic terms such as nitrogen , phosphorus or silicon atoms, e . g ., in place of " alkenyl " , " alkynyl” and the like . Furthermore , as used 45 carbon atoms. Heteroaliphatic moieties may be branched or herein , the terms “ alkyl” , “ alkenyl” , “ alkynyl” and the like linear unbranched . In certain embodiments , heteroaliphatic encompass both substituted and unsubstituted groups. In moieties are substituted (“ substituted heteroaliphatic ” ) by certain embodiments , as used herein , “ lower alkyl" is used independent replacement of one or more of the hydrogen to indicate those alkyl groups ( substituted , unsubstituted , atoms thereon with one or more moieties including , but not branched or unbranched ) having about 1 - 6 carbon atoms. 50 limited to aliphatic ; heteroaliphatic ; cycloalkyl ; heterocy “ Alkenyl” : the term alkenyl denotes a monovalent group cloalkyl; aryl; heteroaryl ; alkylaryl; alkylheteroaryl; alkoxy ; derived from a hydrocarbon moiety having at least one aryloxy ; heteroalkoxy ; heteroaryloxy ; alkylthio ; arylthio ; carbon - carbon double bond by the removal of a single heteroalkylthio ; heteroarylthio ; F ; Cl; Br; I ; - N02 ; CN ; hydrogen atom .“ Substituted alkenyl” groups are substituted CF3 ; - CH2Cp3; CHC12 ; CH2OH ; CH2CH2OH ; with one or more functional groups . Substituents include , 55 CH2NH2 ; _ Ch2S02CH3; - or -GRG1 wherein G is O - , but are not limited to , any of the substituents mentioned S — , - NRG2 — , - C ( O ) - , - S ( O ) - , - S027 , below , i. e ., the substituents recited below resulting in the CeO ) O - , - C ( O )NRG2 — , - OC = 0 ) , - NRG2C formation of a stable compound . Alkenyl groups include , for FO ) ; OCTO) O - , - OCEO )NRG2 — , - NRG2C example , ethenyl, propenyl, butenyl, 1 -methyl - 2 -buten - 1 - yl, ( O ) O - , - NRG2C ( = O ) NRG2 — , CES) , CES) and the like . 60 S , SCS) , SCS) S — , -CNRG2 ) , “ Alkynyl” : the term alkynyl as used herein refers to a C ( = NRG2 ) 0 Ce= NRG2) NRG3 — OC monovalent group derived from a hydrocarbon having at GNRG2) — , - NRG2C = NRG ) , - NRG2S02 – , least one carbon - carbon triple bond by the removal of a - NRG2S02NRG3 — , or S . NRG2 — , wherein each occur single hydrogen atom . “ Substituted alkenyl” groups are rence of RG1, RG2 and RG3 independently includes , but is not substituted with one or more functional groups . Substituents 65 limited to , hydrogen , halogen , or an optionally substituted include, but are not limited to , any of the substituents aliphatic , heteroaliphatic , cycloalkyl, heterocycloalkyl, aryl , mentioned below , i. e ., the substituents recited below result heteroaryl, alkylaryl , or alkylheteroaryl moiety . Additional US 9 ,943 ,609 B2 45 46 examples of generally applicable substituents are illustrated In the case of multicyclic aromatic rings , only one of the by the specific embodiments shown in the Examples that are rings needs to be aromatic ( e . g ., 2 , 3 - dihydroindole ) , described herein . although all of the rings may be aromatic ( e . g ., quinoline ). " Cycloalkyl” : as used herein , the term cycloalkyl refers to The second ring can also be fused or bridged . a saturated or unsaturated nonaromatic hydrocarbon mono - 5 “ Carbocycle ” or “ carbocyclic moiety ” as used herein , is or multi -ring system having 3 to 30 carbon atoms ( e. g ., intended to include any stable monocyclic , bicyclic or C3- C10 ). Suitable cycloalkyls include , but are not limited to tricyclic ring having the specified number of carbons, any of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohep whiwhich may be saturated , unsaturated, or aromatic . Carbo tyl, cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cycle includes cycloalkyl and aryl . For example , a C3 -C14 cycloheptynyl, adamantyl, and the like . U carbocycle is intended to include a monocyclic , bicyclic or " Heterocycloalkyl” as used herein refers to a saturated or tricyclic ring having 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 or 14 unsaturated nonaromatic 3 - 8 membered monocyclic , 8 - 12 carbon atoms. Examples of carbocycles include , but are not membered bicyclic , or 11 - 19 membered tricyclic ring sys limited to , cyclopropyl, cyclobutyl, cyclobutenyl, cyclopen tem having one or more heteroatoms ( such as O , N , S , or Se) , 15 tyl , cyclopentenyl, cyclohexyl, cycloheptenyl, cycloheptyl, unless specified otherwise . In certain embodiments , the term cycloheptenyl, cyclooctyl, cyclooctenyl, cyclooctadienyl, " heterocycloalkyl” refers to a non -aromatic 5 -, 6 -, 7 - or fluorenyl, phenyl , naphthyl, indanyl, adamantyl and tetra 8 -membered ring or a polycyclic group , including, but not hydronaphthyl. Bridged rings are also included in the defi limited to a bi- or tri -cyclic group comprising fused six - nition of carbocycle , including , for example, [3 .3 . 0 ]bicy membered rings having between one and three heteroatoms 20 clooctane, [ 4 .3 .0 ]bicyclononane , [4 . 4 .0 ]bicyclodecane and independently selected from oxygen , sulfur and nitrogen , [ 2 . 2 . 2 ] bicyclooctane . A bridged ring occurs when one or wherein ( i ) each 5 -membered ring has 0 to 2 double bonds more carbon atoms link two non - adjacent carbon atoms. In and each 6 -membered ring has 0 to 2 double bonds, ( ii ) the one embodiment, bridge rings are one or two carbon atoms. nitrogen and sulfur heteroatomsmay optionally be oxidized , It is noted that a bridge always converts a monocyclic ring ( iii ) the nitrogen heteroatom may optionally be quaternized , 25 into a tricyclic ring . When a ring is bridged , the substituents and ( iv ) any of the above heterocycloalkyl; rings may be recited for the ring may also be present on the bridge . Fused fused to an aryl or heteroaryl ring. Examples of heterocy - (e . g ., naphthyl, tetrahydronaphthyl) and spiro rings are also cloalkyl groups include , but are not limited to , piperidinyl, included . piperazinyl, pyrrolidinyl, dioxanyl , tetrahydrofuranyl, tetra “ Heterocycle ” or " heterocyclic moiety ” as used herein , hydrothienyl, isoindolinyl, indolinyl, imidazolidinyl, pyra - 30 includes any ring structure (saturated , unsaturated , or aro zolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl, tet- matic ) which contains at least one ring heteroatom ( e . g ., N , rahyrofuranyl, oxiranyl, azetidinyl, oxetanyl, thietanyl, 1 , 2 , Oor S ) . Heterocycle includes heterocycloalkyl and het 3 , 6 - tetrahydropyridinyl, tetrahydro -2H -pyranyl , 3 , 6 eroaryl. Examples of heterocycles include , but are not dihydro - 2H -pyranyl , morpholinyl, and the like. limited to , morpholine , pyrrolidine , tetrahydrothiophene , “ Aryl” : as used herein , refers to groups with aromaticity, 35 piperidine , piperazine and tetrahydrofuran . including “ conjugated ,” or multicyclic systems with at least Examples of heterocyclic groups include, but are not one aromatic ring and do not contain any heteroatom in the limited to , acridinyl, azocinyl, benzimidazolyl, benzofura ring structure . Examples include phenyl, benzyl, 1 , 2 , 3 ,4 - nyl , benzothiofuranyl , benzothiophenyl, benzoxazolyl, ben tetrahydronaphthalenyl, etc . zoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, “ Heteroaryl” : as used herein , refers to aryl groups, as 40 benzisoxazolyl, benzisothiazolyl , benzimidazolinyl, carba defined above , except having from one to four heteroatoms z olyl , 4aH - carbazolyl, carbolinyl, chromanyl, chromenyl, in the ring structure , and may also be referred to as “ aryl cinnolinyl, decahydroquinolinyl, 2H ,6H - 1 , 5 , 2 - dithiazinyl , heterocycles” or “ heteroaromatics. ” As used herein , the term dihydrofuro [ 2 , 3 - b ]tetrahydrofuran , furanyl, furazanyl, imi " heteroaryl” is intended to include a stable 5 -, 6 - , or 7 -mem - dazolidinyl, imidazolinyl, imidazolyl, 1H - indazolyl, indole bered monocyclic or 7 -, 8 - , 9 -, 10 -, 11- or 12 -membered 45 nyl , indolinyl , indolizinyl, indolyl, 3H - indolyl, isatinoyl, bicyclic aromatic heterocyclic ring which consists of carbon isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl , atoms and one or more heteroatoms, e . g . , 1 or 1 - 2 or 1 - 3 or isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methyl 1 -4 or 1 -5 or 1- 6 heteroatoms, or e. g ., 1 , 2 , 3 , 4 , 5, or 6 enedioxyphenyl, morpholinyl , naphthyridinyl , octahy heteroatoms, independently selected from the group consist - droisoquinolinyl, oxadiazolyl , 1 , 2 , 3 -oxadiazolyl , 1 ,2 , 4 -ox ing of nitrogen , oxygen and sulfur. The nitrogen atom may 50 adiazolyl , 1 , 2 , 5 -oxadiazolyl , 1 , 3 , 4 -oxadiazolyl , 1 , 2 , 4 be substituted or unsubstituted ( i . e . , N or NR wherein Ris o xadiazo15 ( 4H ) -one , oxazolidinyl, oxazolyl, oxindolyl, H or other substituents , as defined ) . The nitrogen and sulfur pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl , heteroatoms may optionally be oxidized ( i. e ., N and phenothiazinyl, phenoxathinyl, phenoxazinyl , phthalazinyl, S ( O ) , where p = 1 or 2 ). It is to be noted that total number piperazinyl, piperidinyl, piperidonyl, 4 -piperidonyl , pipero of S and O atoms in the aromatic heterocycle is not more 55 nyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl , than 1 . Examples of heteroaryl include pyridyl , pyrazinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole , pyri pyrimidinyl , pyrrolyl , pyrazolyl, imidazolyl , thiazolyl, iso - doimidazole , pyridothiazole , pyridinyl, pyridyl , pyrimidi thiazolyl, tetrazolyl, oxazolyl, isooxazolyl, thiadiazolyl, nyl, pyrrolidinyl, pyrrolinyl , 2H -pyrrolyl , pyrrolyl, qui oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, nazolinyl, quinolinyl, 4H -quinolizinyl , quinoxalinyl, tetrazolyl, pyridazinyl, quinazolinyl, dihydroquinazolyl, and 60 quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinazolyl and the like . tetrahydroquinolinyl, tetrazolyl, 6H - 1 , 2 , 5 - thiadiazinyl, 1 , 2 , Furthermore , the terms " aryl” and “heteroaryl ” include 3 - thiadiazolyl, 1, 2 , 4 - thiadiazolyl, 1 , 2 , 5 - thiadiazolyl, 1 , 3 ,4 multicyclic aryl and heteroaryl groups, e. g ., tricyclic , bicy - thiadiazolyl , thianthrenyl, thiazolyl, thienyl, thienothiazolyl, clic , e .g ., naphthalene , benzoxazole , benzodioxazole , ben - thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1, 2 , zothiazole , benzoimidazole , benzothiophene , methylenedi- 65 3 - triazolyl, 1 , 2 , 4 - triazolyl, 1 , 2 , 5 - triazolyl, 1 , 3 , 4 - triazolyl oxyphenyl, quinoline, isoquinoline, naphthrydine , indole , and xanthenyl . Multiple - ring heterocycle can include fused , benzofuran , purine , benzofuran , deazapurine , indolizine . bridged or spiro rings . US 9 ,943 ,609 B2 47 48 The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring embodiments , the alkyl group contains about 1 - 4 aliphatic (or the carbocyclic or heterocyclic group ) can be substituted carbon atoms. Examples of alkylamino include, but are not at one or more ring positions ( e . g . , the ring - forming carbon limited to , methylamino , ethylamino , iso - propylamino and or heteroatom such as N ) with such substituents as described the like. above , for example , aliphatic ; heteroaliphatic ; cycloalkyl; 5 “ Alkylthio ” (or “ thioalkyl” ) means an alkyl group as heterocycloalkyl; aryl ; heteroaryl; alkylaryl; alkylhet - defined herein with the indicated number of carbon atoms eroaryl; alkoxy ; aryloxy ; heteroalkoxy ; heteroaryloxy ; alky attached through a sulfur atom . C1- 6 alkylthio , is intended to Ithio ; arylthio ; heteroalkylthio ; heteroarylthio ; F ; Cl; Br ; 1 ; include C1, C2, C3, C4, C5, and C6 alkylthio groups . C1- 8 - NO2; CN ; CF3 ; CH2CF3; CHC12 ; CH2OH ; alkylthio , is intended to include C1, C2, C3, C4, C5, C6, C7, - CH2CH2OH ; CH NH2; - CH2SO , CHz; - or -GRGI 10 and Cg alkylthio groups . The thioalkyl groups can be sub wherein G is O , S , - NRG2 — , C O ) , stituted with groups such as alkyl, alkenyl, alkynyl , halogen , - S ( O ) - , - S02 — , - CEO ) O - , - CEO )NRG2 — , hydroxyl, alkylcarbonyloxy, arylcarbonyloxy , alkoxycarbo - OCCO) - , - NRG2C ( O ) - , - OC = O ) 0 — , - OC nyloxy , aryloxycarbonyloxy, carboxylate , carboxyacid , GO )NRG2 — — NRG2C = 00 , NRG2C = O ) alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbo NRG2 — , CCS) — , CES) S — , - SCES) - , - SC 15 nyl , alkylaminocarbonyl, dialkylaminocarbonyl , alkylthio (ES ) S — , CENRG2 ) , CE NRG2 ) , carbonyl , alkoxyl, amino ( including alkylamino , dialky - CÍNRG2NR63 __ - OC ( = NRG2) — , - NRG2C lamino , arylamino , diarylamino and alkylarylamino ), ENR63 ) - , - NRG280 , — — NRG2SO NRG3 — , or acylamino ( including alkylcarbonylamino , arylcarbo - SO NRG2 — , wherein each occurrence of RG1, RG2 and nylamino , carbamoyl and ureido ), amidino , imino , sulfhy RG3 independently includes , but is not limited to , hydrogen , 20 dryl, alkylthio , arylthio , thiocarboxylate , sulfates , alkylsulfi halogen , or an optionally substituted aliphatic , heteroali - nyl, sulfonato , sulfamoyl , sulfonamido , nitro , phatic , cycloalkyl, heterocycloalkyl; aryl, heteroaryl, alky - trifluoromethyl, cyano , azido , heterocyclyl alkylaryl, or an laryl, or alkylheteroaryl moiety . Aryl and heteroaryl groups aryl or heteroaryl moieties . can also be fused or bridged with cycloalkyl or heterocyclic “ Thiocarbonyl” or “ thiocarboxy ” includes compounds rings , which are not aromatic so as to form a multicyclic 25 and moieties which contain a carbon connected with a system ( e . g ., tetralin , methylenedioxyphenyl ) . double bond to a sulfur atom . “ Alkoxy ” (or “ alkyloxy ” ) : as used herein , the term alkoxy “ Thioether” includes moieties which contain a sulfur ( or alkyloxy ) refers to an alkyl group , as previously defined , atom bonded to two carbon atoms or heteroatoms. Examples attached to the parent molecular moiety through an oxygen of thioethers include , but are not limited to alkthioalkyls , atom ( alkoxy ' ) . In certain embodiments, the alkyl group 30 alkthioalkenyls and alkthioalkynyls . The term “ alkthio contains about 1 - 20 aliphatic carbon atoms. In certain other alkyls ” include moieties with an alkyl, alkenyl or alkynyl embodiments , the alkyl group contains about 1 - 10 aliphatic group bonded to a sulfur atom which is bonded to an alkyl carbon atoms. In yet other embodiments , the alkyl group group . Similarly, the term “ alkthioalkenyls ” refers to moi contains about 1 - 8 aliphatic carbon atoms. In still other eties wherein an alkyl, alkenyl or alkynyl group is bonded to embodiments , the alkyl group contains about 1 - 6 aliphatic 35 a sulfur atom which is covalently bonded to an alkenyl carbon atoms. In yet other embodiments , the alkyl group group ; and alkthioalkynyls ” refers to moieties wherein an contains about 1 - 4 aliphatic carbon atoms. Examples of alkyl, alkenyl or alkynyl group is bonded to a sulfur atom alkoxy groups, include but are not limited to , methoxy, which is covalently bonded to an alkynyl group . ethoxy , propoxy , isopropoxy, n -butoxy , tert - butoxy, neopen - “ Arylthio ” ( or “ thioaryl” ) means an aryl group as defined toxy and n -hexoxy . 40 herein with the indicated number of carbon atoms attached “ Aryloxy " : as used herein , the term aryloxy refers to an through a sulfur atom . aryl group , as defined herein , attached to the parent molecu “ Carboxylic acid ” as used herein refers to a compound lar moiety through an oxygen atom . Examples of aryloxy comprising a group of formula — CO , H . groups include but are not limited to phenoxy and napth - “ Dicarboxylic acid ” refers to a compound comprising two yloxy . 45 groups of formula - CO , H . “ Heteroaryloxy ” : as used herein , the term heteroaryloxy “ Halo , halide and halogen " : The terms halo , halide and refers to a heteroaryl group , as defined herein , attached to the halogen as used herein refer to an atom selected from parent molecular moiety through an oxygen atom . Examples fluorine , chlorine , bromine , and iodine . of heteroaryloxy groups include but are not limited to , “ Methylol” : The term methylol as used herein refers to an quinolyloxy and isoquinolizinyloxy. 50 alcohol group of the structure — CH ,OH . " Amine” : the term amine refers to a group having the “ Hydroxyalkyl” : As used herein , the term hydroxyalkyl structure — N ( R ) , wherein each occurrence of R is indepen refers to an alkyl group , as defined above , bearing at least dently hydrogen , or an aliphatic or heteroaliphatic moiety , or one OH group . the R groups, taken together , may form a heterocyclic “Mercaptoalkyl ” : The term mercaptoalkyl as used therein moiety . In certain instances , an amine group can be charged 55 refers to an alkyl group , as defined above , bearing at least ( protonized ) or quarternized , e . g ., HN + ( R ) 2 or — N + ( R ); one SH group " Alkylamino ” : as used herein , the term alkylamino refers “ Acyl” includes moieties that contain the acyl radical to a group having the structure - NHR ' wherein R ' is alkyl, GC( O ) - ) or a carbonyl group . “ Substituted acyl” includes as defined herein . The term “ aminoalkyl” refers to a group acyl groups where one or more of the hydrogen atoms are having the structure NH , R — , wherein R ' is alkyl, as defined 60 replaced by, for example , alkyl groups , alkynyl groups , herein . In certain embodiments, the alkyl group contains halogen , hydroxyl, alkylcarbonyloxy , arylcarbonyloxy , about 1 - 20 aliphatic carbon atoms. In certain other embodi - alkoxycarbonyloxy, aryloxycarbonyloxy , carboxylate , alky ments , the alkyl group contains about 1 - 10 aliphatic carbon lcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl , atoms. In yet other embodiments , the alkyl, alkenyl, and alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbo alkynyl groups employed in the invention contain about 1 - 8 65 nyl, alkoxyl, phosphate , phosphonato , phosphinato , amino aliphatic carbon atoms. In still other embodiments , the alkyl ( including alkylamino , dialkylamino , arylamino , diary group contains about 1 -6 aliphatic carbon atoms. In yet other lamino and alkylarylamino ), acylamino ( including alkylcar US 9 ,943 ,609 B2 49 50 bonylamino , arylcarbonylamino , carbamoyl and ureido ), animals . Preferably , the non -human animal is a mammal amidino , imino , sulfhydryl , alkylthio , arylthio , thiocarboxy ( e . g ., a rodent, a mouse , a rat , a rabbit, a monkey , a dog , a late , sulfates , alkylsulfinyl, sulfonato , sulfamoyl, sulfona cat, a primate , or a pig ) . An animal may be a transgenic mido , nitro , trifluoromethyl, cyano , azido , heterocyclyl, animal or a human clone . The term “ subject” encompasses alkylaryl, or an aryl or heteroaryl moiety . animals . “ Hydrocarbon ” : The term hydrocarbon , as used herein , “ Efficient amount” : In general, as it refers to an active refers to any chemical group comprising hydrogen and agent or drug delivery device , the term “ efficient amount” carbon . The hydrocarbon may be substituted or unsubsti refers to the amount necessary to elicit the desired biological tuted . The hydrocarbon may be unsaturated , saturated , branched , unbranched , cyclic , polycyclic , or heterocyclic . 10 response . As will be appreciated by those of ordinary skill in Illustrative hydrocarbons include , for example , methyl, this art, the efficient amount of an agent or device may vary ethyl, n - propyl, iso -propyl , cyclopropyl, allyl, vinyl, n -butyl , depending on such factors as the desired biological end tert - butyl, ethynyl, cyclohexyl, methoxy, diethylamino , het point , the agent to be delivered , the composition of the erocycloalkyl, aryl, heteroaryl , thioalkyl , and the like . As encapsulating matrix , the target tissue , etc . For example , the would be known to one skilled in this art, all valencies must 15 efficient amount of microparticles containing an antigen to be satisfied in making any substitutions. be delivered to immunize an individual is the amount that “ Alkylaryl” as used herein refers to an aryl group substi - results in an immune response sufficient to prevent infection tuted with one or more alkyl groups ( e .g ., methylphenyl) . with an organism having the administered antigen . “ Alkylarylamino ” as used herein refers to N RG4R5, “ Natural amino acid ” as used herein refers to any one of wherein RG4 is alkyl, as defined herein , and RG5 is an aryl, 20 the common , naturally occurring L - amino acids found in as defined herein , or at least one of RG4 and RG5 is an naturally occurring proteins : glycine (Gly ) , alanine ( Ala ) , alkylaryl as defined herein . valine ( Val) , leucine (Leu ) , isoleucine ( Ile ) , lysine (Lys ) , “ Substituted ” : The terms substituted , whether preceded arginine (Arg ) , histidine (His ) , proline (Pro ) , serine (Ser ) , by the term “ optionally ” or not, and substituent, as used threonine ( Thr ), phenylalanine ( Phe ) , tyrosine ( Tyr ) , tryp herein , refers to the replacement of hydrogen radicals in a 25 tophan ( Trp ), aspartic acid ( Asp ), glutamic acid (Glu ), given structure with the radical of a specified substituent. asparagine (Asn ), glutamine (Gln ), cysteine (Cys ) and When more than one position in any given structure may be methionine (Met ). substituted with more than one substituent selected from a “ Unnatural amino acid ” as used herein refers to any specified group , the substituent may be either the same or amino acid which is not a natural amino acid . This includes, different at every position . As used herein , the term “ sub - 30 for example , amino acids that comprise a - , B - , w - , D - , stituted ” is contemplated to include all permissible substitu - L -amino acyl residues . More generally , the unnatural amino ents of organic compounds. In a broad aspect , the permis - acid comprises a residue of the general formula sible substituents include acyclic and cyclic , branched and unbranched , carbocyclic and heterocyclic , aromatic and nonaromatic substituents of organic compounds . Heteroa - 35 toms such as nitrogen may have hydrogen substituents mm and / or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroa mor toms. Examples of substituents include , but are not limited to aliphatic ; heteroaliphatic ; cycloalkyl; heterocycloalkyl; 40 aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy ; aryloxy ; wherein the side chain R is other than the amino acid side heteroalkoxy ; heteroaryloxy ; alkylthio ; arylthio ; heteroalky - chains occurring in nature. Exemplary unnatural amino Ithio ; heteroarylthio ; F ; Cl; Br; 1; — NO2; CN ; CFz; acids, include, but are not limited to , sarcosine ( N -methyl - CH2CF3 ; CHC12 ; CH2OH ; CH2CH2OH ; glycine ) , citrulline ( cit) , homocitrulline , B - ureidoalanine , - CH2NH2; _ CH _ SOZCHz; - or -GRG1 wherein G is 45 thiocitrulline, hydroxyproline, allothreonine, pipecolic acid - 0 % , s - , - NRG2 — , C ) - , - S ( = O ) , (homoproline ), a - aminoisobutyric acid , tert - butylglycine , - S0 , - , CC00 — , CEO) NRG2 — , OC tert- butylalanine , allo - isoleucine, norleucine, -methylleu ( O ) - , - NRG2C0 - , - OC ( 0 ) 0 — , - OC (= O ) cine, cyclohexylglycine, ß -cyclohexylalanine , B -cyclopen NRG2 — , - NRG2C4010 — , - NRG2CEO) NRG2 _ , tylalanine , a -methylproline , phenylglycine, a - methylphe - CCS) , CES) S , SCCS) , - SCS ) S , 50 nylalanine and homophenylalanine . - C1= NRG2) , - C ( = NRG2 ) , - C = NRG2NR63 — “ Amino acyl” : More generally , the term amino acyl, as - OCENRG2) — , - NRG2CE= NR63) — , - NRG250 , – , used herein , encompasses natural amino acid and unnatural - NRG2SO2NR63 — , or SO NRG2 — , wherein each amino acids . occurrence of RGI, RG2 and RG3 independently includes , but “ Polyamide” : refers to homo - or hetero -polymers of natu is not limited to , hydrogen , halogen , or an optionally sub - 55 ral amino acid and unnatural amino acids . Illustrative homo stituted aliphatic , heteroaliphatic , cycloalkyl , heterocy - polymers include, but are not limited to , poly - lysine , poly cloalkyl, aryl, heteroaryl, alkylaryl, or alkylheteroaryl moi arginine , poly - y - glutaric acid , and the like . Illustrative ety . Additional examples of generally applicable substituents hetero -polymers include , but are not limited to , polymers are illustrated by the specific embodiments shown in the comprising peptides fragments selected from peptidases , Examples that are described herein . 60 lysozymes , metalloproteinases , and the like . The following are more general termsused throughout the “ PHF ” refers to poly ( 1 -hydroxymethylethylene present application : hydroxymethyl- formal) . “ Animal ” : The term animal , as used herein , refers to As used herein , the terms “ polymer unit” , “ monomeric humans as well as non -human animals , at any stage of unit ” , “ monomer ” , “ monomer unit ” , “ unit ” all refer to a development , including, for example , mammals , birds, rep - 65 repeatable structural unit in a polymer . tiles , amphibians , fish , worms and single cells . Cell cultures The present invention is intended to include all isotopes of and live tissue samples are considered to be pluralities of atoms occurring in the present compounds . Isotopes include US 9 ,943 ,609 B2 51 52 those atoms having the same atomic number but different lysis / cleavage of the polymer acetal/ ketal groups ) will result mass numbers . By way of general example and without in fragmentation of the polyal conjugate to low molecular limitation , isotopes of hydrogen include tritium and deute weight components ( i. e ., degradation ). rium . Isotopes of carbon include C - 13 and C - 14 . In certain embodiments , biodegradable biocompatible The present invention is intended to include all isomers of 5 polymer carriers, used for preparation of polymer conjugates the compound , which refers to and includes , optical isomers, of the invention , are naturally occurring polysaccharides , and tautomeric isomers , where optical isomers include glycopolysaccharides, and synthetic polymers of polygly enantiomers and diastereomers , chiral isomers and non chiral isomers, and the optical isomers include isolated coside , polyacetal, polyamide , polyether , and polyester ori optical isomers as well as mixtures of optical isomers 10 gin and products of their oxidation , fictionalization , modi including racemic and non -racemic mixtures ; where an fication , cross - linking , and conjugation . isomer may be in isolated form or in a mixture with one or In certain other embodiments , the carrier is a hydrophilic more other isomers. biodegradable polymer selected from the group consisting of Polymeric Carriers carbohydrates , glycopolysaccharides , glycolipids , glyco In certain exemplary embodiments , the conjugates of the 1615 conjugates , polyacetals , polyketals, and derivatives thereof. invention find use in biomedical applications , such as drug In certain exemplary embodiments, the carrier is a natu delivery and tissue engineering , and the carrier is biocom rally occurring linear and / or branched biodegradable bio patible and biodegradable . In certain embodiments , the compatible homopolysaccharide selected from the group carrier is a soluble polymer, nanoparticle , gel , liposome, consisting of cellulose , amylose , dextran , levan , fucoidan , micelle , suture , implant, etc . In certain embodiments , the 20 carraginan , inulin , pectin , amylopectin , glycogen and lix term " soluble polymer” encompasses biodegradable bio enan . compatible polymer such as a polyal (e . g ., hydrophilic In certain other exemplary embodiments , the carrier is a polyacetal or polyketal) . In certain other embodiments , the naturally occurring linear and branched biodegradable bio carrier is a fully synthetic , semi- synthetic or naturally compatible heteropolysaccharide selected from the group occurring polymer . In certain other embodiments , the carrier 25 consisting of agarose , hyluronan , chondroitinsulfate , derma is hydrophilic . tansulfate , keratansulfate , alginic acid and heparin . In certain exemplary embodiments , the carriers used in In yet other exemplary embodiments , the polymeric car the present invention are biodegradable biocompatible poly rier comprises a copolymer of a polyacetal/ polyketal and a als comprising at least one hydrolysable bond in each hydrophilic polymer selected from the group consisting of monomer unit positioned within the main chain . This 30 polyacrylates, polyvinyl polymers, polyesters , polyorthoe ensures that the degradation process ( via hydrolysis / cleav sters , polyamides , polypeptides, and derivatives thereof. age of the monomer units ) will result in fragmentation of the In yet another embodiment, the polymeric carrier is polymer conjugate to the monomeric components (i . e ., dextrin that is produced by the hydrolysis of a starch degradation ) , and confers to the polymer conjugates of the obtained from various natural products such as , for example , invention their biodegradable properties . The properties 35 wheat , rice , maize and tapioca . Depending on the structure ( e .g ., solubility , bioadhesivity and hydrophilicity ) of biode of the starch starting material each dextrin comprises a gradable biocompatible polymer conjugates can be modified unique distribution of a - 1 , 4 linkages and a - 1 , 6 linkages. by subsequent substitution of additional hydrophilic or Since the rate of biodegradability of a - 1, 6 linkages is hydrophobic groups . Examples of biodegradable biocom typically less than that for a - 1 , 4 linkages , preferably the patible polymers suitable for practicing the invention can be 40 percentage of a - 1 , 6 linkages is less than 10 % and more found inter alia in U . S . Pat. Nos. 5 ,811 , 510 ; 5 , 863 , 990 ; preferably less than 5 % . In one embodiment the molecular 5 ,958 , 398 ; 7 ,838 ,619 and 7 , 790 , 150 ; and U . S . Publication weight of the dextrin is in the range of about 1 kDa to about No . 2006 / 0058512 ; each of the above listed patent docu 200 kDa , more preferably from about 2 kDa to about 55 ments is incorporated herein by reference in its entirety. kDa. Guidance on the significance , preparation , and applications 45 In certain embodiments , the carrier comprises polysac of this type of polymers may be found in the above - cited charides activated by selective oxidation of cyclic vicinal documents . In certain embodiments , it is anticipated that the diols of 1 , 2 - , 1 , 4 - , 1 , 6 - , and 2 , 6 - pyranosides , and 1 , 2 - , 1 , 5 - , present invention will be particularly useful in combination 1, 6 - furanosides , or by oxidation of lateral 6 -hydroxy and with the above - referenced patent documents , as well as U . S . 5 ,6 - diol containing polysaccharides prior to conjugation Pat . Nos . 5 , 582 , 172 and 6 ,822 ,086 , each of the above listed 50 with drug molecules or PBRMs. patent documents is incorporated herein by reference in its In still other embodiments , the polymeric carrier com entirety . prises a biodegradable biocompatible polyacetal wherein at The conjugates of this invention are hydrophilic , hydro least a subset of the polyacetal repeat structural units have lysable and comprise drug molecules ( e . g ., vinca alkaloids the following chemical structure: or derivatives, non -natural camptothecin compounds or 55 derivatives, auristatins, tubulysins, duocarmycins, PI3 RiR3 RS kinases, MEK inhibitors , KSP inhibitors , and analogs thereof) and antibodies ( e . g . , Trastuzumab, Cetuximab , to - -0¢ - 0 – 42 - R * 1, Rituximab , Bevacizumab , Epratuzumab , Veltuzumab , Labetuzumab ) or peptides (LHRH receptor targeting pep - 60 R2 R4 Ro tides , EC - 1 peptide ) covalently attached to the polymer carrier via linkages that contain one or more biodegradable wherein for each occurrence of the n bracketed structure , bonds. Thus , in certain exemplary embodiments , carriers one of R1 and R , is hydrogen , and the other is a biocom suitable for practicing the present invention are polyals patible group and includes a carbon atom covalently having at least one acetal/ ketal oxygen atom in each mono - 65 attached to C '; R * is a carbon atom covalently attached to mer unit positioned within the main chain . As discussed C ? ; n " is an integer ; each occurrence of R3, R4, R , and Ro above, this ensures that the degradation process ( via hydro - is a biocompatible group and is independently hydrogen or US 9 ,943 ,609 B2 53 54 an organic moiety ; and for each occurrence of the bracketed partially oxidized dextran acetal units ( B ) and exhaustively structure n , at least one of R1, R2, Rz, R4, Rs and Ro dextran acetal units ( C ) of the following structures : comprises a functional group suitable for coupling . In cer tain embodiments , the functional group is a hydroxyl moi ety . 3 In one embodiment, the polymeric carrier comprises activated hydrophilic biodegradable biocompatible poly mers comprising from 0 . 1 % to 100 % polyacetal moieties HO ?? whose backbone is represented by the following chemical 10 structure : ?? ( CH2CHRO CHR30 % ) . @ Wherein : R , and Rg are independently hydrogen , hydroxyl, hydroxy 15 alkyl (e . g. , CH2OH , CH (OH ) CH2OH ) , _ CHO , HO OH - CH (OH ) - CHO or -carbonyl ; and o is an integer from 20 to 2000 . OH In yet other embodiments , the polymeric carrier com @ prises a biodegradable biocompatible polyketal wherein at 20 least a subset of the polyketal repeatable structural units have the following chemical structure : HO TOH ?? Ri R3 Rs 25 - ? or to - O - O-C2 - R * t or - RA R6

30 HO HO R3 DT + 0 - ¢ - 0 – ¢? In another embodiment, the polymeric carrier comprises RA R4 unmodified acetal units , i. e . , polyacetal segments . In some embodiments , the polyacetals can be derived from exhaus tively oxidized dextran followed by reduction . These poly wherein each occurrence of R , and R , is a biocompatible mers have been described in U .S . Pat. No. 5 ,811 , 510 , which group and R , R3, R4, Rs, RG and are as defined herein is hereby incorporated by reference for its description of In certain embodiments , the ketal units are monomers of polyacetals at column 2 , line 65 to column 8 , line 55 and Formula ( Ila ) or ( IIb ): 40 their synthesis at column 10 , line 45 to column 11, line 14 . In one embodiment, the unmodified polyacetal polymer is a (IIa ) poly (hydroxymethylethylene hydroxymethyl formal) poly OH mer (PHF ) . In addition to poly (hydroxymethylethylene hydroxym 45 ethyl formal ) polymers , the backbone of the polymeric Or carrier can also comprise co -polymers of poly (hydroxym teft?? ethylethylene hydroxymethyl formal ) blocks and other ( II ) acetal or non - acetal monomers or polymers . For example , polyethylene glycol polymers are useful as a stealth agent in 50 the polymer backbone because they can decrease interac tions between polymer side chains of the appended func tional groups. Such groups can also be useful in limiting interactions such as between serum factors and the modified 55 polymer . Other stealth agent monomers for inclusion in the polymer backbone include , for example , ethyleneimine , methacrylic acid , acrylamide , glutamic acid , and combina OH OH tions thereof. The acetal or ketal units are present in the modified Biodegradable , biocompatible polyketal polymers and 60 polymer in an amount effective to promote biocompatibility . their methods of making have been described in U . S . Pat. The unmodified acetal or ketal unit can be described as a Nos . 5 , 811 ,510 , 7 ,790 , 150 and 7 , 838 ,619 , which are hereby “ stealth agent” that provides biocompatibility and solubility incorporated by reference in their entirety . to the modified polymers . In addition , conjugation to a In one embodiment, the polymeric carrier can be obtained polyacetal or polyketal polymer can modify the susceptibil from partially oxidized dextran (616 ) -D - glucose ) fol- 65 ity to metabolism and degradation of the moieties attached lowed by reduction . In this embodiment, the polymer com - to it , and influence biodistribution , clearance and degrada prises a random mixture of the unmodified dextran ( A ) , tion . US 9 , 943, 609 B2 55 56 The unmodified acetal units are monomers of Formula other formulae described herein , each polyacetal unit has a ( III ) : single hydroxyl group attached to the glycerol moiety of the unit and an X ' group ( or another substituent such as -LP - D ) attached to the glycolaldehyde moiety of the unit. This is for 5 convenience only and it should be construed that the poly ( III) mer having units of Formula ( IV ) and other formulae described herein can contain a random distribution of units having a X ' group ( or another substituent such as -LP - D ) attached to the glycolaldehyde moiety of the units and those OH OH ] having a single X ' group (or another substituent such as -Lº - D ) attached to the glycerol moiety of the units as well as units having two X ' groups ( or other substituents such as The molar fraction , n , of unmodified polyacetal units is -LP - D ) with one attached to the glycolaldehyde moiety and the molar fraction available to promote biocompatibilitytiny, , 1615 the other attached to the glycerol moiety of the units . solubility and increase half -life , based on the total number of In one embodiment, biodegradable biocompatible polyals polymer units in the modified polymer. The molar fraction n suitable for practicing the present invention have a molecu may be the minimal fraction of unmodified monomer acetal lar weight of between about 0 .5 and about 300 kDa. In a units needed to provide biocompatibility , solubility , stability , preferred embodiment of the present invention , the biode or a particular half - life , or can be some larger fraction . The 20 gradable biocompatible polyals have a molecular weight of most desirable degree of cytotoxicity is substantially none, between about 1 and about 300 kDa ( e . g ., between about 1 i. e . , the modified polymer is substantially inert to the sub - and about 200 kDa , between about 2 and about 300 kDa, ject. However, as is understood by those of ordinary skill in between about 2 and about 200 kDa , between about 5 and the art , some degree of cytotoxicity can be tolerated depend - 25 about 100 kDa, between about 10 and about 70 kDa, ing on the severity of disease or symptom being treated , the between about 20 and about 50 kDa, between about 20 and efficacy of the treatment, the type and degree of immune about 300 kDa, between about 40 and about 150 kDa, response , and like considerations. between about 50 and about 100 kDa, between about 2 and In one embodiment, the modified polymer backbone about 40 kDa, between about 6 and about 20 kDa, or comprises units of Formula (IV ): 30 between about 8 and about 15 kDa ) . In one embodiment , the biodegradable biocompatible polyals suitable for practicing the present invention are modified before conjugating with a drug or a PBRM . For 35 example , the polyals may contain subunits of linkers LP or ( IV ) L ' , such as CEO) — X — (CH2 ) , C ( = O ) — with X being CH2, O , or NH , and v being an integer from 1 to 6 . Table A below provides some examples of the modified polyals suitable for conjugating with a drug or PBRM or OH X Ja 40 derivatives thereof . Unless otherwise specified , reference numbers in Tables A through E below correspond to the wherein X ' indicates the substituent for the hydroxyl group Example numbers described herein ; the term “ ND ” means of the polymer backbone. As shown in Formula ( IV ) and the not determined ; and X is CH2, O , or NH . TABLE A

Ref # Polymer Scaffold

Ex 2

“ ?? ?? OH

?? US 9 ,943 ,609 B2 57 58 TABLE A - continued RefRef # # Polymer Scaffold Ex 1 tyy tym

. ?? . HO . HO

NH

OHH

X = CH , Ex 5 betHO HO . HO Wat, HO 4 HN

Š

OH OH . HO . HO

?? HN

SH US 9 ,943 ,609 B2 59 60 TABLE A - continued Ref # Polymer Scaffold

X = CH2, Ex 12

H HO HO O ?? ?? in

HO HN

X = CH2 Ex 71

OH OH OH HO

HO

HN

NO2 US 9 ,943 ,609 B2 61 TADITABLE A - continued Ref # Polymer Scaffold tyy of tygym tyg

OH OH 0

?? HN

HN

H3C

NO

X = CH Ex 68 684 ° °N ° ° °

??? “ ?? ?? ??

HN

NO2 US 9 ,943 ,609 B2 63 64 TABLE A - continued

Ref # Polymer Scaffold tyy tyy ty

HO HO OH OH

?? NH

NH

tyy tyy tyy HO HO . HO BrynjaTypington, HO

OH HN

NH US 9 ,943 ,609 B2 65 TABLE A - continued Ref # Polymer Scaffold tyy tyy tyy HO HO LOH. HO LOH

OH HN

1 - 12

tyy tyg

HO HO . HO .OH HO

?? HN ??????1 - 12 ON US 9 ,943 ,609 B2

TABLE A - continued Ref # Polymer Scaffold ^ y tyay

OH OH OH OH O

HN

HN

fya Tyy tyy

?? OH?? OH OH

HO HN

Therapeutic Agents capable of being linked to a polymer carrier ) include cyto In certain embodiments , the therapeutic agent is a small toxic compounds ( e. g ., broad spectrum ), angiogenesis molecule having a molecular weight preferably sabout 5 inhibitors , cell cycle progression inhibitors, PI3K / m - TOR / kDa, more preferably sabout 4 kDa , more preferably sabout 60 AKT pathway inhibitors , MAPK signaling pathway inhibi 3 kDa, most preferably sabout 1 .5 kDa or sabout 1 kDa t ors , kinase inhibitors , protein chaperones inhibitors , HDAC In certain embodiments , about 0 . 1 to about 25 % mono - inhibitors , PARP inhibitors , Wnt/ Hedgehog signaling path mers comprise a therapeutic agent , more preferably about way inhibitors and RNA polymerase inhibitors . 0 . 5 to about 20 % , more preferably about 1 to about 15 % , and Broad spectrum cytotoxins include , but are not limited to , even more preferably about 2 to about 10 % . 65 DNA -binding or alkylating drugs , microtubule stabilizing The small molecule therapeutic agents used in this inven - and destabilizing agents , platinum compounds, and topoi tion ( e . g . , antiproliferative ( cytotoxic and cytostatic ) agents somerase I inhibitors . US 9 ,943 ,609 B2 69 70 Exemplary DNA -binding or alkylating drugs include, 0305149, and in U . S . Pat. No . 7 , 303, 749 B1, the disclosures CC - 1065 and its analogs , anthracyclines (doxorubicin , epi of which are incorporated herein by reference in their rubicin , idarubicin , daunorubicin ) and its analogs , alkylating entirety . agents , such as calicheamicins, dactinomycines , mitromy Exemplary epothilone compounds include epothilone A , cines, pyrrolobenzodiazepines, and the like . 5 B , C , D , E and F , and derivatives thereof. Suitable epothilone Exemplary CC - 1065 analogs include duocarmycin SA . compounds and derivatives thereof are described , for duocarmycin Ci , duocarmycin C2 , duocarmycin B2 , example , in U . S . Pat. Nos. 6 ,956 ,036 ; 6 , 989 ,450 ; 6 , 121 , 029 ; DU - 86 , KW -2189 , bizelesin , seco -adozelesin , and those 6 , 117 ,659 ; 6 ,096 ,757 ; 6 ,043 , 372 ; 5 , 969 ,145 ; and 5 , 886 ,026 ; described in U . S . Pat . Nos . 5 ,475 , 092 ; 5 , 595 ,499 ; 5 , 846 , and WO 97 / 19086 ; WO 98 /08849 ; WO 98 /22461 ; WO 545 . 6 534 660 . 6586 618. 6756 397 and 7 049 316 Doxo 10 98 / 25929; WO 98738192 ; WO 99701124; WO 99702514 : WO 99/ 03848 ; WO 99/ 07692 ; WO 99/ 27890 ; and WO rubicin and its analogs include those described in U . S . Pat . 99 / 28324 ; the disclosures of which are incorporated herein No . 6 ,630 ,579 . Calicheamicins include those described in by reference in their entirety . U . S . Pat . Nos . 5 , 714 ,586 and 5 , 739 , 116 . Duocarmycins Exemplary cryptophycin compounds are described in include those described in U . S . Pat . Nos . 5 , 070 , 092 ; D1015 , 101, 15 U . S . Pat . Nos . 6 ,680 ,311 and 6 ,747 ,021 . 038 ; 5 ,187 ,186 ; 6 ,548 , 530 ; 6 ,660 ,742 ; and 7, 553 ,816 B2 ; Exemplary platinum compounds include cisplatin (PLA and Li et al. , Tet Letts ., 50 :2932 - 2935 ( 2009 ). Pyrroloben TINOL® ), carboplatin ( PARAPLATIN® ) , oxaliplatin zodiazepines include those described in Denny , Exp . Opin . ( ELOXATINE® ) . iproplatin , ormaplatin , and tetraplatin . Ther. Patents . , 10 ( 4 ) : 459 - 474 ( 2000 ) . Exemplary topoisomerase I inhibitors include camptoth Exemplary microtubule stabilizing and destabilizing 20 ecin , camptothecin , derivatives , camptothecin analogs and agents include taxane compounds, such as paclitaxel , doc non - natural camptothecins , such as , for example , CPT- 11 etaxel; maytansinoids, auristatins and analogs thereof, ( irinotecan ), SN -38 , topotecan , 9 - aminocamptothecin , tubulysin A and B derivatives, vinca alkaloid derivatives, rubitecan , gimatecan , karenitecin , silatecan , lurtotecan , epothilones and cryptophycins . exatecan , diflomotecan , belotecan , lurtotecan and S39625 . Exemplary maytansinoids or maytansinoid analogs 25 Other camptothecin compounds that can be used in the include maytansinol and maytansinol analogs , maytansine present invention include those described in , for example , J . or DM - 1 and DM - 4 are those described in U . S . Pat. Nos . Med . Chem . , 29 : 2358 - 2363 ( 1986 ) ; J . Med . Chem . , 23 :554 5 , 208 ,020 ; 5 ,416 ,064 ; 6 ,333 . 410 ; 6 , 441, 163 ; 6 , 716 ,821 ; ( 1980 ) ; J . Med . Chem ., 30 : 1774 ( 1987 ) . RE39 , 151 and 7 ,276 ,497 . In certain embodiments , the cyto Angiogenesis inhibitors include , but are not limited , toxic agent is a maytansinoid , another group of anti -tubulin 30 MetAP2 inhibitors . Exemplary MetAP2 inhibitors include agents ( ImmunoGen , Inc .; see also Chari et al. , 1992 , Cancer fumagillol analogs, meaning any compound that includes Res . 52 : 127 - 131) , maytansinoids or maytansinoid analogs. the fumagillin core structure, including fumagillamine , that Examples of suitable maytansinoids include maytansinol inhibits the ability of MetAP -2 to remove NH2- terminal and maytansinol analogs. Suitable maytansinoids are dis - methionines from proteins as described in Rodeschini et al. , closed in U . S . Pat . Nos . 4 , 424 ,219 ; 4 ,256 , 746 ; 4 ,294 ,757 ; 35 J . Org . Chem ., 69 , 357 - 373 , 2004 and Liu , et al. , Science 4 , 307 ,016 ; 4 , 313 , 946 ; 4 ,315 , 929 ; 4 ,331 ,598 ; 4 , 361, 650 ; 282 , 1324 - 1327 , 1998 . Non limiting examples of “ fum 4 , 362 , 663 ; 4 , 364, 866 ; 4 ,450 , 254 ; 4 , 322 , 348 ; 4 , 371, 533 ; agillol analogs ” are disclosed in J . Org . Chem ., 69 , 357 , 6 ,333 ,410 ; 5 , 475 ,092 ; 5 ,585 , 499 ; and 5 ,846 ,545 . 2004 ; J . Org . Chem ., 70 , 6870 , 2005 ; European Patent Exemplary auristatins include auristatin E (also known as Application 0 354 787 ; J. Med . Chem . , 49, 5645 , 2006 ; a derivative of dolastatin - 10 ) , auristatin EB ( AEB ) , aurista - 40 Bioorg . Med . Chem . , 11 , 5051, 2003; Bioorg . Med . Chem . , tin EFP ( AEFP ) , monomethyl auristatin E (MMAE ) , 14 , 91, 2004 ; Tet. Lett. 40 , 4797 , 1999 ; W099 /61432 ; U . S . monomethyl auristatin F (MMAF ) , auristatin F and dolas - Pat. Nos. 6 ,603 ,812 ; 5 , 789, 405 ; 5 , 767 , 293 ; 6 , 566 ,541 ; and tatin . Suitable auristatins are also described in U .S . Publi - 6 ,207 ,704 . cation Nos. 2003 /0083263 , 2011 /0020343 , and 2011/ Exemplary cell cycle progression inhibitors include CDK 0070248 ; PCT Application Publication Nos . WO 45 inhibitors such as , for example , BMS - 387032 and 09 / 117531 , WO 2005 /081711 , WO 04 /010957 ; WO PD0332991; Rho -kinase inhibitors such as , for example 02 /088172 and Wo01/ 24763 , and U . S . Pat . Nos. 7 ,498 , 298 ; GSK429286 ; checkpoint kinase inhibitors such as , for 6 ,884 , 869 ; 6 , 323 ,315 ; 6 ,239 , 104 ; 6 , 124 ,431 ; 6 , 034 ,065 ; example , AZD7762 ; aurora kinase inhibitors such as , for 5 , 780 , 588 ; 5 , 767 , 237 ; 5 ,665 , 860 ; 5 ,663 , 149 ; 5 ,635 ,483 ; example , AZD1152 , MLN8054 and MLN8237 ; PLK inhibi 5 ,599 ,902 ; 5 ,554 ,725 ; 5 ,530 , 097 ; 5 ,521 , 284 ; 5 ,504 , 191 ; 50 tors such as, for example , BI 2536 , B16727 (Volasertib ) , 5 ,410 ,024 ; 5 , 138 ,036 ; 5 ,076 , 973 ; 4 , 986 ,988 ; 4 , 978 ,744 ; GSK461364 , ON -01910 ( Estybon ) ; and KSP inhibitors such 4 ,879 ,278 ; 4 ,816 ,444 ; and 4 , 486 ,414 , the disclosures of as , for example , SB 743921 , SB 715992 ( ispinesib ) , which are incorporated herein by reference in their entirety. MK -0731 , AZD8477 , AZ3146 and ARRY -520 . Exemplary tubulysin compounds include compounds Exemplary PI3K / m - TOR / AKT signaling pathway inhibi described in U . S . Pat . Nos. 7 ,816 ,377 ; 7 ,776 ,814 ; 7 ,754 , 55 tors include phosphoinositide 3 -kinase (PI3K ) inhibitors , 885 ; U . S . Publication Nos . 2011/ 0021568 ; 2010 / 004784 ; GSK - 3 inhibitors, ATM inhibitors , DNA -PK inhibitors and 2010 /0048490 ; 2010 /00240701 ; 2008 /0176958 ; and PCT PDK - 1 inhibitors . Application Nos . WO 98 / 13375 ; WO 2004 /005269 ; WO Exemplary PI3 kinases are disclosed in U . S . Pat. No. 2008 / 138561 ; WO 2009 /002993 ; WO 2009 /055562 ; WO 6 ,608 , 053 , and include BEZ235 , BGT226 , BKM120 , 2009 /012958 ; WO 2009 /026177 ; WO 2009 / 134279 ; WO 60 CAL101 , CAL263, demethoxyviridin , GDC -0941 , 2010 / 033733 ; WO 2010 /034724 ; WO 2011/ 017249 ; WO GSK615 , IC87114 , LY294002, Palomid 529 , perifosine , 2011/ 057805 ; the disclosures of which are incorporated by PF -04691502 , PX - 866 , SAR245408 , SAR245409 , SF1126 , reference herein in their entirety. Wortmannin , XL147 and XL765 . Exemplary vinca alkaloids include vincristine , vinblas- Exemplary AKT inhibitors include, but are not limited to tine, vindesine , and navelbine ( vinorelbine ) . Suitable Vinca 65 AT7867. alkaloids that can be used in the present invention are also Exemplary MAPK signaling pathway inhibitors include disclosed in U . S . Publication Nos. 2002 / 0103136 and 2010 / MEK , Ras , JNK , B -Raf and p38 MAPK inhibitors. US 9 ,943 ,609 B2 71 72 Exemplary MEK inhibitors are disclosed in U . S . Pat . No . compounds and platinum compounds; PARP inhibitors and 7 ,517 , 994 and include GDC -0973 , GSK1120212 , platinum compounds; broad spectrum cytotoxic compounds MSC1936369B , AS703026 , RO5126766 and R04987655 , and PARP inhibitors . PD0325901 , AZD6244 , AZD 8330 and GDC -0973 . In one embodiment, the Vinca alkaloid is a compound of Exemplary B - raf inhibitors include CDC - 0879 , PLX - 5 Formula ( V ) : 4032 , and SB590885 . Exemplary B p38 MAPK inhibitors include BIRB 796 , LY2228820 and SB 202190 ( V) Receptor tyrosine kinases (RTK ) are cell surface receptors which are often associated with signaling pathways stimu - " lating uncontrolled proliferation of cancer cells and neoan giogenesis . Many RTKs, which over express or have muta tions leading to constitutive activation of the receptor , have HN JUIN been identified , including , but not limited to , VEGFR , 16 R16 EGFR , FGFR , PDGFR , EphR and RET receptor family H3CO2C receptors . Exemplary RTK specific targets include ErbB2 , CH30 0111111 Y .. . 111R17 111 FLT - 3 , c - Kit , c -Met , HIF . R18 Exemplary inhibitors of ErbB2 receptor ( EGFR family ) include but not limited to AEE788 (NVP - AEE 788 ), 20 BIBW2992 , (Afatinib ) , Lapatinib , Erlotinib ( Tarceva ), and R15 Gefitinib ( Iressa ) . Exemplary RTK inhibitors targeting more then one sig HN naling pathway (multitargeted kinase inhibitors ) include ?111111 R19Río AP24534 (Ponatinib ) that targets FGFR , FLT - 3 , VEGFR - 25 Ö HO PDGFR and Ber - Abl receptors ; ABT- 869 (Linifanib ) that OR 14 targets FLT- 3 and VEGFR - PDGFR receptors; AZD2171 that targets VEGFR - PDGFR , Flt - 1 and VEGF receptors ; CHR 258 (Dovitinib ) that targets VEGFR -PDGFR , FGFR , Flt- 3 , wherein : and c - Kit receptors . 30 R14 is hydrogen , C ( O ) - C1- 3 alkyl or C ( O ) - chloro Exemplary protein chaperon inhibitors include HSP90 substituted C1- 3 alkyl; inhibitors . Exemplary HSP90 inhibitors include 17AAG Ris is hydrogen , CHz or CHO ; derivatives, BIIBO21, BIIB028 , SNX - 5422 , NVP - AUY - 922 when R17 and Rig are taken independently , R1, is hydro and KW - 2478 . Exemplary HDAC inhibitors include Belinostat 35 gen , and either R16 or R17 is ethyl and the other is hydroxyl ; (PXD101 ) , CUDC - 101 , Droxinostat , ITF2357 (Givinostat , when R?, and Rig are taken together with the carbon to Gavinostat) , JNJ- 26481585 , LAQ824 (NVP - LAQ824 , which they are attached to form an oxiran ring , R16 is ethyl; Dacinostat ) , LBH - 589 (Panobinostat ) , MC1568, Rig is hydrogen , OH , amino group , alkyl amino or — [ C MGCD0103 (Mocetinostat ), MS- 275 ( Entinostat) , PCI- an 112012111a22 24781, Pyroxamide (NSC 696085 ), SB939 , Trichostatin A each ofR20 and R21 independently is hydrogen , C1- 6 alkyl, and Vorinostat (SAHA ) . C6- 10 aryl, hydroxylated C6- 10 aryl, polyhydroxylated C6- 10 Exemplary PARP inhibitors include iniparib (BSI 201) , aryl, 5 to 12 -membered heterocycle , C3- 8 cycloalkyl, olaparib (AZD -2281 ) . ABT- 888 (Veliparib ). AG014699 . hydroxylated C3 -8 cycloalkyl, polyhydroxylated Cze CEP 9722 , MK 4827 , KU -0059436 (AZD2281 ) , LT- 673 , 45 cycloalkyl or a side chain of a natural or unnatural amino 3 - aminobenzamide , A - 966492 , and AZD2461 acid ; Exemplary Wnt/ Hedgehog signaling pathway inhibitors R22 is OH , - NH2, COOH , - R32 - C ( O ) ( CH2) . include vismodegib (RG3616 /GDC -0449 ) , cyclopamine C (H )( R23 ) — N ( H ) (R23 ), - R32 - C (O )( CH2 ) (O CH2 ( 11 -deoxojervine ) ( Hedgehog pathway inhibitors ) and XAV CH2) N ( H ) (R23 ) or R32 — ( C ( O ) - CH (X² ) NH ) . 939 (Wnt pathway inhibitor ) 50 R77 ; Exemplary RNA polymerase inhibitors include amatox - each R , , independently is hydrogen , C , alkyl, Ca 1 aryl, ins . Exemplary amatoxins include a -amanitins , ß -amanitins , y -amanitins , e -amanitins , amanullin , amanullic acid , amani C3- 8 cycloalkyl, COOH , or COO _ C1- alkyl; namide, amanin , and proamanullin . X² is a side chain of a natural or unnatural amino acid ; In one embodiment the drug of the invention is a non - 55 R77 is hydrogen or X² and NR , 7 form a nitrogen contain natural camptothecin compound , vinca alkaloid , kinase ing heterocyclic moiety ; inhibitor ( e . g . , PI3 kinase inhibitor (GDC -0941 and R . , is — NH or oxygen ; PI- 103 )) , MEK inhibitor, KSP inhibitor, RNA polymerse inhibitor, PARP inhibitor , docetaxel, paclitaxel, doxorubicin , a is an integer from 1 to 6 ; duocarmycin , tubulysin , auristatin or a platinum compound . 60 c is an integer from 0 to 3 ; In specific embodiments , the drug is a derivative of SN - 38 , d is an integer from 1 to 3; and vindesine , vinblastine , PI- 103 , AZD 8330 , auristatin E , auristatin F , a duocarmycin compound , tubulysin compound , f is an integer from 1 to 12 . or ARRY- 520 . Further examples of Vinca alkaloids are described in US In another embodiment, the drug used in the invention is 65 2010 /0305149 and US 2002 / 0103136 . a combination of two or more drugs , such as , for example , In one embodiment the Vinca alkaloid of Formula ( V ) is P13 kinases and MEK inhibitors ; broad spectrum cytotoxic a compound of Formula ( VI) : US 9 ,943 ,609 B2 74 73 -continued ( VI) CH ; 0 nu NH2; HN ( 10 ) ????N OH H3C02C NH2; CH30 111111 - Xii ( 11)

NH : HCV w

HN (12 ) R40 lil ?? HOYHO ?? mm NH2; Xile ( 13 ) CH3 wherein : R40 is hydrogen , OH , - NH2, or any of the following NH ; structures : 25 Xabier ( 14 ) mm „ NH2; CH3 ( 15 ) Xtil CH , 0 w OH ; mm foroffoxCH3 ( 16 ) mi OH ; * Holter 11- 12NH2 ; ( 17) OH ; – NH2 ; CH3 ( 18 ) min NH2; porenteryourLethycho– C( H )( CH3) - - chuoti ( CH2 ) 2NH2 ; CH3 ( 19 )

m NH2; ; and

CH (20 ) 0 CH3

NH ) ; CH3; tlfox tw NH2 NH2; wherein : mm 65 a is an integer from 1 to 6 ; and X . merec is an integer from 0 to 3 . US 9 , 943 ,609 B2 75 76 In one embodiment, R40 is In one embodiment the non -natural camptothecin com pound of Formula (VII ) is a compound of Formula (VIII ) or Formula (XXV ): OH . 5 mi (VIII )

In another embodiment , non -natural camptothecin is a R3 compound of Formula (VII ) : 10 10 CO ( VII ) - OH ; or R24 15

R25 (XXV ) HO R26R26 20 R27 N wherein : R30 R24 is - H , - C1, - F , - OH or alkyl ; or R24 and R25 , 25 may be taken together to form a five - or six -membered ring ; R25 is H , F , OH , CH3, - CH = N / O -t - Butyl , CH?CH, Si ( CH3) 3 , Si ( CH?) ) -t -butyl , JOC ( O ) R29; wherein R30 is — NH2, R28 - C1- 6 alkyl- R22 , 5 to R29 is - NH2, R28 - C1- 6 alkyl -R22 , 5 to 12 -membered 30 12 -membered heterocycloalkyl, R28 - C5- 12 heterocy heterocycloalkyl , R28 - C5- 12 heterocycloalkyl- C1 - talkyl cloalkyl -C1 - 6 alkyl -R22 or — R28 - C1- 6 alkyl- C6 - 12 aryl R22 or - R28 - C1- 6 alkyl- C6- 12 aryl -C1 - 6 alkyl- R22 ; C1- galkyl - R22 ; R26 is — H , - CH2- N (CH3 ) 2 , NH2, or NO2; R28 is absent, NH or oxygen ; R27 is ethyl, N -methyl piperidine , cycloalkyl, R22 is OH , - NH2, COOH , - R82 - C ( O ) (CH2 ) . - CH2CH NHCH (CH3 ) 2 , or — N - 4 -methylcyclohexylam - 35 C ( H ) (R23 ) — N ( H ) (R23 ) , - R92 - C ( O ) ( CH2) - ( O CH2 ine ; CH3) N ( H ) ( R23 ) or — R 2 — ( C ( O ) - CH ( X² ) NH ) . R79 is – H or - C ( O ) - R28 — [ C (R20R21 ) la - R22 ; R77 ; each of R20 and R21 independently is hydrogen , C1- 6 alkyl, each Rzz independently is hydrogen , C1- 6 alkyl, C6- 10 aryl , C6 - 10 aryl, hydroxylated C6- 10 aryl, polyhydroxylated C - 10 C3- 8 cycloalkyl, - COOH , or — COO _ C1- talkyl ; aryl , 5 to 12 -membered heterocycle , C3- 8 cycloalkyl, X is a side chain of a natural or unnatural amino acid ; hydroxylated C3- 8 cycloalkyl, polyhydroxylated C3- 8 Rz7 is a hydrogen or X² and NR77 form a nitrogen cycloalkyl or a side chain of a natural or unnatural amino containing cyclic compound ; acid ; Rg2 is — NH or oxygen ; R , , is — OH , — NH2, – COOH , — R92 - C ( O ) ( CH2) . - 45 c is an integer from 0 to 3 ; C ( H ) (R23 ) — N ( H ) (R23 ) , — R82 - C ( O ) ( CH2) . ( O CH2 — d is an integer from 1 to 3 ; and CH3) N ( H ) (R23 ) , or R82 — ( C ( O ) CH (XP ) NH ) f is an integer from 1 to 12 . R77; each R23 independently is hydrogen ,C1 - 6 alkyl, C6 -10 aryl, In some embodiments R30 is any one of the following C3 -8 cycloalkyl, COOH , or COO _ C1- alkyl; 50 structures : X2 is a side chain of a natural or unnatural amino acid ; R77 is a hydrogen or X² and NR77 form a nitrogen containing cyclic compound ; @ R32 is — NH or oxygen ; or R26 and R27 when taken together with the two carbon 55 $ _ NH ( CH2) g - NH ; atoms to which they attach and the third carbon atom connecting the two carbon atoms form an optionally sub @ stituted six -membered ring ; pormun R28 is absent, NH or oxygen ; ? NH — (CH2 ) , - OH ; a is an integer from 1 to 6 ; 60 == C609 01 c is an integer from 0 to 3 ; @ d is an integer from 1 to 3 ; NH2; f is an integer from 1 to 12 ; u is an integer 0 or 1 ; w is an integer 0 or 1 ; and 65 with the proviso that the compound of Formula (VII ) must mi contain at least one of R29 and R79 . VIDmuseos US 9 ,943 ,609 B2 77 78 - continued wherein (4 ) R47 is an amino group , R , [C (R2R22 ) la - R10 , 5 - R , C3- 12 heterocycloalkyl -C4 - galkyl - R10 or 5 to 12- membered heterocycloalkyl; each of R20 and R21 independently is hydrogen , C1- 6 alkyl, to C6- 10 aryl , hydroxylated C6- 10 aryl , polyhydroxylated C6- 10 – O – ( CH2) , — NH2; 10 aryl, 5 to 12- membered heterocycle , C3- 8 cycloalkyl , min hydroxylated C3- 8 cycloalkyl, polyhydroxylated C3- 8 cycloalkyl or a side chain of a natural or unnatural amino 15 acid ; NH2; Rio is OH , NHR33 , - N - (R23 )R11 , - COOH , mu - R32 C (O ) (CH2 ) . - C ( H ) (R23 ) N ( H ) (R23 ), R32C 20 ( O ) (CH2 ) - ( 0 CH2CH2) N ( H ) (R23 ) , - R82 — ( C - ( CH2) - NH2; (O ) CH (X2 ) NH ) R77 or - R82C( 0 ) [ C (R20 R21) la - R32 - R83; ann (8 ) each R23 independently is hydrogen , C1 -6 alkyl, Co- 10 aryl , – C( H ) ( CH3) - ( CH2) NHg ; and 25 C3- 8 cycloalkyl, COOH , or - COO C1- 6 alkyl ; X² is a side chain of a natural or unnatural amino acid ; m R77 is a hydrogen or XP and NR. 77 form a nitrogen na ; 30 containing cyclic compound ; Rg2 is — NH or oxygen ; HN R , is absent, N ( R 3 ) or oxygen ; Rg3 is hydrogen or CH3; R11 is:

NH ) min NH CNH - R13 helR12 pingCutty upmy birthinto wherein : each R12 independently is hydrogen , chloride, — CHz or a is an integer from 1 to 6 ; c is an integer from 0 to 3 ; and OCHZ; g is an integer from 2 to 6 . R13 is hydrogen or - C (O ) (CH2 ) . (O CH In another embodiment the PI3 kinase is a compound of 50 Formula ( IX ): X4 is the side chain of lysine , arginine, citrulline, alanine ( IX ) 55 Or glycine; X , is the side chain of phenylalanine , valine , leucine, isoleucine or tryptophan ; each of Xo and X , is independently the side chain of 60 glycine , alanine, serine, valine or proline; IN a is an integer from 1 to 6 ; R47 c is an integer from 0 to 3 ; d is an integer from 1 to 3 ; 65 f is an integer from 1 to 12 ; and each u independently is an integer 0 or 1 . US 9 ,943 ,609 B2 79 80 In some embodiments 79 is citrulline -valine ; lysine- phenylalanine ; citrulline -phenyl Insome embodime alanine; citrulline- leucine ; citrulline -valine - glycine - glycine; X5 glycine- phenylalanine - glycine - glycine ; valine ; proline ; leu 5 cine or isoleucine . Hyyping In another embodiment, R11 is any one of the following structures: Hronteroit ( 1 ) mm ?????NH IZ " NH2;

NH

O NH2

mothanthi NH , NH

0 NH2

NH2; and MT III NH2; they thatNH NH

HN 0 NH o CNH ) US 9439 ,943 , ,609 B2 B2 - continued 82 mi NH2 NH

HN

0 NH2NH

In some embodiments R47 is any one of the following structures : man – NH?esi( CH2 )g - NH ; mfanowana –x NH? =( CH2 ), — OH; NH2; LOH ;

mu magariim

- O - (CH2 ) , — NH2; We ya NH : ? ( CH2) . - NH2; ?wi - ??????CH ( CH3) - (??????? CH2) 2NH2 ; ( 10 ) CH3 mi mfumoinform- N - ( CH2) , — OH ;

HN

( 12 ) CH3 CH3 mine —2014 N — (CH2 ) g -- NH2 ; porno— N — (CH2 ) , — 0 - - ( CH2) , — NH2; ( 14 ) CH3 mowa CH3 = win ComboonN — ( CH2) , — O - C - ( CH2) , - OH - N - ( CH2) , - 0 - ! CH3 O=0 in - N - ( CH2) ; - O - O . ? ??????????? NH2? US 9 ,943 ,609 B2 8383 US 9, 943 , 609 B2 84 - continued continued ( 16 ) mi thylhagytaNH2;

HN

0 NH2

( 17 )

mi N thylaysayanN NH2; and HN

07 NHNH2

( 18 )

mm IIII. wory NHL

HN

o NH2 wherein : R33 is hydrogen , C1- 8 alkyl , C3- 8 carbocycle , C6- 10 aryl , a is an integer from 1 to 6 ; 50 C1- 8 alkyl- C6- 10 aryl, x ! - ( C3- 8 carbocycle ), C3- 8 hetero c is an integer from 0 to 3 ; and cycle or X ! (C3 - 8 heterocycle ); g is an integer from 2 to 6 . R34 is hydrogen , C1- 8 alkyl, C3- 8 carbocycle , C6- 10 aryl , In another embodiment the auristatin is a compound of X ? _ C6- 10 aryl, X ? — (C3 - 8 carbocycle ) , C3- 8 heterocycle or Formula ( X ) : 55 . X ' = (C3 - 8 heterocycle ); R35 is hydrogen or methyl; or R34 and R35 , together with the carbon atom to which they attach form a carbocyclic ring having the formula R33 0 R37 Riga - CH, R44 — ( CR55R41) n - wherein each of R35 and R41 independently R31R3iaNYX , i 1 6 0 is hydrogen or C1- 8 alkyl and b is an integer from 3 to 7 ; Rz? © R34 R35 R36 R38 Ö RS3 R36 is hydrogen or C1- 8 alkyl ; R38 0 Rz7 is hydrogen , C1- 8 alkyl , C3- 8 carbocycle , C6- 10 aryl, - X - C6- 10 aryl, - X ? - (C3 - 8 carbocycle ), C3- 8 hetero wherein : 65 cycle or — X7— (C3 - 8 heterocycle ) ; each of R31 and R32 independently is hydrogen or C1- 8 each Rzg independently is hydrogen , OH , C1- 3 alkyl, C3- 8 alkyl and at most one of R31 and R32 is hydrogen ; carbocycle or 0 — (C1 - 8 alkyl) ; US 9 ,943 ,609 B2 85 86 RegisR53 is : 85 R39 is benzyl or

us mm R45 or R 54 R39 R39 is H , C1- 8 alkyl, C6- 10 aryl, - X ! – C6- 10 aryl, C38- 100 carbocycle , C3- 8 heterocycle , — X — C3- 8 heterocycle , mm C1- galkylene -NH2 , or ( CH2) 2SCH and Serveieach X ' independently is C1- 10 alkylene or C3- 10 Run is hydrogen ; cycloalkylene; 15 In one embodiment the auristatin of Formula ( X ) is a R44 is hydrogen or C1- 8 alkylsel; akykeneos Csom compounder en represents of Formula are(XI ) , Formula (XII ) or Formula R45 is X - R42 or NH _ R19 ; (XIII ) : many reasons X3 is O or S ; wherein the compound of Formula (XI ) is :

H3C CH3 H3C . - CH3 CH3 H3C . .IIlllll CH3 CH3 CH3 Ö ??? | H?c CCHC 7 0- R42

R1, is hydrogen , OH , amino group , alkyl amino or — [C30 wherein R42 is CH , or any one of the following struc (R20 R21 )] a - R22 ; tures : R42 is an amino group , C1- 6 alkyl amino or — [ C (R20 R21 )] a - R22 ; each of R20 and R21 independently is hydrogen , C1- 6 alkyl, @ C6- 10 aryl , hydroxylated C6- 10 aryl, polyhydroxylated C6- 10 aryl , 5 to 12 -membered heterocycle , C3- 8 cycloalkyl, un OH ; hydroxylated C3- 8 cycloalkyl, polyhydroxylated C3- 8 cycloalkyl or a side chain of a natural or unnatural amino acid ; OH ; R22 is — OH , — NHR23, – COOH , – R82 - C (O ) 40 men (CH2 ) . C (H )( R23 ) N (H )( R23 ), - R32 - C (O ) CH3 (CH ). ( O CH , CH2) , N ( H .)( R23 com ) or .- R32 h (CO .co ) ; * * CH( X ) - NHOM - R77 ; each R23 independently is hydrogen , C1- 6 alkyl , C6- 10 aryl, 45 C3- 8 cycloalkyl, COOH , orc00 C1- galkyl ; X² is a side chain of a natural or unnatural amino acid ; OH ; R77 is a hydrogen or X² and NR77 form a nitrogen ni containing cyclic compound ; @ Rg2 is — NH or oxygen ; 50 R54 is _ C (R56 )2 - C (R56 )2 - C6- 10 aryl, C (R56 )2 - C (R56 ) 2 - C3- 8 heterocycle or — C (R56 ) 2 – C (R56 ) 2 – C3- 8 car bocycle ; Rso is independently selected from H , OH , C1- 8 alkyl , C3- 8 CHZ carbocycle , O _ C1- 8 alkyl, O _ C ( O ) - R29 and 55 - 0 – R230 C1- galkyl - NH2 ; @ R29 is an amino group , 5 to 12 -membered heterocy cloalkyl , - R28 C1- 6 alkyl- R22 , R28 - C5 - 12 heterocy NH : cloalkyl- C1 - 6alkyl -R22 , [ C (R20 R21 ) la - R22 , or - R28 — C1- 6 alkyl- C6- 12 aryl- C1 - 6 alkyl- R22 ; 60 R28 is absent, NH or oxygen ; mu CH3 a is an integer from 1 to 6 ; c is an integer from 0 to 3 ; d is an integer from 1 to 3 ; and NH2; fis an integer from 1 to 12 . 65 nim In some embodiments , in the auristatin compound of Formula ( X ) : US 9, 943, 609 B2 87 88 - continued -continued (8 ( 15 ) xri NH2; a CH , CH3 0 NH2; xil tilmu . " 10 ( 16 ) w o ( 10 ) XL„ NH2. ; " . Hulst1- 2 11- 12NH2 ; ( 17 )

20 , NH2; — (CH2 ) , - NH2; m X CH3 "stom (11 ) 25 ( 18 ) CH3 w - CH ( CH3) - ( CH2 )2NH2 ; theirNH2; fameurende ( 19 ) ( 12) O CH3 mi XileNH2 ; Xindy ; and

CH3 (20 ) 0 CH3 NH2; CH3; Xem NH2 (14 ) that CH 0 NH2; wherein : 50 a is an integer from 1 to 6 ; and CH3 c is an integer from 0 to 3 ; Xem wherein the compound of Formula (XII ) is :

( XII) H3C H39 O H3C , -111111 CH3 CH3 CH3 Ö OCH3 0 H3C CH3 07 N - R40 US 9 ,943 ,609 B2 68 90 wherein R40 is hydrogen , OH , - NH2, or any of the - continued following structures :

NH2; a X

OH ; CH3 ( 11 ) ma 0 m CH3 NH2; OH ; " the mi CH ? ( 12 )

NH2; ? mu min xer ( 13) NH ) ; Xem ( 14 ) HO mm NH2; MyCH3 ** * XiCH3 ( 15 )

NH2; mun CH3 .: xeit ( 16 ) mmHarith 1 - 2 -- NH ( 17 ) NH2; - ( CH2) , — NH2; the ofsuportalesque (18 ) –cances C ( H) ( CH3) - ( enCH2 ) NHels ; Ximu NH2 ; ) 61( and CH3 0 (20 ) NH2; ethit0 CH3 mt Y CH3; the CH3; mu NH2 (10 ) CH3 0 NH2; wherein : xemn 65 a is an integer from 1 to 6 ; and o c is an integerde from pat 0 to 3 . US 9 , 943, 609 B2 91 wherein the compound of Formula (XIII ) is :

(XIII ) H3CC H3C _R29 CH3 H3C Illlll CH ; CHz OCH Ö OCH 0 CH3 H3C CHz

wherein R29 is an amino group , 5 to 12 -membered het - 15 R77 is a hydrogen or X2 and NR77 form a nitrogen erocycloalkyl , - R28 - C1- 6 alkyl- R22 ,R28 - C5- 12 heterocy - containing cyclic compound ; cloalkyl- C1- 6 alkyl- R22 , — R28 [ C (R20R21 ] a - R22 , or Re, is — NH or oxygen ; - R28 - C1- 6 alkyl- C6 - 12 aryl- C1 - 6 alkyl- R22 ; each ofR20 and R21 independently is hydrogen , C1- 6 alkyl, c is an integer from 0 to 3 ; C6- 10 aryl, hydroxylated C6- 10 aryl, polyhydroxylated C6- 10 20 d is an integer from 1 to 3 ; and aryl, 5 to 12 -membered heterocycle , C3- 8 cycloalkyl , f is an integer from 1 to 12 . hydroxylated C3- gcycloalkyl, polyhydroxylated C3- 3cy cloalkyl or a side chain of a natural or unnatural amino acid ; In yet another embodiment, R29 is any one of the follow R22 is OH , NHR23 , COOH , - R32 - C ( O ) . ing structures : (CH2 ) . C (H )( R23 ) — N (H )( R23 ), - R32 - C ( O ) (CH ) ( O CH , CH2) , N ( H )( R23 ) or R $2 ( C (O ) CH ( X ^) … NH ) - R77 ; each R23 independently is hydrogen , C1- 6 alkyl, C6- 10 aryl, C3- 8 cycloalkyl, — COOH , or COO _ C1- alkyl; 30 5 – NH?( CH2 ) g – NH2 ; X2 is a side chain of a natural or unnatural amino acid ; wanan Rz7 is a hydrogen or X2 and NR ,, form a nitrogen w containing cyclic compound ; Rg2 is — NH or oxygen ; ? NH — (CH2 ) , - OH01 ; R9, is absent, NH or oxygen ; 35 09 a is an integer from 1 to 6 ; c is an integer from 0 to 3 ; NH2; d is an integer from 1 to 3 ; and f is an integer from 1 to 12 . In one embodiment, in Formula (XII ) , R40 is 40 min ton2OH ; mwi OL 45 min noun NH2, or. CH3 50 B0 — (CH2 ) , — NH2; OH

mm CH3 55 In one embodiment in the compound of Formula (XIII ), NH2; R29 is — NH2, 5 membered heterocycloalkyl , R28 - C1- 6 alkyl -R22 , R28 - C5- 12 heterocycloalkyl -C1 - 6 alkyl- R22 or - R28 - C1- 6 alkyl -C6 - 12 aryl- C1 - 6 alkyl- R22 ; R2g is absent, NH or oxygen ; 60 (CH2 ) . - NH2; R22 is — OH , NHR23, COOH , R32 - C (O ) (CH2 ) . — C (H ) (R23 ) — N ( H ) (R23 ), - R32 - Cco ( O ) win C1) NH2 (CH ) ( O CH , CH2) , N ( H ) (R23 ) or R32 ( C ( O ) CH ( X ^ ) NH) Ram, each R23 independently is hydrogen , C1- 6 alkyl, C6- 10 aryl , 65 ? - C ( H ) (CH3 ) - (CH2 ) NH2; and C3- 8 cycloalkyl, COOH , or COO _ C1- talkyl ; ary, as marian CMCH) – cih . Nh; and X2 is a side chain of a natural or unnatural amino acid ; US 9 , 943 ,609 B2 93 94 -continued In some embodiments R43 is C ( O ) ( CH2) - NH2, or - C (O ) - C (H ) (CH3 ) ( CH2) . - NH2; in which a is an inte ger from 1 to 6 ; and c is an integer from 0 to 3 . 5 In another embodiment , the duocarmycin compound is a min compound of Formula (XV ):

HN 10 (XV ) wherein : a is an integer from 1 to 6 ; R49 ? c is an integer from 0 to 3 ; and R48 R51 g is an integer from 2 to 6 . In one embodiment, the MEK inhibitor is a compound of N R52R52 Formula ( XIV ) : AA 20

R47

25 ((XIVXIV ) LOR43 F L OR wherein : R47 is as defined herein ; 30 Rag is hydrogen , COOC - alkyl, - COOH , NH , or CH3; R49 is C1, Br or OH ; 35 Rso is hydrogen , OCHZ , wherein R43 is H or - R46 R47 ; each of R2, and Rz, independently is hydrogen , C1- 6 alkyl, C6- 10 aryl , hydroxylated C6- 10 aryl, polyhydroxylated C6- 10 aryl, 5 to 12 -membered heterocycle , C3- cycloalkyl, hydroxylated C3- gcycloalkyl, polyhydroxylated C3- scy cloalkyl or a side chain of a natural or unnatural amino acid ; R22 is OH , — NH2, COOH , - R32 - C ( O )( CH2) . — mu C ( H ) (R23 ) - N ( H ) (R23 ) , - R32 - C (O ) (CH2 ) ( O CH2— ; or CH2) - N (H )( R23 ) or R $2 (C (O ) CH (X² ) NH ) R77; each R23 independently is hydrogen , C1- 6 alkyl , C6- 10 aryl, H3C C3- 8 cycloalkyl , COOH , or COO _ C1- galkyl ; X² is a side chain of a natural or unnatural amino acid ; Rz7 is a hydrogen or X² and NR. 77 form a nitrogen containing cyclic compound ; OH e 55 tyre R 2 is NH or oxygen ; 55 R46 is C ( 0 ) ; C (O ) , - C ( O )- NH , or absent ; each of Rs1 and Rs2 independently is hydrogen or R47 is as defined herein ; OCHz; and a is an integer from 1 to 6 ; 60 ring AA is either a phenyl or pyrrolyl ring . c is an integer from 0 to 3 ; Further examples of duocarmycin compounds are dis d is an integer from 1 to 3 ; and closed in U .S . Pat. No. 7 ,553 ,816 . f is an integer from 1 to 12 . In one embodiment the duocarmycin compound of For Further examples of the MEK inhibitor are disclosed in mula (XV ) is a compound of Formula (XVI ) , (XVII ) , U . S . Pat. No. 7 ,517 , 994 B2 . ( XVIII ) or (XIX ) : US 9, 943, 609 B2 95 96

(XVI ) OCH3 KO

- OCH ; H300 N OCH3 - IZ DevelopmentR47

OCH3 (XVII )

- OCH3

IZ OCH ;

O R47 02

(XVIII ) al HiC

NH

H3C ??

sportsR 47

( XIX )

H3C N

< d US 9, 943, 609 B2 97 98 wherein : - continued R49 is C1, Br or OH ; and R4, is as defined herein . OH ; In another embodiment, the duocarmycin compound is a duocarmycin SA compound of Formulacourtesy (XX ) : ofU . S . protes Pat . No . 3 t min CH3 5 , 101, 038 ; or (XXI ) : mm OCH ; 10 OH ; - OCH ; mer 15 CH3 OCHZ R42 - 0 NH NH2; 20 (XXI ) mu CH3 H3C - 0 - R42 NH2; Br 25 HN mi OCH3 CH3

H CN NCOO - OCH3 30 NH ; OCH tot wherein : 3535 NH2; R42 is C1- 6 alkyl amino or — [C (R20R21 ]. - R22; each ofR20 and R21 independently is hydrogen , Cl- , alkyl, Xin C6- 10 aryl , hydroxylated C6- 10 aryl, polyhydroxylated C6- 10 o aryl, 5 to 12 -membered heterocycle , C3- 8 cycloalkyl, hydroxylated C3- 8 cycloalkyl, polyhydroxylated C3- 8 40* NH2; cycloalkyl or a side chain of a natural or unnatural amino acid ; mmthie R22 is OH , - NH2, COOH , R32 - C ( O ) ( CH2) . 0 C (H )( R23 ) N ( H )( R23 ), - R82 - C (O ) ( CH2) - ( O CH2 – 45 CH2) - N (H ) (R23 ), or R 2 — ( C ( O ) - CH (X² ) — NH ). " NH2 R77; XL each R23 independently is hydrogen , C1- 6 alkyl , C6- 10 aryl, 0 CH3 C3- 8 cycloalkyl, - COOH , or — COO _ C1- alkyl; X2 is a side chain of a natural or unnatural amino acid ; 50 NH) ; Rz7 is a hydrogen or X2 and NR77 form a nitrogen containing cyclic compound ; mm Rg2 is — NH or oxygen ; 0 CH3 a is an integer from 1 to 6 ; 55 c is an integer from 0 to 3 ; XLmu NH2; d is an integer from 1 to 3 ; and f is an integer from 1 to 12 . CH3 In some embodiments , R42 is any one of the following structures: 60 XiiNH2 ; CH2 0 @ mm NH2; cm CH3 US 9 ,943 ,609 B2 99 100 - continued R64 is : CHO (15 ) R71 5 (16 ) R73 or mi NH2 10 (17 ) min - ( CH2) a - NH2; R68 15 ( 18 ) R72

( CH3 )2NH2 ; R73; m (19 ) 20 mm 7 ; and 25 wherein : ( 20 ) Rog is hydrogen or C . -C . alkyl ; R69 is CO R70 , C ( O ) - R73 , CONHNH2, OH , NH , SH or optionally substituted alkyl, cycloalkyl , heteroalkyl or het CH3; erocycloalkyl group ; 30 R70 is an optionally substituted alkyl ( i. e . C1- 6 alkyl mi NH2 amine) , heteroalkyl or heterocycloalkyl group ; each of R71 and R73 independently is hydrogen , halo , - NO , , - CN , — NHR 4 , C , alkyl, haloalkyl, alkoxy , and wherein : haloalkoxy ; a is an integer from 1 to 6 ; and 35 R22 is hydrogen , OR43 , alkoxy, halogen , — NHR74 , c is an integer from 0 to 3 . - O - C ( O ) - R47, NO2 , - CN , C6- 10 aryl , C1- 6 alkyl, amino another embodiment the tubulysin is a compound of or dialkylamino ; In another embodiment the tubulysin is a compound of R74 is hydrogen , CHO , - C ( O ) - C1- alkyl, OH , amino Formula (XXII ): group , alkyl amino or — [ C (R20R21 ) la - R22 ; R43 is H or - R46 R47; 40 R46 is C (O ) ; C (O ) O - , - C ( O ) - NH — , or (XXII ) absent; O R61 R62 R63 R4, is as defined herein ; T R64R64 R78 is X - R75 or NH — R19 ; Z X3 is O or S ; N A5 R19 is hydrogen , OH , amino group , alkyl amino or — [ C 0 R59 Roo (R20 R21 )] a - R22 ; R75 is a hydrogen , an amino group , C1- 6 alkyl amino or — [ C (R20R21 R22; wherein : each ofR20 and R21 independently is hydrogen , C1- 6 alkyl, 50 C6- 10 aryl, hydroxylated C6- 10 aryl, polyhydroxylated C6- 10 Rs7 is C1- 4 alkyl or - C ( O )R58 ; aryl, 5 to 12 -membered heterocycle , C3- cycloalkyl, R58 is C1- 6 alkyl, CF3 or C6- 10 aryl ; hydroxylated C3- 8 cycloalkyl, polyhydroxylated C3 -8 Rsg is C1- 6 alkyl; cycloalkyl or a side chain of a natural or unnatural amino Roo is hydrogen , C1 -6 alkyl, C2- 7 alkenyl , CH2- phenyl , acid ; CH OR6 or CH OCOR66 ; 55 R22 is — OH , — NH2, COOH , - R32 - C (O ) (CH2 ) . - Ros is hydrogen , C1- 6 alkyl, C2- 7 alkenyl, C6- 10 aryl or C ( H ) (R23 ) N ( H ) (R23 ), - R32 - C ( O ) (CH2 ) . ( O CH2 C ( O )R67 ; CH ) , N ( H ) (R23 ), or — R92 — ( C ( O ) - CH (X² ) NH ) . R67 is C1- 6 alkyl , C2- 6 alkenyl , C6- 10 aryl or heteroaryl; R77each ; R23 independently is hydrogen , C1- 6 alkyl , C6- 10 aryl, R66 is C1- 6 alkyl, CoHz or CH2- phenyl ; 60 C3- 8 cycloalkyl , COOH , or — COO C1- 6 alkyl; X2 is a side chain of a natural or unnatural amino acid ; R61 is C1- 6 alkyl; Rz7 is a hydrogen or X2 and NR77 form a nitrogen R62 is hydrogen , OH , O - C1- 4 alkyl or 0 - C ( O ) containing cyclic compound ; C1- alkyl ; Rg2 is — NH or oxygen ; Roz is hydrogen , OH , O _ C1- 4 alkyl, O _ C ( O ) 65 Ray is as defined herein ; C1- alkyl, halogen or C1- 6 alkyl ; a is an integer from 1 to 6 ; e is an integer between 1 and 3 inclusive ; c is an integer from 0 to 3 ; US 9 ,943 ,609 B2 101 102 d is an integer from 1 to 3 ; -continued f is an integer from 1 to 12 ; and with the proviso that when R69 is C ( O ) X - R75 or CH3 0 C ( O ) - NH - R19 , one or both of R71 and R73 are — NHR74 , NH2; and R72 is OR43 , — NHR74 or 40 C ( O ) - R47 , at least one 5 of R19 , R43 , R74 and R75 cannot be hydrogen . mon In some embodiments in the compound of Formula ( 10 ) ( XXII ) : ·Formula 0 R37 is CH3; NH2; Rs9 is sec -butyl ; 10 o L R60 is hydrogen , methyl, ethyl, propyl, iso -propyl or X ( 11 ) iso -butyl ; on methos, olhyl , propy , korpeops on" CH3 Roi is iso -propyl , R62 is hydrogen ; Roz is hydrogen , OH , O C3H ,, O C (O ) CHz; mm h eNH2 ; Ros is hydrogen or — CHZ; ( 12 ) R69 is CO2H , CO R70 or C ( OCH ) - ,R78 0 ;- co - cns ; t O R70 is C1- 6 alkyl amine; mum each of R71 and R73 independently is hydrogen ; 20 ww NH2; R72 is hydrogen , — OR 43 , OH , F , — CHz or — OCHZ; ( 13 ) R78 is OH , OR75 or — NHR 40 ; e is the integer 2 ; NH ; R40 is hydrogen , OH , - NH2, or any of the following m structures : o l im leaving ou, with, or any of the following, ( 14 ) mus XO ( 15 ) C CH 0 wasfound OH : mi mu CH3 D (16 ) mi JOH ; E Harmin hatt2NH , OH ; ( 17 ) men (CH2 ) a - NH2; © win emuat ( 18 ) tofind NH2; – C( H) ( CH3) - ( CH2) NH2; CH3 mifrom coctades ( 19 ) © you NH2; ; and ( 20 ) © O CHZ w NH2; N NH2 DO withoutyouw NH2; wherein : a is an integer from 1 to 6 ; sec is an integer from 0 to 3 ; US 9 , 943 ,609 B2 103 104 R75 is any one of the following structures: - continued ( 11 ) OCH mm " . Men" NH2 ; ( 12 )

NH2; mi CH3 " » XOLI ( 13 ) @

my NH2; Xii ( 14 ) min OH ; NH , CH3 @ ( 15 ) XtCH , mi NH2; men CH3 @ ( 16 ) NH2; min - 12NH . CH3 Holand tul ( 17 ) @

( CH2 ) , NH ) and NH2; twin ( 18 ) - C ( H ) ( CH3) - ( CH2) 2NH2 ; NH ; " . coca colate Xi @ CH3 0 wherein : a is an integer from 1 to 6 ; and NH2; *45 c is an integer from 0 to 3 ; R43 is hydrogen , C ( O ) (CH2 ) a NH2, or - C ( O ) C tuil ( 10 ) ( H ) ( CH? ) ( CH2 ) _ NH ; O wherein : NH2; 5050 alsa is auan integer from 1 to 6 ; c is an integer from 0 to 3 ; and XL R47 is any one of the following structures :

( 1 ) wan – NH?( CH2) – NH2; mun – NH - ( CH2) g – OH ;

- NH2; OH ; tonmm Home US 9 ,943 ,609 B2 105 106 -continued (5 ) - O - (CH2 ) , - NH2; mi NH2; mi - ( CH2) , - NH2; mufu C ( H )( CH3) = (CH2 ) . NH ; ( 10 ) mm mfanomunwan - N - (CH2 ) , - OH ; HN

(12 ) CH3 O = - N mens- (CH2 ) , - NH2 ; mofon N –— cons ( CH2;) 7 — ???? OÖ– — cons ( CH2,) , = — stsNH2 ; ( 13 ) ( 14 ) ? - N ???????????rits- ( CH2) , - 0 – 0 — ( CH2) g - OH man - N ??????????- ( CH2) - O - ( 15 ) CH3 0 HollyN — ( CH2) , – 0 – 0 . NH

( 16 ) mi thylaysanNH2;

HN

O NH2

( 17 )

mm N thingsN NH2; or

HN

0 NH2 US 9 ,943 ,609 B2 107 108 - continued (( 1818 )

IIIII NH NH2;

HN

07 CNHNH2, wherein : with the proviso that if R72 is OH , then R75 cannot be hydrogen ; if R69 is COOH then R72 must be OR43 or a is an integer from 1 to 6 ; 200 C (0 ) R47 . c is an integer from 0 to 3 ; and In some embodiments , the tubulysin of Formula (XXII ) is g is an integer from 2 to 6 ; a compound of Formula (XXIII ) or (XXIV ) :

(XXIII ) R76

11111 CH3 O C P ensiuneaMnr R78 ( XXIIIa ) R76 ;

CH3 0 N Perego1111111 20 — R75

(XXIIIL ) R76 ;

N

CH3 0 NH Ro Become111111* US 9 ,943 ,609 B2 109 110 - continued (XXIV ) R76 IIIIIIII EZ CH3 IIIII CH3 O - R75

? wherein : -continued R76 is hydrogen , OH , OCH3, F , OR43 or O C ( O ) @ R47; OH ; wherein R78 , R75 , R19, R47 and R43 are as defined herein ; 20 $ NH ( CH2 ) , and with the proviso that if Rze is OH , OCHz or F , then R75 NH2; @ and R19 cannot be hydrogen . m In one embodiment, R47 is "25 tar JOH ; win mi @ NH2; In another embodiment, the KSP inhibitor compound is a ?0 — ( CH ). compound of Formula (XXVI ) : @

(XXVI ) 40 - mi NH2; R30 @ NH2; or 2 )

store @ - canC( H )( CH3co ) - ( CH2) 2NH2 ; @

mu . wherein R30 is as defined herein . In some embodiments R30 is : 60 wherein : a is an integer from 1 to 6 ; NH2; c is an integer from 0 to 3 ; and NH?( CH2 ), 65 g is an integer from 2 to 6 . case In another embodiment the KSP inhibitor compound is a winan compound of Formula (XXVII ), (XXVIII ) or (XXIX ): US 9 , 943 ,609 B2 111 112 ( XXVII ) - continued (XXIX ) F

10

RUHN

15 NHRU wherein : Ry is as defined herein . (XXVIII ) One skilled in the art of therapeutic agents will readily understand that each of the therapeutic agents described herein can be modified in such a manner that the resulting compound still retains the specificity and / or activity of the original compound . The skilled artisan will also understand that many of these compounds can be used in place of the 25 therapeutic agents described herein . Thus , the therapeutic agents of the present invention include analogues and derivatives of the compounds described herein . Table B below provides more examples of the therapeutic agents and derivatives thereof suitable for conjugation to 30 form the polymer - drug - protein conjugates or polymer -drug scaffolds of the invention . Spectral data of certain com pounds are also provided (ND in the table means “ not NHR11 determined ” ) . These examples may also be the active form of the drug when it is released from the conjugates in vitro or in vivo . TABLE B ( VI)

HN UN Me02C

Me01

Me

R40 HO OH

Ref # R40 HO mi Ex 6

HN US 9, 943, 609 B2 113 114 TABLE B -continued Ex 22

OH

Ex 23 mi (IX )

R47

Ref # R47 m / z Ex 24 TNH2 ND mi Ex 25 ND min - NH2 Ex 30 ND min NH2

Ex 33 ND III www NH ( XI)

Me

Me OMe OMe Heyyó 0 - R42 Ref # R42 mz

CH3 760 Ex 39 802 . 6

NH US 9, 943, 609 B2 115 116 TABLE B - continued mi NH2 790 Ex 64 804 NH2 . mm ( XII)

Me z

- Me 0 IIIIIII OMe OMe NH "RAD Ref # m / z ?40- - H Ex 48 ? 803. 5 ma OH

789 . 1

OH

Ex 49 974 . 2 win Ex 50 874. 5

nu NH

w NH ,

- OH. 788 Ex 61 803 . 4 . OH

EX 62 803. 4

mm OH

CH3 0 874 . 4 III

CH3 US 9 , 943, 609 B2 117 118 117 TABLE B - continued CH3 0 874 . 4

CH3

NH 900 . 5

900 . 5 mm (XIII )

- R79 H3C CH3 HzC

H3C N Y CH2 CH3 CH3 OCH Ö HzC CH3 yto T - C ( O ) - R29 m / z 903. 2

mm TITUL 803 . 1

790 wth NH

ND Xowwww NH2 829 . 1 mi N

802 US 9, 943, 609 B2 119 120 TABLE B - continued (XIV ) OR43 F

Ref # R43 m / z Ex 36 IIIIIIIII ND NH2 OS IIIIIIIII FO 644 . 9 mm NH ??

(XVII ) OCH3

- OCHZ

NH OCH

R47

R47 m /z 553 . 1 NH2 w

538 . 1 mo „ NH

564. 1

HN 566 . 1 wir H?NANT ma 568 . 1

OH US 9, 943, 609 B2 121 122 TABLE B - continued

ND

min

- NH2 ND

667 . 2

HNH2N ,

622 . 2 w

HN

ND min

N HN IyonetN

H N

ND inn myeHN furrent NH H2N0 US 9, 943, 609 B2 123123 124 TABLE B -continued

ND min maniH2N6o, uonente NH

HN

( XXIII )

R76

Inn> R78

Ref # R76 R78 m / z - OCH3 786 . 4 5 - NH2 771 . 4

HO - NH2 829 . 4 um

n HO ND min NH2

HO ND HN N - OCHZ 900 . 4 - NH2

HO ND

N OCH3 1000 . 5 NHBoc

EX 63 HO 869 . 4 US 9, 943, 609 B2 125 126 TABLE B - continued NH2 - OH ND

OH 871 . 4 EO NH2 Ex 42 F ND tyretnim NH Ex 43 F ND mm - NH2

( XXX )

ITIN NH 111111r.

Ref # - R99 Ex 45

im Ex 46 te HON 55 Protein - Based Recognition Molecules ( PBRMs) polymer to a specific tissue such as the liver, kidney, lung or The protein -based recognition molecule directs the drug - pancreas. The protein - based recognition molecule can target polymer carrier conjugates to specific tissues , cells , or the modified polymer to a target cell such as a cancer cell, locations in a cell . The protein -based recognition molecule such as a receptor expressed on a cell such as a cancer cell, can direct the modified polymer in culture or in a whole 60 a matrix tissue , or a protein associated with cancer such as organism , or both . In each case , the protein -based recogni - tumor antigen . Alternatively , cells comprising the tumor tion molecule has a ligand that is present on the cell surface vasculature may be targeted . Protein -based recognition mol of the targeted cell (s ) to which it binds with an effective ecules can direct the polymer to specific types of cells such specificity , affinity and avidity . In some embodiments , the as specific targeting to hepatocytes in the liver as opposed to protein - based recognition molecule targets the modified 65 Kupffer cells . In other cases , protein - based recognition polymer to tissues other than the liver. In other embodiments molecules can direct the polymer to cells of the reticular the protein - based recognition molecule targets the modified endothelial or lymphatic system , or to professional phago US 9 ,943 ,609 B2 127 128 cytic cells such as macrophages or eosinophils . ( In such cedelizumab , certolizumab , cetuximab (ERBITUX ), citatu cases the polymer itself might also be an effective delivery zumab , cixutumumab , clenoliximab , clivatuzumab , conatu system , without the need for specific targeting ) . mumab , CR6261 , dacetuzumab , daclizumab (ZENAPAX ), In still other embodiments , the protein based recognition daratumumab , denosumab (PROLIA ), detumomab , dorli molecule can target the modified polymer to a location 5 momab , dorlixizumab , ecromeximab , eculizumab (SO within the cell, such as the nucleus, the cytoplasm , or the LIRIS ) , edobacomab , edrecolomab ( PANOREX ), efali endosome, for example. In specific embodiments, the pro zumab (RAPTIVA ), efungumab (MYCOGRAB ) , tein based recognition molecule can enhance cellular bind - elotuzumab , elsilimomab , enlimomab , epitumomab , epratu ing to receptors, or cytoplasmic transport to the nucleus and zumab , erlizumab , ertumaxomab (REXOMUN ), etaraci nuclear entry or release from endosomes or other intracel- 10 zumab ( ABEGRIN ) , exbivirumab , fanolesomab (NEUTRO lular vesicles . SPEC ) , faralimomab , farletuzumab , felvizumab , In specific embodiments the protein based recognition fezakinumab , figitumumab , fontolizumab (HuZAF ) , fora molecules include antibodies , proteins and peptides or pep virumab , fresolimumab , galiximab , gantenerumab , gavili tide mimics . momab , gemtuzumab girentuximab , glembatumumab , goli Exemplary antibodies or antibodies derived from Fab , 15 mumab ( SIMPONI), gomiliximab , ibalizumab , Fab2 , scFv or camel antibody heavy - chain fragments spe - ibritumomab , igovomab ( INDIMACIS - 125 ) , imciromab cific to the cell surface markers , include , but are not limited MYOSCINT( ) , infliximab (REMICADE ) , intetumumab . to , 5T4 , AOC3, C242 , CA - 125 , CCL11, CCR 5 , CD2, CD3, inolimomab , inotuzumab , ipilimumab , iratumumab , kelix CD4, CD5, CD15 , CD18 , CD19 , CD20 , CD22 , CD23 , imab , labetuzumab (CEA - CIDE ) , lebrikizumab , lemale CD25 , CD28 , CD30 , CD31 , CD33 , CD37 , CD38 , CD40 , 20 somab , lerdelimumab , lexatumumab , libivirumab , lintu CD41 , CD44 , CD51, CD52 , CD54 , CD56 , CD62E , CD62P , zumab , lucatumumab , lumiliximab , mapatumumab , CD62L , CD70 , CD74 , CD80 , CD125 , CD138 , CD141 , maslimomab , matuzumab , mepolizumab (BOSATRIA ) , CD147 , CD152 , CD 154 , CD326 , CEA , clumping factor, metelimumab , milatuzumab , minretumomab , mitumomab , CTLA - 4 , EGFR , ErbB2 , ErbB3, EpCAM , folate receptor, morolimumab , motavizumab (NUMAX ) , muromonab -CD3 FAP, GD2 , GD3 , GPNMB , HGF, HER2 , ICAM , IGF - 1 25 (ORTHOCLONE OKT3 ), nacolomab , naptumomab , natali receptor, VEGFR1, EphA2 , TRPV1, CFTR , gpNMB , CA9 , zumab ( TYSABRI) , nebacumab , necitumumab , nereli Cripto , ACE , APP , adrenergic receptor -beta2 , Claudine 3 , momab , nimotuzumab ( THERACIM ), nofetumomab , ocre Mesothelin , IL - 2 receptor, IL -4 receptor, IL - 13 receptor, lizumab , odulimomab , ofatumumab ( ARZERRA ) , integrins ( including 04, QB3, aßs, a , B6 , QB4, C4B1, Außz, olaratumab , omalizumab ( XOLAIR ) , ontecizumab , oportu aß , aoß2,A B , intergins) , IFN - a , IFN - y , IgE , IgE , IGF - 1 30 zumab , oregovomab (OVAREX ) , otelixizumab , pagibax receptor, IL - 1, IL - 12 , IL - 23 , IL - 13, IL - 22 , IL - 4 , IL - 5 , IL - 6 , imab , palivizumab (SYNAGIS ), panitumumab interferon receptor, ITGB2 (CD18 ), LFA - 1 ( CD11a ) , L - se - (VECTIBIX ) , panobacumab , pascolizumab , pemtumomab lectin ( CD62L ), mucin , MUC1 , myostatin , NCA - 90 , NGF, (THERAGYN ), pertuzumab (OMNITARG ) , pexelizumab , PDGFRa , phosphatidylserine, prostatic carcinoma cell , pintumomab , priliximab , pritumumab , PRO 140 , rafi Pseudomonas aeruginosa , rabies , RANKL , respiratory syn - 35 virumab , ramucirumab , ranibizumab (LUCENTIS ) , raxi cytial virus , Rhesus factor, SLAMF7 , sphingosine - 1 -phos - bacumab , regavirumab , reslizumab , rilotumumab , rituximab phate , TAG - 72 , T - cell receptor, tenascin C , TGF - 1 , TGF- B2 (RITUXAN ), robatumumab , rontalizumab , rovelizumab TGF -B , TNF - a , TRAIL -R1 , TRAIL -R2 , tumor antigen (LEUKARREST ), ruplizumab (ANTOVA ), satumomab CTAA16 . 88 , VEGF - A , VEGFR2, vimentin , and the like. pendetide , sevirumab , sibrotuzumab , sifalimumab , siltux In one embodiment the antibodies or antibody derived 40 imab , siplizumab , solanezumab , sonepcizumab , sontu from Fab , Fab2 , scFv or camel antibody heavy - chain frag - zumab , stamulumab , sulesomab (LEUKOSCAN ) , tacatu ments specific to the cell surface markers include CA - 125 , zumab (AFP - CIDE ), tetraxetan , tadocizumab , talizumab , C242 , CD3 , CD19 , CD22 , CD25 , CD30 , CD31 , CD33 , tanezumab , taplitumomab paptox , tefibazumab ( AUR CD37 , CD40 , CD44 , CD51 , CD54 , CD56 , CD62E , CD62P , EXIS ) , telimomab , tenatumomab , teneliximab , teplizumab , CD62L , CD70 , CD138 , CD141, CD326 , CEA , CTLA - 4 , 45 TGN1412 , ticilimumab ( tremelimumab ) , tigatuzumab , EGFR , ErbB2 , ErbB3 , FAP, folate receptor, IGF- 1 receptor, TNX -650 , tocilizumab (atlizumab , ACTEMRA ), torali GD3 , GPNMB , HGF, HER2 , VEGF - A , VEGFR2 , zumab , tositumomab (BEXXAR ) , trastuzumab (HERCEP VEGFR1, EphA2 , EpCAM , 5T4 , TAG -72 , tenascin C , TIN ), tremelimumab , tucotuzumab , tuvirumab , urtoxa TRPV1, CFTR , gpNMB , CA9 , Cripto , ACE , APP, PDGFR zumab , ustekinumab (STELERA ) , vapaliximab , a , phosphatidylserine , prostatic carcinoma cells , adrenergic 50 vedolizumab , veltuzumab , vepalimomab , visilizumab (NU receptor- beta2 , Claudine 3 , mucin , MUC1, Mesothelin , IL - 2 VION ) , volociximab (HUMASPECT ) , votumumab , zalutu receptor , IL -4 receptor, IL - 13 receptor and integrins (includ mumab (HUMEX - EGFr) , zanolimumab (HUMAX -CD4 ) , ing a ,B3 , Q , ßs, a , B6, a ß4, 24ßi, asl?, aoß4 intergins) , ziralimumab and zolimomab . tenascin C , TRAIL - R2 and vimentin . In some embodiments the antibodies are directed to cell Exemplary antibodies include 3F8 , abagovomab , abcix - 55 surface markers for 5T4 , CA - 125 , CEA , CD3, CD19 , CD20 , imab (REOPRO ) , adalimumab (HUMIRA ), adecatumumab , CD22 , CD30 , CD33 , CD40 , CD44 , CD51, CTLA - 4 , afelimomab , afutuzumab , alacizumab , ALD518 , alemtu - EpCAM , HER2, EGFR , FAP , folate receptor, HGF , integrin zumab (CAMPATH ), altumomab , amatuximab , anatu a ß3, integrin asl?, IGF - 1 receptor, GD3, GPNMB ,mucin , momab , anrukinzumab , apolizumab , arcitumomab (CEA - MUC1, phosphatidylserine , prostatic carcinoma cells , SCAN ) , aselizumab , atlizumab ( tocilizumab , Actemra , 60 PDGFR A , TAG -72 , tenascin C , TRAIL -R2 , VEGF- A and RoActemra ), atorolimumab , bapineuzumab , basiliximab VEGFR2 . In this embodiment the antibodies are abagov ( Simulect ) , bavituximab , bectumomab (LYMPHOSCAN ) , omab , adecatumumab , alacizumab , altumomab , anatu belimumab (BENLYSTA ), benralizumab , bertilimumab , momab , arcitumomab , bavituximab , bevacizumab ( AVAS besilesomab (SCINITIMUN ) , bevacizumab ( AVASTIN ) , TIN ) , bivatuzumab , blinatumomab , brentuximab , biciromab (FIBRISCINT ) , bivatuzumab , blinatumomab , 65 cantuzumab , catumaxomab , capromab , cetuximab , citatu brentuximab , briakinumab , canakinumab ( ILARIS ) , cantu - zumab , clivatuzumab , conatumumab , dacetuzumab , edreco zumab , capromab , catumaxomab (REMOVAB ), CC49 , lomab , epratuzumab , ertumaxomab , etaracizumab , farletu US 9 , 943, 609 B2 129 130 zumab , figitumumab , gemtuzumab , glembatumumab , Exemplary proteins comprise insulin , transferrin , fibrino ibritumomab , igovomab , intetumumab , inotuzumab , labetu - gen - gamma fragment, thrombospondin , claudin , apolipo protein E , Affibody molecules such as , for example , ABY zumab , lexatumumab , lintuzumab , lucatumumab , matu 025 , Ankyrin repeat proteins , ankyrin - like repeats proteins zumab , mitumomab , naptumomab estafenatox , necituh 5 and synthetic peptides . mumabm , oportuzumab , oregovomab , panitumumab , 5 al In some embodiments of the invention the protein drug pemtumomab, pertuzumab , pritumumab , rituximab polymer conjugates comprise broad spectrum cytotoxins in (RITUXAN ) , rilotumumab , robatumumab , satumomab , combination with cell surface markers for HER2 such as sibrotuzumab , taplitumomab , tenatumomab , tenatumomab , pertuzumab or trastuzumab ; for EGFR such as cetuximab ; ticilimumab ( tremelimumab ), tigatuzumab , trastuzumab 10 for CEA such as labetuzumab ; for CD20 such as rituximab ; (HERCEPTIN ) , tositumomab , tremelimumab , tucotuzumab for VEGF - A such as bevacizumab ; or for CD - 22 such as celmoleukin , volociximab and zalutumumab . epratuzumab or veltuzumab . In specific embodiments the antibodies directed to cell In other embodiments of the invention the protein -drug surface markers for HER2 are pertuzumab or trastuzumab polymer conjugates or protein -polymer conjugates used in and for EGFR the antibody is cetuximab and for CD20 the 15 the invention comprise combinations of two or more protein antibody is rituximab and for VEGF- A is bevacizumab and based recognition molecules , such as , for example , combi for CD - 22 the antibody is epratuzumab or veltuzumab and nation of bispecific antibodies directed to the EGF receptor for CEA the antibody is labetuzumab . (EGFR ) on tumor cells and to CD3 and CD28 on T cells ; Exemplary peptides or peptide mimics include integrin combination of antibodies or antibody derived from Fab , targeting peptides (RGD peptides ) , LHRH receptor targeting 20 Fab2 , scFv or camel antibody heavy - chain fragments and peptides , ErbB2 (HER2 ) receptor targeting peptides, pros - peptides or peptide mimetics ; combination of antibodies or tate specific membrane bound antigen (PSMA ) targeting antibody derived from Fab , Fab2 , scFv or camel antibody peptides, lipoprotein receptor LRP1 targeting, ApoE protein heavy - chain fragments and proteins; combination of two derived peptides, ApoA protein peptides , somatostatin bispecific antibodies such as CD3XCD19 plus CD28xCD22 receptor targeting peptides , chlorotoxin derived peptides, 25 bispecific antibodies . and bombesin . Table C below provides more examples of the PBRM In specific embodiments the peptides or peptide mimics described hereof, which are suitable for conjugation to form are LHRH receptor targeting peptides and ErbB2 (HER2 ) the polymer- drug -protein conjugates or polymer - PBRM receptor targeting peptides scaffolds of the invention . TABLE C Ref # PBRMPBRM Ex 3 yang-- TRASTUZUMAB Ex 4 i TRASTUZUMAB FinaleNH Ex 53 07 EropaRITUXIMAB

Ex 60 TRASTUZUMAB - Fab - SH

H TRASTUZUMAB -Fab ShyhodneN US 9, 943, 609 B2 131 132 131 TABLE C - continued

Ref # PBRM

Ex 10

NH

NH2

AyggjueHO HN NH HN NH . HNN

Ex 14

HO .

HO OH OH HOHOHN O Im HN

HN NHOL AO NHNH2, NHO $ 0 HONHO JOHN 1

??? ? i _ HT À HON NH

HN US 9, 943, 609 B2 133 134 133 TABLEUS 9, 943 C - continued, 609 B2 Ref # PBRM Ex 16 OH

NH 07 OH NH

NH

NH NH ?? NH sisteminin??

Linkers (LP and L ) In other embodiments , the biocleavable linker LD or L is As described above , the drug or PBRM is connected to the pH -sensitive , i. e ., sensitive to hydrolysis at certain pH polymeric carrier via a linker LD or LP . In some embodi- " values . Typically , the pH -sensitive linker is hydrolysable ments , the linker is biocleavable/ biodegradable under intra - under acidic conditions. For example , an acid - labile linker cellular conditions, such that the cleavage of the linker that is hydrolysable in the lysosome or endosome ( e . g . , a releases the drug or PBRM from the polymer unit in the hydrazone , semicarbazone , thiosemicarbazone, cis - aconitic intracellular environment. 35 amide , orthoester, acetal ,ketal , or the like) can be used. Such A linker is any chemical moiety that is capable of linking linkers are relatively stable under neutral pH conditions, a drug or a PBRM to a polymer backbone through chemical such as those in the blood , but are unstable at below pH 5 . 5 bonds such that the drug or PBRM and the polymer are or 5 .0 , the approximate pH of the lysosome . In certain chemically coupled ( e . g . , covalently bonded ) to each other. embodiments , the hydrolysable linker is a thioether linker In some embodiments , the linker comprises a biodegradable 40 ( such as , e . g . , a thioether attached to the therapeutic agent linker moiety ( e . g ., a biodegradable bond such as an ester or via an acylhydrazone bond . amide bond ) . In other embodiments the linker LP or L is photo -labile In other embodiments , the linker LD or LP is biodegrad - and is useful at the body surface and in many body cavities able under mild conditions, i. e ., conditions within a cell that are accessible to light. Furthermore , LP or L is bio under which the activity of the drug is not affected . 45 cleavable by infrared light which can penetrate tissue . Examples of suitable biodegradable linker moiety include Accordingly, Lor L is useful for both applications on the disulfide linkers, acid labile linkers, photolabile linkers , body surface and in the tissue . peptidase labile linkers, and esterase labile linkers . In some embodiments , the linker LD or LP is biocleavable In some embodiments , the linker LD or LP is biocleavable by a cleaving agent that is present in the intracellular under reducing conditions ( e . g ., a disulfide linker ) . In this environment ( e . g ., within a lysosome or endosome or caveo embodiment the drug or PBRM moiety is linked to the lea ) . The linker can be , for example , a peptidyl linker that is polymer through a disulfide bond . The linker molecule cleaved by an intracellular peptidase or protease enzyme, comprises a reactive chemical group that can react with the including, but not limited to , a lysosomal or endosomal drug . Preferred reactive chemical groups for reaction with 55 protease . the drug or PBRM moiety are N - succinimidyl esters and In some embodiments the linker LD or L is cleaved by N - sulfosuccinimidyl esters . Additionally the linker molecule esterases . Only certain esters can be cleaved by esterases comprises a reactive chemical group , preferably a dithio present inside or outside cells . Esters are formed by the pyridyl group that can react with the drug to form a disulfide condensation of a carboxylic acid and an alcohol. Simple bond . In some embodiments the linker molecules include , 60 esters are esters produced with simple alcohols , such as for example , N -succinimidyl 3 - ( 2 -pyridyldithio )propionate aliphatic alcohols , and small cyclic and small aromatic ( SPDP ) , N - succinimidyl 4 - ( 2 -pyridyldithio )butanoate alcohols . (SPDB ) , N - succinimidyl 4 - ( 2 -pyridyldithio )pentanoate In yet other embodiments , the linker LD or LP is not ( SPP ) , N - succinimidyl- S -acetylthioacetate (SATA ) and biocleavable and the drug is released by antibody degrada N - succinimidyl- oxycarbonyl- alpha -methyl - alpha - ( 2 - 65 tion . See , for example , U . S . Pat. No . 7 ,498 , 298 , which is pyridyl- dithio ) toluene or 2 , 5 -dioxopyrrolidin - 1 - yl 4 - ( 1 - incorporated by reference herein in its entirety and for all (pyridin - 2 -yldisulfanyl ) ethyl) benzoate (SMPT ) . purposes . US 9 ,943 ,609 B2 135 136 Typically , the linker L ' or L is not substantially sensitive able linker moiety selected from the group consisting of to the extracellular environment. As used herein , “ not sub alkyl, alkenyl, alkynyl, cycloalkyl, heteroalkyl, heteroalk stantially sensitive to the extracellular environment, ” in the enyl, heteroalkynyl, heterocycloalkyl, aryl, heteroaryl, and a context of a linker, means that no more than about 20 % , combination thereof and each of Mº , Mº , M ” , M ' Mº , typically no more than about 15 % , more typically no more 5 and M " optionally contains one or more — ( C = 0 ) - but than about 10 % , and even more typically no more than about does not contain any of the biodegradable linker moieties mentioned above . 5 % , no more than about 3 % , or no more than about 1 % of For example , each of MPI, MP2 , MP3 , MP4 , MP1, MP2, the linkers , in a sample of Polymer Drug Conjugate , are MP3 and MP4, independently is C1- 6 alkyl, C1- 6 alkyl -C cleaved when the Polymer Drug Conjugate presents in an ( 0 ) - C0- 6 alkyl, C1- 6 alkyl- NH - C0- 6 alkyl, C1- 6 alkyl- 0 extracellular environment ( e . g . , in plasma ) for 24 hours . 10 Co- 6 alkyl, C1- 6 alkyl- S Co- 6 alkyl, C1- 6 alkyl- C (O ) C1- 6 Whether a linker is not substantially sensitive to the extra alkyl- NH , C1- 6 alkyl- C ( O ) C1- 6 alkyl - O , C1- 6 alkyl- C cellular environment can be determined , for example , by ( O ) C1- 6 alkyl - S , C3- 10 cycloalkyl - C ( O ) - C6 alkyl, 3 - 19 incubating the Polymer Drug Conjugate with plasma for a membered heterocycloalkyl- C ( O ) C1- 6 alkyl, aryl- C ( O ) predetermined time period ( e. g ., 2 , 4 , 8 , 16 , or 24 hours) and CoC - 6 alkyl., ( CHCH2CH2O .CH . O ) ,1 - 12 , and the like . then quantitating the amount of free drug present in the 15 For example , for each Lº ,M i is not absent when XP is plasma . absent. In embodiments , the linker L has the structure : For example , for each LP , MPl is not absent when XP is - R - CEO ) XP -MPI - YP -MP2 -ZP -MP3 - QP -MP4 , absent. with R I connected to an oxygen atom of the polymeric For example , for each LP , at least one of X ” , Yº , ZP , and carrier and MP4 connected to the drug molecule to be 20 PP is not absent. delivered . For example , for each L ’ , at least one of XP, YP , ZP , and In embodiments, the linker L has the structure: is not absent. LP is a linker having the structure : R2- C ( O ) - XP - For example , each of M?l and MPl independently is C1- 6 MP1- YP -MP2 - ZP -MP3 - QP -MP4 , with R12 connected to an alkyl or C1- 6 heteroalkyl. oxygen atom of the polymeric carrier and MP4 connected to 25 For example , each of Mº , MP3, MP4 , MP2 , M ” , and the PBRM . MP4 , independently is absent , C1- 6 alkyl , cycloalkyl , het For example , each of R 1 and R 2 independently is absent, eroalkyl, heterocycloalkyl , or a combination thereof. alkyl, alkenyl, alkynyl, cycloalkyl, heteroalkyl, heteroalk - For example , for each L ” , at most two of MP2, MP3 , and enyl, heteroalkynyl, heterocycloalkyl, aryl, or heteroaryl. M°4 are absent. For example , each ofR1 and R12 independently is absent , 30 For example, for each L ” , atmost two of MP2, MP3, and alkyl, cycloalkyl , heteroalkyl, or heterocycloalkyl. MP4 are absent. For example , R4 is absent. For example , for each LP , one of MP3 and MP3 has one For example , RL2 is absent . of the following structures: For example , each of X and X ” , independently is 0 % , - S — , - N (R ) — , or absent, in which Rl is hydrogen , an 35 aliphatic , heteroaliphatic , carbocyclic , or heterocycloalkyl moiety , CORB, COORB, SO _ R1B or mm - N (R ') — is a heterocycloalkyl moiety , wherein RIB is hydrogen , an aliphatic , heteroaliphatic , carbocyclic , or het IpN erocycloalkyl moiety . 40 munHalit For example , each of Yº , YP, ZP , ZP , PP , and Q ? independently , is absent or a biodegradable linker moiety selected from the group consisting of S - S — , CEO) 0 , C O )NR2 _ , _ OC ( O ) - , - NR2C ( = O ) , - OC ( = O ) O - , - OC ( = O ) NR2 — , - NR²C ( = O ) O - , 45 - NR²CEO) NR — C (OR ) , C (OR ) S , - C (OR )NR3 — , - C (SR2 ) O - , - C (SR² ) S — , - C (SR² ) art NR , C (NR²Rº ) 0 , C (NR²R ) , C (NR²R ) the NR4 — , - C ( O ) - , - SC ( O ) - , - SCO S , - OC ( O ) - , SC00 — , C SS - , SC 50 ( S ) , OCCS) , CES) O - , - SCES) O - , - OCC= S ) S — , OCESS) O - , - SCES) S , - C = NR ) O - , C NRP) S , CNRP) NR3 — , mi - OCC= NR ) , SC = NR2) , - NRC( = NR2) , - NR²50 , - - NR²NR — - C ( O )NR²NR — , 55 - NR²NRCO) , OC = O )NR2NR }, — NR²NRC ( 0 ) 0 , CS)NR²NR3 , NR²NR CES) , -CNR4 ) NR NR3 _ , - NR²NRC NR4 ) , - O ( N = CR3) , (CR = NO , C O )NR2 — mir ( N = CR ) , (CR = N ) - NR²C ( = O ) , S03 — , 60 (CH3 ) 1- 2 - NR²SO ,NR3 — , - SO , NR2 , and polyamide, wherein each occurrence of R², Rº, and R4 independently is hydro gen or an aliphatic , heteroaliphatic , carbocyclic , or hetero cyclic moiety , or each occurrence of NR ? — or ( CH3) 1 - 2 - NR NR — is a heterocycloalkyl moiety . mi For example , each of MPI, MP2, MP3, MP4, MP1, yaMP2, 05 MP3 and MP4 , independently , is absent or a non -biodegrad US 9 ,943 ,609 B2 137 138 - continued137 -continued mm min (CH3 ) 1- 2 m Xw miw 10 minen

CH3 min 20 IpS winn CH3

,> andand 2525 Xornet mm - , and

min . themi most V; 3030 tomt. in which q is an integer from 0 to 12 and each of p and t independently is an integer from 0 to 3 , and the other ofMD2 or MP3 is either absent or a moiety different from the above , 35 such as C1- 6 alkyla. toiminnast For example , for each L ’ , one ofMP2 and MP3 has one of in which q is an integer from 0 to 12 and each of p and t the following structures : independently is an integer from 0 to 3 , and the other ofMP2 or MP is either absent or a moiety different from the above , 40 such as C1- 6 alkyl. antis ir keinen anarman en For example, p is 2 . ? For example , q is 0 or 12 . For example , t is 0 or 1 . 45 For example , each of -MP2 - Z ” , - ZP -MP3 -, - Z ” tayfurt MP2 , or -MP3 - 7 , independently has one of the follow ing structures :

> 50 num @

55 mm titrat @ PS M tayot 60 xxx N = N

Hy mi 65 ni mi (CH3 ) 1 - 2 ve US 9 ,943 ,609 B2 139 140 -continued - continued ( 12 )

mnm in Ni - NH mi

??)

mi ( 13 )

nin mu ( 14 )

RIJ Z min Rit )SI ( t RIJ ni thy ( 16 ) mm RÚ + ( 17 ) NHN = Hitmm ( 18 ) mum H leo RW mm winny - Ph #int test )II ( 09 ( 19 ) tf 65 thatwww " US 9, 943, 609 B2 141 142 -continued - continued142 @(20 ) J wi ma mm (21 ) 10 mm ma ?? www 15 (22 ) R tomon min min 20

@

25 mm RIJ

-7 30

w ; and the (24 ) 35 mi NHN 40 mo mun in which ring A or B independently is cycloalkyl or hetero - u . cycloalkyl; R " is an aliphatic , heteroaliphatic , carbocyclic , or heterocycloalkyl moiety ; R ' is hydrogen , an aliphatic , heteroaliphatic , carbocyclic , or heterocycloalkyl moiety ; and ring D is heterocycloalkyl. mi nim For example, each of -MP2 - Z? — , - ZP -MP3 -, ZP - 50 RIJ MP2 , and -MP3 - Z ? — independently , has one of the follow ing structures : (1) Sg55 mm mm mi 0 Ph ( 11 ) ou toutR I? mm men Sex ; and US 9 ,943 ,609 B2 143 144 -continued For example , Z is 14 ( ore12 ) pe care in

mwa 10 mu in which ring A is cycloalkyl or heterocycloalkyl and Rld is hydrogen , an aliphatic , heteroaliphatic , carbocyclic , or het erocycloalkyl moiety . NN For example , ring A is 5 - 19 membered heterocycloalkyl, e . g . , mm For example, XP is absent , O or NH . 20 For example , XP is absent, O or NH . mm For example , each of XD and XP , independently is

25

For example , ring A is C3- 8 cycloalkyl. For example , ring D is piperazinyl or piperidinyl. O = For example , R " is C1- 6 alkyl. 30 w For example , R ' is hydrogen or C1- 6 alkyl. or For example , ZP is 25 tot mm 35 For example , each of Yº and Y² independently is - S S - , COCO , COO , CONH — or NHCO . We For example , each of Q and Q independently is absent , 40 S - 5 % , OCO , COO , CONH — , — NHCO - , - OCONHNH — , or — NHNHCOO — . For example , -LP - D can have one of the following mm structures below , in which the wavy bond indicates that D ( i. e . Drug ) is either connected to the functional linker directly or via another moiety :

(1 ) @ Drug, ????R80 IZ T ????Rgo ? ininR80 R82 Drug, hayatRgo

no NH Drug , US 9 ,943 ,609 B2 145145 US9943 ,609 B2 146146 -continued (7 ) itoDrug, indeteR80 **R82 R920 Drug , Drug, R80 1 - 11 S N mm

( 10 )

ug , rug, hodetR80 inteR80 initoR80 R82 - Drug,

Drug, R80 ?????? N NH

HN ( 14 )

Drug , R80 Yue soitN HN

HN ( 15 )

Drug , - N * R80and f ork HN

HONH N US 9 ,943 ,609 B2 148 147 - continued ( 16 )

w R80 inityN Drug

HN

H?N So and (17 )

min Drug ; yuyan 01-12 HN

H N wherein Rgo is CH2, — NH , or oxygen ; and 30 For example , polymeric carrier - L - PBRM can have one Rg2 is — NH or oxygen . of the following structures below :

mm iningR80 N - PBRM ,

-0112 - PBRM , frin-R80 IZ unit N S PBRM , R80 - PBRM , my inlineN button baby

S Rso PBRM , R80 ????? N man PBRM , PBRM “ Rigo ml PBRM , Rgo ? US 9 ,943 ,609 B2 149 150 149 -continued

indoR80

N — N

ni

PBRM ( 10 )

R80 IZ - PBMR , ladyhytm (CH3 ) 1 - 2 ( 11 )

PBRM , K80 NH - N Rs13 ( CH3) 1 - 2 inint tot ( 12 )

v R80 S - PBRM ., (CH3 ) 1 - 2 indent ( 13)

N - R81 PBRM . S R80 inbytet(CH3 ) 1- 2 (14 ) CH3 inizinK80 N PBRM , (15 )

N - R813 - PBRM ; IndgaudetR80 ( 16 ) fairlyR80 IZ NH PBRM ; (CH3 ) 1 - 2 mno (17 )

N - R91 - PBRM ; R80 NH N indulyte(CH3 ) 1- 2 US 9 ,943 ,609 B2 151151 US 9943,609 B2 152 - continued 152 ( CH3) 1- 2

R80 index* PBRM ; (CH3 ) 1 - 2 N PBRM ???????Rgo NH - S PBRM ; indoorR80 IZ S PBRM ; - Rigo IZ

follo.

PBRM : dalakR80 / 14 IZ 30 wherein : conjugate , and the drug moiety is cleaved from the protein Rgo is CH2, NH or oxygen ; polymer -drug conjugate when the protein -polymer -drug conjugate does enter the cell . Rg???? , is , ?? ??? In another embodiment, the bioavailability of the protein 35 polymer -drug conjugate or an intracellular metabolite of the protein -polymer - drug conjugate in a subject is improved when compared to a drug compound or conjugate compris ing the drug moiety of the protein -polymer -drug conjugate , 40 or when compared to an analog of the compound not having NH or the drug moiety . In another embodiment, the drug moiety is intracellularly cleaved in a subject from the protein -polymer -drug conju 45 gate , or an intracellular metabolite of the protein - polymer drug conjugate . XY N Conjugates or Polymeric Scaffolds wan Conjugates of the invention comprise one or more occur rences of D , where D is a therapeutic agent, e . g . , a drug , While biocleavable linkers preferably are used in the 50 wherein the one or more occurrences of D may be the same invention , a non -biocleavable linker also can be used to or different. generate the above- described conjugateoniugate . A nonnon -- biocleavable In certain other embodiments , one or more occurrences of linker is any chemical moiety that is capable of linking a PBRM is attached to the polymeric carrier , wherein the one drug or PBRM , to a polymer in a stable , covalent mannerner . 55 or more occurrences of PBRM may be the same or different . Thus , non - biocleavable linkers are substantially resistant to In certain other embodiments , one ormore polymer carriers that contains one or more occurrences of D are connected to acid - induced cleavage , light- induced cleavage, peptidase a PBRM ( e . g . , an antibody) . induced cleavage , esterase -induced cleavage , and / or disul 60 As discussed more generally above , in certain embodi fide bond cleavage, at conditions under which the drug or ou ments , each polymeric carrier independently , has about 0. 1 polymer remains active. to about 25 % monomers comprising a D , more preferably In one embodiment, a substantial amount of the drug about 0 . 5 to about 20 % , more preferably about 1 to about moiety is not cleaved from the conjugate until the protein - s 15 % , and even more preferably about 2 to about 10 % . polymer - drug conjugate enters a cell with a cell - surface In certain embodiments , the conjugate of this invention is receptor specific for the PBRM of the protein -polymer - drug of Formula ( I ) : US 9 , 943 ,609 B2 153 154 wherein : each of n , n , , n2, nz, and nq, is the molar fraction of the corresponding polymer unit ranging between 0 and 1 ; n + n + n2 + nz + n4 = 1 ; provided that none of n , n , , and n is 0 . 5 For example , the ratio between n , and n , is greater than 1 : 1 and 5200 : 1 . OH OH OH For example , the ratio between n , and n4 is between 10 : 1 - n and 50 : 1 . For example , the ratio between n2 and n4 is between 30 : 1 and 50 : 1 . ni For example , the ratio between n , and n4 is about 50 : 1 , 25 : 1, 10 :1 , 5 : 1 or 2 : 1. MD1 In certain embodiments , the conjugates are formed in several steps . These steps include ( 1 ) modifying a polymer MD2 so that it contains a functional group that can react with a WD 15 functional group of the drug or its derivative ; ( 2 ) reacting the 0 . 0 modified polymer with the drug or its derivative so that the drug is linked to the polymer ; ( 3 ) modifying the polymer drug conjugate so that the polymer contains a functional ?? group that can react with a functional group of the PBRM or 20 its derivative ; and ( 4 ) reacting the modified polymer - drug conjugate with the PBRM or its derivative to form the conjugate of this invention . Step ( 3 ) may be omitted if the n2 modified polymer produced by step ( 1) contains a functional MDI group that can react with a functional group of the PBRM or 25 its derivative . In another embodiment the conjugates are formed in MD2 several steps: ( 1 ) modifying a polymer so that it contains a functional group that can react with a functional group of a MD3 first drug or its derivative ; ( 2 ) reacting the modified polymer 30 with the first drug or its derivative so that the first drug is linked to the polymer ; ( 3 ) modifying the polymer - drug MD4 conjugate so that it contains a different functional group that can react with a functional group of a second drug or its derivative ( 4 ) reacting the modified polymer- drug conjugate 35 with the second drug or its derivative so that the second drug is linked to the polymer -drug conjugate ; (5 ) modifying the OL polymer -drug conjugate containing 2 different drugs so that the polymer contains a functional group that can react with a functional group of the PBRM or its derivative ; and ( 6 ) 40 reacting the modified polymer- drug conjugate of step (5 ) with the PBRM or its derivative to form the conjugate of this MP1 invention . Steps ( 5 ) and ( 6 ) may be repeated if 2 different PBRM or its derivatives are to be conjugated to form a MP2 polymer- drug conjugate comprising two different drugs and WP 45 two different PBRMs. In yet another embodiment, the conjugates are formed in several steps. These steps include ( 1 ) modifying a polymer so that it contains a functional group that can react with a functional group of the drug or its derivative ; ( 2 ) further ?? 50 modifying the polymer so that it also contains a functional group that can react with a functional group of the PBRM or its derivative ; ( 3 ) reacting the modified polymer with the drug or its derivative so that the drug is linked to the Jn4 polymer ; and ( 4 ) reacting the modified polymer -drug con MP1 55 jugate with the PBRM or its derivative to form the conjugate of this invention . The sequence of steps ( 1 ) and ( 2 ) or that of steps ( 3 ) and ( 4 ) can be reversed . Further either step ( 1 ) or ( 2 ) may be omitted if the modified polymer contains a functional group that can react with both a functional group 60 of the drug or its derivatives and a functional group of the MP3 PBRM or its derivative. In another embodiment the conjugates are formed in several steps: ( 1) modifying a polymer so that it contains a MP4 functional group that can react with a functional group of a PBRM , 65 first drug or its derivative; ( 2 ) further modifying a polymer so that it contains a functional group that can react with a functional group of the PBRM or its derivative ; (3 ) reacting US 9 ,943 ,609 B2 155 156 the modified polymer with the first drug or its derivative so abundance of alcohol groups may provide low rate of that the first drug is linked to the polymer ; ( 4 ) modifying the polymer recognition by cell receptors , particularly of phago polymer- drug conjugate so that it contains a different func - cytes . The polymer backbones of the present invention tional group that can react with a functional group of a generally contain few , if any , antigenic determinants (char second drug or its derivative ( 5 ) reacting the modified 5 acteristic , for example , for some polysaccharides and poly polymer - drug conjugate with the second drug or its deriva peptides) and generally do not comprise rigid structures tive so that the second drug is linked to the polymer - drug capable of engaging in “ key - and -lock ” type interactions in conjugate ; (6 ) reacting themodified polymer- drug conjugate vivo unless the latter are desirable . Thus, the soluble , containing 2 different drugs so that the polymer with the crosslinked and solid conjugates of this invention are pre PBRM or its derivative to form the conjugate of this 10 dicted to have low toxicity and bioadhesivity , which makes invention . Step ( 6 ) may be repeated if 2 different PBRM or them suitable for several biomedical applications. its derivatives are to be conjugated to form a polymer -drug I n certain embodiments of the present invention , the conjugate comprising two different drugs and two different biodegradable biocompatible conjugates can form linear or PBRMs. Step (4 ) may be carried out after step ( 1 ) so that the branched structures . For example , the biodegradable bio modified polymer contains two different functional groups 15 compatible polyal conjugates of the present invention can be that can react with two different drugs or their derivatives . chiral ( optically active ). Optionally, the biodegradable bio In this embodiment , the modified polymer containing two compatible polyal conjugates of the present invention can be different functional group that can react with two different scalemic . drugs or their derivatives can be further modified so that it In certain embodiments , the conjugates of the invention contains a functional group that can react with a functional 20 are water- soluble . In certain embodiments , the conjugates of group of the PBRM or its derivative ; prior to the reaction of the invention are water - insoluble . In certain embodiments , the modified polymer with either the two different drugs the inventive conjugate is in a solid form . In certain embodi ( step ( 3 ) and step ( 5 ) or PBRM ( step (6 ) . ments , the conjugates of the invention are colloids . In certain The biodegradable biocompatible conjugates of the embodiments , the conjugates of the invention are in particle invention can be prepared to meet desired requirements of 25 form . In certain embodiments , the conjugates of the inven biodegradability and hydrophilicity . For example , under tion are in gel form . physiological conditions , a balance between biodegradabil This invention also features a polymeric scaffold useful ity and stability can be reached . For instance , it is known that for conjugating with a PBRM to form a polymer - drug molecules with molecular weights beyond a certain thresh PBRM conjugate described herein . The scaffold comprises a old ( generally, above 40 - 100 kDa, depending on the physical 30 polymeric carrier, one or more - Lº - D connected to the shape of the molecule ) are not excreted through kidneys , as polymeric carrier, and one or more LP connected to the small molecules are , and can be cleared from the body only polymeric carrier which is suitable for connecting a PBRM through uptake by cells and degradation in intracellular to the polymeric carrier , wherein : compartments , most notably lysosomes. This observation each occurrence of D is independently a therapeutic agent exemplifies how functionally stable yet biodegradable mate - 35 having a molecular weight 55 kDa; rials may be designed by modulating their stability under the polymeric carrier is a polyacetal or polyketal, general physiological conditions (pH = 7 . 5 + 0 . 5 ) and at lyso LP is a linker having the structure : somal pH (pH near 5 ) . For example , hydrolysis of acetal and ketal groups is known to be catalyzed by acids, therefore polyals will be in general less stable in acidic lysosomal 40 environment than , for example, in blood plasma . One can design a test to compare polymer degradation profile at , for - RLI - C ( = 0 ) — IDI _ example, pH = 5 and pH = 7 . 5 at 37° C . in aqueous media , and thus to determine the expected balance of polymer stability in normal physiological environment and in the “ digestive ” 45 with R i connected to an oxygen atom of the polymeric lysosomal compartment after uptake by cells . Polymer carrier and LDI connected to D , and integrity in such tests can be measured , for example , by size exclusion HPLC . One skilled on the art can select other suitable methods for studying various fragments of the degraded conjugates of this invention . In many cases , it will be preferable that at pH = 7 . 5 the effective size of the polymer will not detectably change over min 1 to 7 days, and remain within 50 % from the original for at least several weeks . AtpH = 5 , on the other hand , the polymer denotes direct or indirect attachment of D to LPI, and L ” should preferably detectably degrade over 1 to 5 days, and 55 contains a biodegradable bond so that when the bond is be completely transformed into low molecular weight frag - broken , D is released from the polymeric carrier in an active ments within a two -week to several month time frame. form for its intended therapeutic effect ; Although faster degradation may be in some cases prefer - LDI is a carbonyl- containing moiety ; able , in general it may be more desirable that the polymer L is a linker different from L ” and having the structure : degrades in cells with the rate that does not exceed the rate 60 — R _ 2 _ C ( O )- L l with R _ 2 connected to an oxygen atom of metabolization or excretion of polymer fragments by the of the polymeric carrier and L suitable for connecting cells . Accordingly , in certain embodiments , the conjugates directly or indirectly to a PBRM ; of the present invention are expected to be biodegradable, in each ofR1 and RL2 independently is absent, alkyl, het particular upon uptake by cells , and relatively " inert” in eroalkyl, cycloalkyl, or heterocycloalkyl; and relation to biological systems. The products of carrier deg - 65 LP is a moiety containing a functional group that is radation are preferably uncharged and do not significantly capable of forming a covalent bond with a functional group shift the pH of the environment. It is proposed that the of a PBRM . US 9 ,943 ,609 B2 157 158 For example , LP is a linker having the structure : - continued

w – RLICK = 0 ) < I O2 m2 Jm3- m3 D1 101 in which LP2 is a moiety containing a functional group that 10 is capable of forming a covalent bond with a functional vi group of a PBRM , and

15 wherein : m is an integer from 1 to about 2200 , m , is an integer from 1 to about 660 , my m , is an integer from 1 to about 300 , 20 mz is an integer from 1 to about 110 , and the sum of m , m?, m , and mz ranges from about 15 to denotes direct or indirect attachment of LP2 to LDI. about 2200 For example , the functional group of LP1 or LP2 is For example , when the PHF in Formula ( la ) has a selected from — SRP , S S - LG , maleimido , and halo , in 25 molecular weight ranging from about 2 kDa to about 40 kDa which LG is a leaving group and RP is H or a sulfur 25 ( i. e ., the sum of m , mj, m2, and mz ranging from about 15 protecting group . to about 300 ) , m , is an integer from 1 to about 40 , mz is an For example , LPi comprises — X — ( CH2) , C ( = ) integer from 1 to about 18 , and / or m , is an integer from 1 to with X directly connected to the carbonyl group of R21 — about 140 ( e .g , m , being about 1 - 90 ) . C ( O ), in which X is CH2, O , or NH , and v is an integer 30 For example , when the PHF in Formula ( la ) has a from 1 to 6 . molecular weight ranging from about 6 kDa to about 20 kDa For example , LP or LP2 contains a biodegradable bond . ( i . e ., the sum of m , m , , my, and mz ranging from about 45 to about 150 ) , m , is an integer from 2 to about 20 , mz is an For example , each of R21 and R22 is absent. integer from 1 to about 9 , and/ or m , is an integer from 1 to For example , the polymeric carrier of the scaffold of the 35 about 75 (eg , m , being about 4 - 45 ) . invention is a polyacetal, e . g ., a PHF having a molecular For example , when the PHF in Formula (la ) has a weight ( i. e ., MW of the unmodified PHF) ranging from molecular weight ranging from about 8 kDa to about 15 kDa about 2 kDa to about 300 kDa . The selection of a polymeric ( i. e ., the sum of m , m , , m , , and mz ranging from about 60 carrier with a specific MW range may depend on the size of to about 110 ), m , is an integer from 2 to about 15 , mz is an the PBRM to be conjugated with . 40 integer from 1 to about 7 , and /or m , is an integer from 1 to For example , for conjugating a PBRM having a molecular about 55 ( e . g , m , being about 4 - 30 ) . weight of 40 kDa or greater ( e. g ., 80 kDa or greater ) , the For example, when the PHF in Formula ( la ) has a polymeric carrier of the scaffold of the invention is a molecular weight ranging from 20 kDa to 300 kDa (i . e ., the polyacetal, e . g ., a PHF having a molecular weight ( i. e ., MW - sum of m , m , , m , , and mz ranging from about 150 to about of the unmodified PHF ) ranging from about 2 kDa to about » 2200 ) , m , is an integer from 3 to about 300 , mz is an integer 40 kDa ( e . g . , about 6 - 20 kDa or about 8 - 15 kDa ). from 1 to about 110 , and / or m , is an integer from 1 to about For example , for conjugating a PBRM having a molecular 660 ( e . g , m , being about 10 - 250 ) . weight of 200 kDa or less ( e . g . , 80 kDa or less ) , the For example , when the PHF in Formula (la ) has a polymeric carrier of the scaffold of the invention is a 50 molecular weight ranging from 40 kDa to 150 kDa ( i . e ., the polyacetal , e . g . , a PHF having a molecular weight ( i. e . , MW sum of m , m , , m , , and m , ranging from about 300 to about of the unmodified PHF ) ranging from about 20 kDa to about 1100 ) , m , is an integer from 4 to about 150 , mz is an integer from 1 to about 75 , and /or m , is an integer from 1 to about 300 kDa (e . g. , about 40 - 150 kDa or about 50 - 100 kDa) . 330 ( e . g , m , being about 15 - 100 ) . For example , the scaffold is of Formula ( la ): 55 For example , when the PHF in Formula (la ) has a molecular weight ranging from about 50 kDa to about 100 kDa ( i. e . , the sum of m , m?, m2 , and mz ranging from about (La ) 370 to about 740 ) , m , is an integer from 5 to about 100 , mz is an integer from 1 to about 40 , and /or m , is an integer from 60 1 to about 220 ( e . g , m , being about 15 - 80 ) . For example , the scaffold further comprises a PBRM OH OH OH connected to the polymeric carrier via L . For example , one or more PBRMs are connected to one 65 drug -carrying polymeric carrier. rette 1711 LDL For example , the scaffold (e . g . , a PBRM -polymer - drug conjugate ) is of Formula ( lb ): US 9 , 943 ,609 B2 159 160 kDa (i . e ., the sum of m , m?, m2 , mz, and m4 ranging from (Ib ) about 370 to about 740 ) , m , is an integer from 5 to about 100 , mz is an integer from 1 to about 40 , m , is an integer from 1 to about 20 , and / or m? is an integer from 1 to about 220 ( e . g , m , being about 15 - 80 ) . Alternatively or additionally , one or more drug -carrying OH polymeric carriers are connected to one PBRM . For ?? example , the scaffold ( e . g . , a PBRM - polymer - drug conju OH In 1 gate ) comprises a PBRM with a molecular weight of greater jDi 10 than 40 kDa and one or more D - carrying polymeric carriers connected to the PBRM , in which each of the D -carrying polymeric carrier independently is of Formula ( Ic ):

OH OH ( Ic ) m22 m3 TDI[ DI TDI

nin 20 P2 OH O OH OH ]m mi LDI 207 25 OH

DI OH OH

m 30 m2 Jm3 30 DI I DI P2 mun mu 1 P2 PBRM , 35 wherein :

40 Ö 40 L m4 TDI between LP2 and PBRM denotes direct or indirect attach - 45 | P2 ment of PBRM to LP2, each occurrence of PBRM independently has a molecular min weight of less than 200 kDa , m is an integer from 1 to about 2200 , m , is an integer from 1 to about 660 , 50 wherein : m , is an integer from 3 to about 300 , terminal mz is an integer from 0 to about 110 , m , is an integer from 1 to about 60 ; and the sum of m , m?, m , mz and m , ranges from about 150 55 to about 2200 . For example , in Formula ( Ib ), m , is an integer from about 10 to about 660 ( e . g , about 10 - 250 ) . win For example , when the PHF in Formula! (Ib 1b ) , hashas aa attached to LP2 denotes direct or indirect attachment of LP2 molecular weight ranging from 40 kDa to 150 kDa ( i. e . , the 60 to PBRM such that the D - carrying polymeric carrier is sum of m , mj, m2 , mz, and my ranging from about 300 to connected to the PBRM , about 1100 ) , m , is an integer from 4 to about 150 , mz is an integer from 1 to about 75 , m , is an integer from 1 to about m is an integer from 1 to 300 , 30 , and / or m , is an integer from 1 to about 330 ( e . g , m , being m , is an integer from 1 to 140 , about 10 - 330 or about 15 - 100 ) . 65 m2 is an integer from 1 to 40 , For example , when the PHF in Formula ( lb ) has a mz is an integer from 0 to 18 , molecular weight ranging from about 50 kDa to about 100 m4 is an integer from 1 to 10 ; and US 9 , 943, 609 B2 161 162 the sum of m , m , m ,, mz, and m4 ranges from 15 to 300 ; For example , the functional group of LP or LP2 is provided that the total number of LP2 attached to the PBRM selected from — SRP, S - S - LG , maleimido , and halo , in is 10 or less . which LG is a leaving group and RP is H or a sulfur For example , in Formula ( Ic ), m , is an integer from 1 to protecting group . about 120 ( e .g , about 1 - 90 ) and / or mz is an integer from 1 5 For example , LDI comprises — X — (CH , ) — C40 to about 10 ( e . g , about 1 - 8 ) . with X directly connected to the carbonyl group of RA For example , when the PHF in Formula ( Ic ) has a CEO ) , in which X is CH2, O , or NH , and v is an integer molecular weight ranging from about 6 kDa to about 20 kDa from 1 to 6 . (i . e . , the sum of m , m , m , mz, and m4 ranging from about For example, LP1 or LP2 contains a biodegradable bond. 45 to about 150 ) , m2 is an integer from 2 to about 20 , mz is 10 For example , each of Rll and RL2 is absent. an integer from 1 to about 9 , and /or m , is an integer from 1 to about 75 ( e . g , m , being about 4 -45 ) . For example , the polymeric carrier of the D - free scaffold For example , when the PHF in Formula ( Ic ) has a is a polyacetal , e . g ., a PHF having a molecular weight ( i. e ., molecular weight ranging from about 8 kDa to about 15 kDa MW of the unmodified PHF) ranging from about 2 kDa to ( i . e . , the sum of m , mim , m2m , mzm , andand m4m rangingranging from about 15154 about 300 kDa. 60 to about 110 ), m , is an integer from 2 to about 15 , mz is The D -free scaffold is of Formula ( Id ): an integer from 1 to about 7 , and / or my is an integer from 1 to about 55 ( e . g , m , being about 4 - 30 ) . In another aspect , the invention features a polymeric ( Id ) scaffold useful to conjugate with both a protein based 20 recognition -molecule (PBRM ) and a therapeutic agent ( D ) . The D - free scaffold comprises a polymeric carrier, one or more L connected to the polymeric carrier which is suitable for connecting a PBRM to the polymeric carrier , and one or OH more — R - I _ C ( O ) - LP connected to the polymeric car- 25 OH rier via R41, wherein : OH OH , L m1 the polymeric carrier is a polyacetal or polyketal, LDL R4 is connected to an oxygen atom of the polymeric carrier , Di is a linker suitable for connecting a D molecule to the 30 polymeric carrier , in which each occurrence of D is inde pendently a therapeutic agent having a molecular weight 55 OH O kDa; 03 m3 LP is a linker different from - RI C ( O ) -LDI , and IDI having the structure : R22 - C ( O )- LP1 with RL2 con - 35 nected to an oxygen atom of the polymeric carrier and L1 minn suitable for connecting to a PBRM ; each of R41 and R 2 independently is absent , alkyl, het L P2 eroalkyl, cycloalkyl, or heterocycloalkyl; LP is a moiety containing a functional group that is 40 wherein : capable of forming a covalent bond with a functional group m is an integer from 1 to about 2200 , of D , and m , is an integer from 1 to about 660 , L ' is a moiety containing a functional group that is mz is an integer from 1 to about 110 , and capable of forming a covalent bond with a functional group the sum of m , m ] , and mz ranges from about 15 to about of a PBRM . 45 2200 . For example , the D - free scaffold useful to conjugate with For example , when the PHF in Formula ( Id ) has a a PBRM and a D can have one or more of the following molecular weight ranging from about 2 kDa to about 40 kDa features . ( i . e . , the sum of m , m , , and mz ranging from about 15 to For example , L is a linker having the structure : about 300 ) , mz is an integer from 1 to about 18 , and/ or m , 50 is an integer from 1 to about 140 ( e . g , m , being about 2 - 120 ) . For example , when the PHF in Formula ( Id ) has a molecular weight ranging from about 6 kDa to about 20 kDa — RLI - C ( = O ) — IDI TP2 ( i . e ., the sum of m , m , , and m , ranging from about 45 to _ p¢= c = o = L about 150 ), mz is an integer from 1 to about 9 , and / or m , is mm 55 an integer from 1 to about 75 ( e . g , m , being about 6 -60 ) . For example , when the PHF in Formula ( Id ) has a in which LP2 is a moiety containing a functional group that molecular weight ranging from about 8 kDa to about 15 kDa is capable of forming a covalent bond with a functional ( i. e ., the sum of m , m , , and mz ranging from about 60 to group of a PBRM , and about 110 ), mz is an integer from 1 to about 7 , and /or m , is 60 an integer from 1 to about 55 ( e . g , m , being about 6 -45 ) . For example , when the PHF in Formula ( Id ) has a molecular weight ranging from 20 kDa to 300 kDa ( i. e ., the sum of m , m , , and mz ranging from about 150 to about 2200 ) , mz is an integer from 1 to about 110 , and /or m , is an 65 integer from 1 to about 660 ( e . g , m , being about 13 - 550 ) . For example , when the PHF in Formula ( Id ) has a denotes direct or indirect attachment of LP2 to LDI. molecular weight ranging from 40 kDa to 150 kDa (i . e ., the US 9 ,943 ,609 B2 163 164 sum of m , my, and mz ranging from about 300 to about kDa ( i. e ., the sum of m , m?, mz, and m4 ranging from about 1100 ) , mz is an integer from 1 to about 75 , and / or m , is an 370 to about 740 ) , mz is an integer from 1 to about 40 , m , integer from 1 to about 330 ( e . g , m , being about 20 - 250 ) . is an integer from 1 to about 20 , and / or m , is an integer from For example, when the PHF in Formula ( Id ) has a 1 to about 220 ( e . g , m , being about 20 - 180 ) . molecular weight ranging from about 50 kDa to about 100 5 Alternatively or additionally , one or more D - free poly kDa ( i. e ., the sum of m , m , , and m , ranging from about 370 meric carriers are connected to one PBRM . For example , the to about 740 ) ,mz is an integer from 1 to about 40 , and / or m scaffold comprises a PBRM with a molecular weight of is an integer from 1 to about 220 ( e . g , m , being about greater than 40 kDa and one or more polymeric carriers 20 - 180 ) . connected to the PBRM , in which each of the polymeric For example , the D - free scaffold further comprises a in carrier independently is of Formula ( 1h ) : PBRM connected to the polymeric carrier via L . For example , one or more PBRMs are connected to one D - free polymeric carrier . (Ih ) For example , the D - free scaffold is of Formula ( le ) : 15 ( le) OH ?? ohn L = - 1 20 LDI OH

OH OH - m 0 = am 1 25 25 LOHOH

m3 j Di

?? OH 30 nen O m3 - m4 L P2 DI TDI

min ni 35 IP2 1. P2

mi m4 PBRM , TDI 40 wherein : min B ma

45 wherein : terminal between LP2 and PBRM denotes direct or indirect attach ment of PBRM to LP2, 50 PBRM has a molecular weight of less than 200 kDa , m is an integer from 1 to 2200 , m , is an integer from 1 to 660 , minen mz is an integer from 0 to 110 , m4 is an integer from 1 to about 60 ; and 55 attached to LP2 denotes direct or indirect attachment of LP2 the sum of m , m?, m?, mz and m4 ranges from about 150 to PBRM such that the D -carrying polymeric carrier is to about 2200 . connected to the PBRM , For example, in Formula ( le ) , m , is an integer from about m is an integer from 1 to 300 , 10 to about 660 ( e . g , about 14 -550 ) . m , is an integer from 1 to 140 , For example , when the PHF in Formula (le ) has a 60 mz is an integer from 0 to 18 , molecular weight ranging from 40 kDa to 150 kDa ( i . e ., the m , is an integer from 1 to 10 ; and sum of m , m , mz, and m , ranging from about 300 to about the sum of m , m , mz, and m , ranges from 15 to 300 ; 1100 ), mz is an integer from 1 to about 75 , m4 is an integer provided that the total number of LP2 attached to the PBRM from 1 to about 30 , and / or m , is an integer from 1 to about is 10 or less . 330 ( e . g , m , being about 20 - 250 ) . 65 For example , in Formula ( Ih ) , m , is an integer from 2 to For example, when the PHF in Formula ( le ) has a about 130 ( e . g , about 3 - 120 ) and /or mz is an integer from 1 molecular weight ranging from about 50 kDa to about 100 to about 10 (e . g, about 1 -8 ) . US 9 ,943 ,609 B2 165 166 For example , when the PHF in Formula ( Ih ) has a molecular weight ranging from about 6 kDa to about 20 kDa ( i . e . , the sum of m , m , , mz, and m , ranging from about 45 to about 150 ) , mz is an integer from 1 to about 9 , and / or m? is an integer from 6 to about 75 ( e . g , m , being about 7 -60 ) . 5 For example , when the PHF in Formula ( Ih ) has a molecular weight ranging from about 8 kDa to about 15 kDa ( i. e ., the sum of m , m , , mz, and m4 ranging from about 60 to about 110 ) , mz is an integer from 1 to about 7 , and / or m , is an integer from 6 to about 55 ( e . g , m , being about 7 - 45 ) . 10 PBRM - drug -polymer conjugates, drug carring -polymeric scaffolds, or PBRM -carring polymer scaffolds can be puri fied (i .e ., removal of residual unreacted drug , PBRM , or polymeric starting materials ) by extensive diafiltration . If necessary , additional purification by size exclusion chroma- 15 tography can be conducted to remove any aggregated PBRM -drug polymer conjugates . In general , the PBRM drug polymer conjugates as purified typically contain < 5 % aggregated PBRM - drug polymer conjugates as determined by SEC or SDS- PAGE ; < 1 % polymer -drug conjugate as 20 determined by SEC and < 2 % unconjugated PBRM as deter mined by RP HPLC . Tables D and E below provide examples of the drug carrying polymeric scaffolds and the polymer -drug -protein conjugates of the invention respectively . US 9, 943, 609 B2 167 ??168

HO

IIII ? HN ? OHOOH Structure ? TABLED ? HO ? OHO ?

? ? HO?HO?

Drug: PHF Ratio

Ref# Ex9 US 9 , 943 ,609 B2 169 170

HO.

IIIII NH FO HO.

NH

Structure OHOHO. TABLED-continued

tyytyyt HO

HOHO.

HO SoHoHoHoHO

Drug: PHF Ratio

Ref# Ex9 US 9 , 943 ,609 B2 171 172172

HO

OH ?? WUN 11 HOOH HN Me02C Me0 Me

HN

HO. HN

SH O NH HO Structure SOHOHO. TABLED-continued * tyøyttyyttyyntyy O HO OH HO

HOHO. * ?????? HOHO HOHO

Drug: 11:1 15:1 PHF Ratio 7:1to 11:1to

Ref# US 9 ,943 , 609 B2 173 174

H

(

OH Niiii }HO ??HN Me0gC ??? Me

-NH

H0.

Structure SH TABLED-continued 0 NH ???HO

HO HO

H0H0.

Drug: PHF Ratio Ref# Ex6 US 9 ,943 , 609 B2 175 176

H

(

OH Niiii ??? }HO ??HN Me0gC ??? Me

-NH

H0. *

Structure SH TABLED-continued 0 NH ??????OH

HÓ ?????{ ???OH *?OH OH

* Drug: PHF Ratio

Ref# Ex18 US 9 ,943 , 609 B2 177 178

NIIIII HOOHY ??HN MeO2C Me

-NH

0.

OH Structure TABLED-continued HN 0H0. r \

HO HO

H0H0.

Drug: 28:1 PHF Ratio 24:1to

Ref# Ex13 US 9 ,943 , 609 B2 17179 ? 180

OH IIIN IIIIII 11111 !HO Ve HN MeO2C Me0

-NH

HO.

( Structure : TABLED-continued NH ?????OH

?OH HO OHOH

?? OHOH

OH

Drug: 15:1 PHF Ratio 11:1to

Ref# Ex59 US 9 ,943 , 609 B2 181 182

H

(

HO Niiii OH

??HN Me0gC ??? Me

-NH

H0. *

Structure SH TABLED-continued 0 NH OH *????? HO ??????????[ OHoOH *OHOH ??? OHOH Drug: PHF Ratio

Ref# Ex20 US 9 ,943 , 609 B2 183183 ? 184

OH IIIN IIIIII !HO Ve HN MeO2C Me0

-NH

HO.

( Structure : TABLED-continued HN H0

HO

0H0.HO

H0 ???HO Drug: PHF Ratio 4:1to8

Ref# US 9 ,943 , 609 B2 185 186

HO

OHOH IIIIN HO

HN Me0gC ?[ Me0

HN:

H0. Structure NO2 TABLED-continued ????? NH HN

H0. ??????? HO *OH HO

Drug: 15:1 PHF Ratio 11:1to

Ref#