Protein-polymer-drug conjugates

[0001] This application is a continuation of U.S. patent application Ser. No. 13/710,355, filed Dec. 10, 2012, which is a continuation-in-part of U.S. patent application Ser. No. 13/493,899, filed Jun. 11, 2012, which claims the benefit of and priority under 35 USC §119(e) to U.S. Patent Application Nos. 61/495,771, filed Jun. 10, 2011; 61/501,000, filed Jun. 24, 2011; 61,513,234, filed Jul. 29, 2011; 61/566,935, filed Dec. 5, 2011; 61/605,618, filed Mar. 1, 2012; and 61/618,499, filed Mar. 30, 2012. The contents of each of these applications are hereby incorporated by reference in their entireties.

[0002] Traditionally, pharmaceuticals have primarily consisted of small molecules that are dispensed orally (as solid pills and liquids) or as injectables. Over the past three decades, formulations (i.e., compositions that control the route and/or rate of drug delivery and allow delivery of the therapeutic agent at the site where it is needed) have become increasingly common and complex. Nevertheless, many questions and challenges regarding the development of new treatments as well as the mechanisms with which to administer them remain to be addressed. For example, many drugs exhibit limited or otherwise reduced potencies and therapeutic 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.

[0003] 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, or both.

[0004] Antibody-drug conjugates have been developed as target-specific therapeutic agents. Antibodies against various cancer cell-surface antigens have been conjugated with different cytotoxic agents that inhibit various essential cellular targets such as microtubules (maytansinoids, auristatins, taxanes: U.S. Pat. Nos. 5,208,020; 5,416,064; 6,333,410; 6,441,163; 6,340,701; 6,372,738; 6,436,931; 6,596,757; and 7,276,497); DNA (calicheamicin, doxorubicin, CC-1065 analogs; U.S. Pat. Nos. 5,475,092; 5,585,499; 5,846,545; 6,534,660; 6,756,397; and 6,630,579). Antibody conjugates with some of these cytotoxic drugs are actively being investigated in the clinic for cancer therapy (Ricart, A. D., and Tolcher, A. W., 2007, Nature Clinical Practice, 4, 245-255; Krop et al., 2010, J. Clin. Oncol., 28, 2698-2704). However, existing antibody-drug conjugates have exhibited a few limitations. A major limitation is their inability to deliver a sufficient 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, daunorubicin, maytansinoids, taxanes, and vincristine. One approach to achieving significant cytotoxicity is by linkage of a large 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 vivo clearance from the blood stream. Therefore, there is a need to improve the ability to deliver a sufficient concentration of a drug to the target such that maximum cytotoxicity for the drug is achieved.

[0005] The present invention relates to a protein-polymer-drug conjugate that is biodegradable, biocompatible and exhibits high drug load as well as strong binding to target antigen. The present invention also relates to a polymeric scaffold useful to conjugate with a protein based recognition-molecule (PBRM) so as to obtain the protein-polymer-drug conjugate.

[0006] In one aspect, the invention relates to a polymeric scaffold of Formula (Ia) useful to conjugate with a protein based recognition-molecule (PBRM) that has a molecular weight of less than 80 kDa, wherein the scaffold comprises a polymeric carrier:

[0007]
further wherein:

[0008] the polymeric carrier is PHF having a molecular weight ranging from 20 kDa to 150 kDa;

[0009] each occurrence of D is independently a therapeutic agent having a molecular weight of ≦5 kDa;

[0010]
is a linker that contains a biodegradable bond so that when the bond is broken, D is released from the polymeric carrier in an active form for its intended therapeutic effect; in which L^D1 is a carbonyl-containing moiety, and the

[0011]
between L^D1 and D denotes direct or indirect attachment of D to L^D1;

[0012]
is a linker distinct from the linker

[0013]
in which L^P2 is a moiety containing a functional group that is capable of forming a covalent bond with a functional group of a PBRM, and the

[0014]
between L^D1 and L^P2 denotes direct or indirect attachment of L^P2 to L^D1;

[0015] m is an integer from 1 to 1100,

[0016] m₁ is an integer from 1 to 330,

[0017] m₂ is an integer from 3 to 150,

[0018] m₃ is an integer from 1 to 55, and

[0019] the sum of m, m₁, m₂ and m₃ ranges from 150 to about 1100.

[0020] The scaffold of (Ia) can include one or more of the following features:

[0021] The PHF has a molecular weight ranging from 30 kDa to 100 kDa, m₂ is an integer from 3 to about 100, m₃ is an integer from 1 to 40, and m₁ is an integer from 1 to 220.

[0022] The functional group of L^P2 is selected from —SR^p, —S—S-LG, maleimido, and halo, in which LG is a leaving group and R^p is H or a sulfur protecting group.

[0023] L^D1 comprises —X—(CH₂)_v—C(═O)— with X directly connected to the carbonyl group of

[0024]
in which X is CH₂, O, or NH, and v is an integer from 1 to 6.

[0025] L^P2 contains a biodegradable bond.

[0026] The scaffold further comprises a PBRM connected to the polymeric carrier via L^P.

[0027] The scaffold is of Formula (Ib):

[0028]
wherein:

[0029]
between L^P2 and PBRM denotes direct or indirect attachment of PBRM to L^P2,

[0030] each occurrence of PBRM independently has a molecular weight of less than 80 kDa,

[0031] m is an integer from 1 to 1100,

[0032] m₁ is an integer from 1 to 330,

[0033] m₂ is an integer from 3 to 150,

[0034] m₃ is an integer from 0 to 55,

[0035] m₄ is an integer from 1 to 30; and

[0036] the sum of m, m₁, m₂, m₃ and m₄ ranges from 150 to 1100.

[0037] The scaffold of Formula (Ib) can include one or more of the following features:

[0038] The PHF has a molecular weight ranging from 30 kDa to 100 kDa, m₁ is an integer from 1 to 220, m₂ is an integer from 3 to 100, m₃ is an integer from 0 to 40, and m₄ is an integer from 1 to 20, and the sum of m₁ and m₂ is an integer from 18 to 220, and the sum of m₃ and m₄ is an integer from 1 to 40.

[0039] m₂ is an integer from 3 to about 150.

[0040] m₄ is an integer from 1 to about 10.

[0041] The sum of m₁ and m₂ is an integer from 14 to 330.

[0042] The sum of m₃ and m₄ is an integer from 1 to 55.

[0043] Each occurrence of PBRM independently has a molecular weight of about 30-70 kDa, and m₂ is an integer from 3 to 100.

[0044] Each occurrence of PBRM independently has a molecular weight of about 20-30 kDa, and m₂ is an integer from 3 to 150.

[0045] Each occurrence of PBRM independently has a molecular weight of about 4-20 kDa, and m₂ is an integer from 3 to 150.

[0046] The ratio of m₂ to m₄ is between 5:1 and 40:1.

[0047] Other features of scaffold of Formula (Ia) or (Ib) include those described herein where applicable.

[0048] In another aspect, the invention features a polymeric scaffold useful to conjugate with a PBRM. The scaffold comprises a polymeric carrier, one or more -L^D-D connected to the polymeric carrier, and one or more L^P connected to the polymeric carrier which is suitable for connecting a PBRM to the polymeric carrier, wherein:

[0049] each occurrence of D is independently a therapeutic agent having a molecular weight ≦5 kDa;

[0050] the polymeric carrier is a polyacetal or polyketal,

[0051] L^D is a linker having the structure:

[0052]
with R^L1 connected to an oxygen atom of the polymeric carrier and L^D1 connected to D, and

[0053]
denotes direct or indirect attachment of D to L^D1, and L^D contains a biodegradable bond so that when the bond is broken, D is released from the polymeric carrier in an active form for its intended therapeutic effect;

[0054] L^D1 is a carbonyl-containing moiety;

[0055] L^P is a linker different from L^D and having the structure: —R^L2—C(═O)-L^P1 with R^L2 connected to an oxygen atom of the polymeric carrier and L^P1 suitable for connecting directly or indirectly to a PBRM;

[0056] each of R^L1 and R^L2 independently is absent, alkyl, heteroalkyl, cycloalkyl, or heterocyclo alkyl; and

[0057] L^P1 is a moiety containing a functional group that is capable of forming a covalent bond with a functional group of a PBRM.

[0058] The polymeric scaffold can include one or more of the following features.

[0059] L^P is a linker having the structure:

[0060]
in which L^P2 is a moiety containing a functional group that is capable of forming a covalent bond with a functional group of a PBRM, and

[0061]
denotes direct or indirect attachment of L^P2 to L^D1.

[0062] The functional group of L^P1 or L^P2 is selected from —SR^p, —S—S-LG, maleimido, and halo, in which LG is a leaving group and R^p is H or a sulfur protecting group.

[0063] L^D1 comprises —X—(CH₂)_v—C(═O)— with X directly connected to the carbonyl group of R^L1—C(═O), in which X is CH₂, O, or NH, and v is an integer from 1 to 6.

[0064] L^P1 or L^P2 contains a biodegradable bond.

[0065] Each of R^L1 and R^L2 is absent.

[0066] The polymeric carrier of the scaffold of the invention is a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF) ranging from about 2 kDa to about 300 kDa.

[0067] For conjugating a PBRM having a molecular weight of 40 kDa or greater (e.g., 80 kDa or greater), the polymeric carrier of the scaffold of the invention is a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF) ranging from about 2 kDa to about 40 kDa (e.g., about 6-20 kDa or about 8-15 kDa).

[0068] For conjugating a PBRM having a molecular weight of 200 kDa or less (e.g., 80 kDa or less), the polymeric carrier of the scaffold of the invention is a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF) ranging from about 20 kDa to about 300 kDa (e.g., about 40-150 kDa or about 50-100 kDa).

[0069] The scaffold is of Formula (Ia):

[0070]

[0071] wherein:

• • [0072] m is an integer from 1 to about 2200,
  • [0073] m₁ is an integer from 1 to about 660,
  • [0074] m₂ is an integer from 1 to about 300,
  • [0075] m₃ is an integer from 1 to about 110, and
  • [0076] the sum of m, m₁, m₂ and m₃ ranges from about 15 to about 2200.

[0077] When the PHF in Formula (Ia) has a molecular weight ranging from about 2 kDa to about 40 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging from about 15 to about 300), m₂ is an integer from 1 to about 40, m₃ is an integer from 1 to about 18, and/or m₁ is an integer from 1 to about 140 (e.g., m₁ being about 1-90).

[0078] When the PHF in Formula (Ia) has a molecular weight ranging from about 6 kDa to about 20 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging from about 45 to about 150), m₂ is an integer from 2 to about 20, m₃ is an integer from 1 to about 9, and/or m₁ is an integer from 1 to about 75 (e.g., m₁ being about 4-45).

[0079] When the PHF in Formula (Ia) has a molecular weight ranging from about 8 kDa to about 15 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging from about 60 to about 110), m₂ is an integer from 2 to about 15, m₃ is an integer from 1 to about 7, and/or m₁ is an integer from 1 to about 55 (e.g., m₁ being about 4-30).

[0080] When the PHF in Formula (Ia) has a molecular weight ranging from 20 kDa to 300 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging from about 150 to about 2200), m₂ is an integer from 3 to about 300, m₃ is an integer from 1 to about 110, and/or m₁ is an integer from 1 to about 660 (e.g., m₁ being about 10-250).

[0081] When the PHF in Formula (Ia) has a molecular weight ranging from about 50 kDa to about 100 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging from about 370 to about 740), m₂ is an integer from 5 to about 100, m₃ is an integer from 1 to about 40, and/or m₁ is an integer from 1 to about 220 (e.g., m₁ being about 15-80).

[0082] The scaffold further comprises a PBRM connected to the polymeric carrier via L^P.

[0083] One or more PBRMs are connected to one drug-carrying polymeric carrier.

[0084] The scaffold (e.g., a PBRM-polymer-drug conjugate) is of Formula (Ib):

[0085]
wherein:

[0086]

[0087] between L^P2 and PBRM denotes direct or indirect attachment of PBRM to L^P2,

[0088] each occurrence of PBRM independently has a molecular weight of less than 200 kDa,

[0089] m is an integer from 1 to about 2200,

[0090] m₁ is an integer from 1 to about 660,

[0091] m₂ is an integer from 3 to about 300,

[0092] m₃ is an integer from 0 to about 110,

[0093] m₄ is an integer from 1 to about 60; and

[0094] the sum of m, m₁, m₂, m₃ and m₄ ranges from about 150 to about 2200.

[0095] In Formula (Ib), m₁ is an integer from about 10 to about 660 (e.g., about 10-250).

[0096] When the PHF in Formula (Ib) has a molecular weight ranging from about 50 kDa to about 100 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ ranging from about 370 to about 740), m₂ is an integer from 5 to about 100, m₃ is an integer from 1 to about 40, m₄ is an integer from 1 to about 20, and/or m₁ is an integer from 1 to about 220 (e.g., m₁ being about 15-80).

[0097] Alternatively or additionally, one or more drug-carrying polymeric carriers are connected to one PBRM. The scaffold (e.g., a PBRM-polymer-drug conjugate) comprises a PBRM with a molecular weight of greater 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):

[0098]
wherein:

[0099] terminal

[0100]
attached to L^P2 denotes direct or indirect attachment of L^P2 to PBRM such that the D-carrying polymeric carrier is connected to the PBRM,

[0101] m is an integer from 1 to 300,

[0102] m₁ is an integer from 1 to 140,

[0103] m₂ is an integer from 1 to 40,

[0104] m₃ is an integer from 0 to 18,

[0105] m₄ is an integer from 1 to 10; and

[0106] the sum of m, m₁, m₂, m₃, and m₄ ranges from 15 to 300; provided that the total number of L^P2 attached to the PBRM is 10 or less.

[0107] In Formula (Ic), m₁ is an integer from 1 to about 120 (e.g., about 1-90) and/or m₃ is an integer from 1 to about 10 (e.g., about 1-8).

[0108] When the PHF in Formula (Ic) has a molecular weight ranging from about 6 kDa to about 20 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ ranging from about 45 to about 150), m₂ is an integer from 2 to about 20, m₃ is an integer from 1 to about 9, and/or m₁ is an integer from 1 to about 75 (e.g., m₁ being about 4-45).

[0109] When the PHF in Formula (Ic) has a molecular weight ranging from about 8 kDa to about 15 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ ranging from about 60 to about 110), m₂ is an integer from 2 to about 15, m₃ is an integer from 1 to about 7, and/or m₁ is an integer from 1 to about 55 (e.g., m₁ being about 4-30).

[0110] Each occurrence of D independently is selected from vinca alkaloids, auristatins, tubulysins, duocarmycins, kinase inhibitors, MEK inhibitors, KSP inhibitors, and analogs thereof.

[0111] L^D is —R^L1—C(═O)—X^D-M^D1Y^D-M^D2-Z^D-M^D3-Q^D-M^D4- with M^D4 directly connected to D, in which

[0112] X^D is —O—, —S—, —N(R¹)—, or absent, in which R¹ is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety, —C(═O)R^1B, —C(═O)OR^1B, or —SO₂R^1B, or —N(R¹)— is a heterocycloalkyl moiety, wherein R^1B is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety;

[0113] each of Y^D, Z^D, and Q^D, independently, is absent or a biodegradable linker moiety selected from the group consisting of —S—S—, —C(═O)O—, —C(═O)NR²—, —OC(═O)—, —NR²C(═O)—, —OC(═O)O—, —OC(═O)NR²—, —NR²C(═O)O—, —NR²C(═O)NR³—, —C(OR²)O—, —C(OR²)S—, —C(OR²)NR³—, —C(SR²)O—, —C(SR²)S—, C(SR²)NR³—, —C(NR²R³)O—, —C(NR²R³)S—, —C(NR²R³)NR⁴—, —C(═O)S—, —SC(═O)—, —SC(═O)S—, —OC(═O)S—, —SC(═O)O—, —C(═S)S—, —SC(═S)—, —OC(═S)—, —C(═S)O—, —SC(═S)O—, —OC(═S)S—, —OC(═S)O—, —SC(═S)S—, —C(═NR²)O—, —C(═NR²)S—, —C(═NR²)NR³—, —OC(═NR²)—, —SC(═NR²)—, —NR³C(═NR²)—, —NR²SO₂—, NR²NR³—, —C(═O)NR²NR³—, —NR²NR³C(═O)—, —OC(═O)NR²NR³—, —NR²NR³C(═O)O—, —C(═S)NR²NR³—, —NR²NR³C(═S)—, —C(═NR⁴)NR²NR³—, —NR²NR³C(═NR⁴)—, —O(N═CR³)—, —(CR³═N)O—, —C(═O)NR²—(NR³)—, —(CR³═N)—NR²C(═O)—, —SO₃—, —NR²SO₂NR³—, —SO₂NR²—, and polyamide, wherein each occurrence of R², R³, and R⁴ independently is hydrogen or an aliphatic, heteroaliphatic, carbocyclic, or heterocyclic moiety, or each occurrence of —NR²— or —NR²NR³— is a heterocycloalkyl moiety; and each of M^D1, M^D2, M^D3, and M^D4 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 heterocyclic moiety, and a combination thereof, and each of M^D1, M^D2, and M^D3 optionally contains one or more —(C═O)— but does not contain any said biodegradable linker moiety;

[0114] provided that for each L^D1, at least one of X^D, Y^D, Z^D, and Q^D is not absent.

[0115] Each

[0116]
when not connected to PBRM, independently comprises a terminal group W^P, in which each W^P independently is:

[0117]
in which R^1K is a leaving group (e.g., halide or RC(O)O— in which R is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety), R^1A is a sulfur protecting group, and ring A is cycloalkyl or heterocycloalkyl, and R^1J is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

[0118] Each R^1A independently is

[0119]
in which r is 1 or 2 and each of R^s1, R^s2, and R^s3 is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

[0120] Each

[0121]
when connected to PBRM, independently is —X^P-M^P1-Y^P-M^P2−-Z^P-M^P3-Q^P-M^P4-, with X^P directly connected to the carbonyl group of R^L1—C(═O) and M^P4 directly connected to PBRM, in which:

[0122] X^P is —O—, —S—, —N(R¹)—, or absent, in which R¹ is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety, —C(═O)R^1B, —C(═O)OR^1B, or —SO₂R^1B, or —N(R¹)— is a heterocycloalkyl moiety, wherein R^1B is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety;

[0123] each of Y^P, Z^P, and Q^P, independently, is absent or a biodegradable linker moiety selected from the group consisting of —S—S—, —C(═O)O—, —C(═O)NR²—, —OC(═O)—, —NR²C(═O)—, —OC(═O)O—, —OC(═O)NR²—, —NR²C(═O)O—, —NR²C(═O)NR³—, —C(OR²)O—, —C(OR²)S—, —C(OR²)NR³—, —C(SR²)O—, —C(SR²)S—, C(SR²)NR³—, —C(NR²R³)O—, —C(NR²R³)S—, —C(NR²R³)NR⁴—, —C(═O)S—, —SC(═O)—, —SC(═O)S—, —OC(═O)S—, —SC(═O)O—, —C(═S)S—, —SC(═S)—, —OC(═S)—, —C(═S)O—, —SC(═S)O—, —OC(═S)S—, —OC(═S)O—, —SC(═S)S—, —C(═NR²)O—, —C(═NR²)S—, —C(═NR²)NR³—, —OC(═NR²)—, —SC(═NR²)—, —NR³C(═NR²)—, —NR²SO₂—, NR²NR³—, —C(═O)NR²NR³—, —NR²NR³C(═O)—, —OC(═O)NR²NR³—, —NR²NR³C(═O)O—, —C(═S)NR²NR³—, —NR²NR³C(═S)—, —C(═NR⁴)NR²NR³—, —NR²NR³C(═NR⁴)—, —O(NR³)—, —(CR³═N)O—, —C(═O)NR²—(N═CR³)—, —(CR³═N)—NR²C(═O)—, —SO₃—, —NR²SO₂NR³—, —SO₂NR²—, and polyamide, wherein each occurrence of R², R³, and R⁴ independently is hydrogen or an aliphatic, heteroaliphatic, carbocyclic, or heterocyclic moiety, or each occurrence of —NR²— or —NR²NR³— is a heterocycloalkyl moiety; and

[0124] each of M^P1, M^P2, M^P3, and M^P4 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 heterocyclic moiety, and a combination thereof, and each of M^P1, M^P2, and M^P3 optionally contains one or more —(C═O)— but does not contain any said biodegradable linker moiety;

[0125] provided that for each

[0126]
connected to PBRM, at least one of X^P, Y^P, Z^P, and Q^P is not absent.

[0127] Each of M^D1 and M^P1 independently is C_1-6 alkyl or C_1-6 heteroalkyl.

[0128] Each of M^D2, M^D3, M^D4, M^P2, M^P3, and M^P4, independently is absent, C_1-6 alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, or a combination thereof.

[0129] In each

[0130]
at most one of M^P2 and M^P3 has one of the following structures:

[0131]
in which q is an integer from 0 to 12 and each of p and t independently is an integer from 0 to 3.

[0132] Also within the scope of the invention is a method of preparing a scaffold described above. The method comprises providing a polymeric carrier that is substituted with one or more -L^D-D and one or more —R^L1—C(═O)-L^D1, and reacting the polymeric carrier with a compound containing an L^P2 moiety to produce the scaffold comprising a polymeric carrier substituted both with one or more -L^D-D and with one or more

[0133]
Alternatively, the method comprises providing a polymeric carrier that is substituted with one or more

[0134]
and one or more —R^L1—C(═O)-L^D1, and reacting the polymeric carrier with D containing a functional group that is capable of forming a covalent bond with —R^L1—C(═O)-L^D1 to produce the scaffold comprising a polymeric carrier substituted both with one or more -L^D-D and with one or more

[0135]

[0136] The invention also features a compound of Formula (XII) or (XIIa):

[0137]
or a pharmaceutically acceptable salt thereof,
wherein:

[0138] R₄₀ is selected from the group consisting of

[0139]

[0140] a is an integer from 1 to 6; and

[0141] c is an integer from 0 to 3.

[0142] R₄₀ can be

[0143]

[0144] In another aspect, the invention features a polymeric scaffold useful to conjugate with both a protein based recognition-molecule (PBRM) and a therapeutic agent (D). The scaffold (i.e., the one free of any D) comprises a polymeric carrier, one or more L^P connected to the polymeric carrier which is suitable for connecting a PBRM to the polymeric carrier, and one or more —R^L1—C(═O)-L^D1 connected to the polymeric carrier via R^L1, wherein:

[0145] the polymeric carrier is a polyacetal or polyketal,

[0146] R^L1 is connected to an oxygen atom of the polymeric carrier,

[0147] L^D1 is a linker suitable for connecting a D molecule to the polymeric carrier, in which each occurrence of D is independently a therapeutic agent having a molecular weight ≦5 kDa;

[0148] L^P is a linker different from —R^L1—C(═O)-L^D1, and having the structure: —R^L2—C(═O)-L^P1 with R^L2 connected to an oxygen atom of the polymeric carrier and L^P1 suitable for connecting to a PBRM;

[0149] each of R^L1 and R^L2 independently is absent, alkyl, heteroalkyl, cycloalkyl, or heterocycloalkyl;

[0150] L^D1 is a moiety containing a functional group that is capable of forming a covalent bond with a functional group of D, and

[0151] L^P1 is a moiety containing a functional group that is capable of forming a covalent bond with a functional group of a PBRM.

[0152] The D-free scaffold useful to conjugate with a PBRM and a D can have one or more of the following features.

[0153] L^P is a linker having the structure:

[0154]
in which L^P2 is a moiety containing a functional group that is capable of forming a covalent bond with a functional group of a PBRM, and

[0155]
denotes direct or indirect attachment of L^P2 to L^D1.

[0156] The functional group of L^P1 or L^P2 is selected from —SR^p, —S—S-LG, maleimido, and halo, in which LG is a leaving group and R^p is H or a sulfur protecting group.

[0157] L^D1 comprises —X—(CH₂)_v—C(═O)— with X directly connected to the carbonyl group of R^L1—C(═O), in which X is CH₂, O, or NH, and v is an integer from 1 to 6.

[0158] L^P1 or L^P2 contains a biodegradable bond.

[0159] Each of R^L1 and R^L2 is absent.

[0160] The polymeric carrier of the D-free scaffold is a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF) ranging from about 2 kDa to about 300 kDa.

[0161] For conjugating a PBRM having a molecular weight of 40 kDa or greater (e.g., 80 kDa or greater), the polymeric carrier of the D-free scaffold is a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF) ranging from about 2 kDa to about 40 kDa (e.g., about 6-20 kDa or about 8-15 kDa).

[0162] For conjugating a PBRM having a molecular weight of 200 kDa or less (e.g., 80 kDa or less), the polymeric carrier of the D-free scaffold of the invention is a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF) ranging from about 20 kDa to about 300 kDa (e.g., about 40-150 kDa or about 50-100 kDa).

[0163] The D-free scaffold is of Formula (Id):

[0164]

[0165] wherein:

• • [0166] m is an integer from 1 to about 2200,
  • [0167] m₁ is an integer from 1 to about 660,
  • [0168] m₃ is an integer from 1 to about 110, and
  • [0169] the sum of m, m₁, and m₃ ranges from about 15 to about 2200.

[0170] When the PHF in Formula (Id) has a molecular weight ranging from about 2 kDa to about 40 kDa (i.e., the sum of m, m₁, and m₃ ranging from about 15 to about 300), m₃ is an integer from 1 to about 18, and/or m₁ is an integer from 1 to about 140 (e.g., m₁ being about 2-120).

[0171] When the PHF in Formula (Id) has a molecular weight ranging from about 6 kDa to about 20 kDa (i.e., the sum of m, m₁, and m₃ ranging from about 45 to about 150), m₃ is an integer from 1 to about 9, and/or m₁ is an integer from 1 to about 75 (e.g., m₁ being about 6-60).

[0172] When the PHF in Formula (Id) has a molecular weight ranging from about 8 kDa to about 15 kDa (i.e., the sum of m, m₁, and m₃ ranging from about 60 to about 110), m₃ is an integer from 1 to about 7, and/or m₁ is an integer from 1 to about 55 (e.g., m₁ being about 6-45).

[0173] When the PHF in Formula (Id) has a molecular weight ranging from 20 kDa to 300 kDa (i.e., the sum of m, m₁, and m₃ ranging from about 150 to about 2200), m₃ is an integer from 1 to about 110, and/or m₁ is an integer from 1 to about 660 (e.g., m₁ being about 13-550).

[0174] When the PHF in Formula (Id) has a molecular weight ranging from about 50 kDa to about 100 kDa (i.e., the sum of m, m₁, and m₃ ranging from about 370 to about 740), m₃ is an integer from 1 to about 40, and/or m₁ is an integer from 1 to about 220 (e.g., m₁ being about 20-180).

[0175] The D-free scaffold further comprises a PBRM connected to the polymeric carrier via L^P.

[0176] One or more PBRMs are connected to one D-free polymeric carrier.

[0177] The D-free scaffold is of Formula (Ie):

[0178]
wherein:

[0179]
between L^P2 and PBRM denotes direct or indirect attachment of PBRM to L^P2,

[0180] PBRM has a molecular weight of less than 200 kDa,

[0181] m is an integer from 1 to 2200,

[0182] m₁ is an integer from 1 to 660,

[0183] m₃ is an integer from 0 to 110,

[0184] m₄ is an integer from 1 to about 60; and

[0185] the sum of m, m₁, m₂, m₃ and m₄ ranges from about 150 to about 2200.

[0186] In Formula (Ie), m₁ is an integer from about 10 to about 660 (e.g., about 14-550).

[0187] When the PHF in Formula (Ie) has a molecular weight ranging from about 50 kDa to about 100 kDa (i.e., the sum of m, m₁, m₃, and m₄ ranging from about 370 to about 740), m₃ is an integer from 1 to about 40, m₄ is an integer from 1 to about 20, and/or m₁ is an integer from 1 to about 220 (e.g., m₁ being about 20-180).

[0188] Alternatively or additionally, one or more D-free polymeric carriers are connected to one PBRM. The scaffold comprises a PBRM with a molecular weight of greater than 40 kDa and one or more polymeric carriers connected to the PBRM, in which each of the polymeric carrier independently is of Formula (Ih):

[0189]
wherein:

[0190] terminal

[0191]
attached to L^P2 denotes direct or indirect attachment of L^P2 to PBRM such that the D-carrying polymeric carrier is connected to the PBRM,

[0192] m is an integer from 1 to 300,

[0193] m₁ is an integer from 1 to 140,

[0194] m₃ is an integer from 0 to 18,

[0195] m₄ is an integer from 1 to 10; and

[0196] the sum of m, m₁, m₃, and m₄ ranges from 15 to 300; provided that the total number of L^P2 attached to the PBRM is 10 or less

[0197] In Formula (Ih), m₁ is an integer from 2 to about 130 (e.g., about 3-120) and/or m₃ is an integer from 1 to about 10 (e.g., about 1-8).

[0198] 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, m₁, m₃, and m₄ ranging from about 45 to about 150), m₃ is an integer from 1 to about 9, and/or m₁ is an integer from 6 to about 75 (e.g., m₁ being about 7-60).

[0199] 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, m₁, m₃, and m₄ ranging from about 60 to about 110), m₃ is an integer from 1 to about 7, and/or m₁ is an integer from 6 to about 55 (e.g., m₁ being about 7-45).

[0200] As used herein, the terms “polymeric scaffold” or simply “scaffold” and “conjugate” are used interchangeably when the scaffold comprises one or more PBRM and one or more D molecules.

[0201] In yet another aspect, the invention encompasses a conjugate comprising a polymeric carrier, one or more -L^D-D connected to the polymeric carrier, and a protein based recognition-molecule (PBRM) connected to the polymeric carrier via L^P, wherein:

[0202] each occurrence of D is independently a therapeutic agent (e.g., a drug) having a molecular weight ≦5 kDa;

[0203] the polymeric carrier is a polyacetal or polyketal,

[0204] L^D is a linker having the structure: —R^L1—C(═O)—X^D-M^D1-Y^D-M^D2-M^D2-Z^D-M^D3-Q^D-M^D4-, with R^L1 connected to an oxygen atom of the polymeric carrier and M^D4 connected to D;

[0205] L^P is a linker having the structure: —R^L2—C(═O)—X^P-M^P1-Y^P-M^P2-M^P2-Z^P-M^P3-Q^P-M^P4-, with R^L2 connected to an oxygen atom of the polymeric carrier and M^P4 connected to the protein based recognition-molecule;

[0206] each of R^L1 and R^L2 independently is absent, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl;

[0207] each of X^D and X^P, independently is —O—, —S—, —N(R¹)—, or absent, in which R¹ is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety, —C(═O)R^1B, —C(═O)OR^1B, —SO₂R^1B or —N(R¹)— is a heterocycloalkyl moiety, wherein R^1B is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety;

[0208] each of Y^D, Y^P, Z^D, Z^P, Q^D, and Q^P, independently, is absent or a biodegradable linker moiety selected from the group consisting of —S—S—, —C(═O)O—, —C(═O)NR²—, —OC(═O)—, —NR²C(═O)—, —OC(═O)O—, —OC(═O)NR²—, —NR²C(═O)O—, —NR²C(═O)NR³—, —C(OR²)O—, —C(OR²)S—, —C(OR²)NR³—, —C(SR²)O—, —C(SR²)S—, C(SR²)NR³—, —C(NR²R³)O—, —C(NR²R³)S—, —C(NR²R³)NR⁴—, —C(═O)S—, —SC(═O)—, —SC(═O)S—, —OC(═O)S—, —SC(═O)O—, —C(═S)S—, —SC(═S)—, —OC(═S)—, —C(═S)O—, —SC(═S)O—, —OC(═S)S—, —OC(═S)O—, —SC(═S)S—, —C(═NR²)O—, —C(═NR²)S—, —C(═NR²)NR³—, —OC(═NR²)—, —SC(═NR²)—, —NR³C(═NR²)—, —NR²SO₂—, NR²NR³—, —C(═O)NR²NR³—, —NR²NR³C(═O)—, —OC(═O)NR²NR³—, —NR²NR³C(═O)O—, —C(═S)NR²NR³—, —NR²NR³C(═S)—, —C(═NR⁴)NR²NR³—, —NR²NR³C(═NR⁴)—, —O(N═CR³)—, —(CR³═N)O—, —C(═O)NR²—(NR³)—, —(CR³═N)—NR²C(═O)—, —SO₃—, —NR²SO₂NR³—, —SO₂NR²—, and polyamide, wherein each occurrence of R², R³, and R⁴ independently is hydrogen or an aliphatic, heteroaliphatic, carbocyclic, or heterocyclic moiety, or each occurrence of —NR²— or —NR²NR³— is a heterocycloalkyl moiety; and

[0209] each of M^D1, M^D2, M^D3, M^D4, M^P1, M^P2, M^P3 and M^P4, 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 heterocyclic moiety, and a combination thereof, and each of M^D1, M^D2, M^D3, M^P1, M^P2, and M^P3 optionally contains one or more —(C═O)— but does not contain any said biodegradable linker moiety;

[0210] provided that for each L^D, at least one of X^D, Y^D, Z^D, and Q^D is not absent, and for each L^P, at least one of X^P, Y^P, Z^P, and Q^P is not absent.

[0211] The conjugate can include one or more of the following features.

[0212] The polymeric carrier can be a polyacetal, e.g., PHF.

[0213] For each L^D, M^D1 is not absent when X^D is absent.

[0214] For each L^P, M^P1 is not absent when X^P is absent.

[0215] The polymeric carrier can be further substituted with one or more —R^L1—C(═O)—X^D-M^D1-Y^D-M^D2-W^D, in which each W^D independently is:

[0216]
in which R^1A is a sulfur protecting group, each of ring A and B, independently, is cycloalkyl or heterocycloalkyl, R^W is an aliphatic, heteroaliphatic, carbocyclic or heterocycloalkyl moiety; ring D is heterocycloalkyl; R^1J is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety; and R^1K is a leaving group (e.g., halide or RC(O)O— in which R is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety).

[0217] The polymeric carrier can be further substituted with one or more —R^L2—C(═O)—X^P-M^P1-Y^P-M^P2-W^P, in which each W^P independently is:

[0218]
in which R^1K is a leaving group (e.g., halide or RC(O)O— in which R is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety), R^1A is a sulfur protecting group, and ring A is cycloalkyl or heterocycloalkyl, and R¹ is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety. For example, R^1A is

[0219]
in which r is 1 or 2 and each of R^s1, R^s2, and R^s3 is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

[0220] Ring A can be C_3-8 cycloalkyl or 5-19 membered heterocycloalkyl.

[0221] Ring A can be

[0222]

[0223] Ring B can be C_3-8 cycloalkyl or 3-12 membered heterocycloalkyl.

[0224] Ring D can be piperazinyl or piperidinyl.

[0225] Each of R^s1, R^s2, and R^s3 can be hydrogen or C_1-6 alkyl.

[0226] Each PBRM independently can be a peptide, a peptide mimetic, an antibody, or an antibody fragment.

[0227] Each of M^D1 and M^P1 independently can be C_1-6 alkyl or C_1-6 heteroalkyl.

[0228] Each of M^D2, M^D3, M^D4, M^P2, M^P3, and M^P4, independently can be absent, C_1-6 alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, or a combination thereof.

[0229] For each L^D, at most two of M^D2, M^D3, and M^D4 can be absent.

[0230] For each L^P, at most two of M^P2, M^P3, and M^P4 can be absent.

[0231] For each L^D, at most one of M^D2 and M^D3 can have one of the following structures:

[0232]
in which q is an integer from 0 to 12 and each of p and t independently is an integer from 0 to 3.

[0233] For each L^P, at most one of M^P2 and M^P3 can have one of the following structures:

[0234]
in which q is an integer from 0 to 12 and each of p and t independently is an integer from 0 to 3.

[0235] For each L^D, each of -M^D2-Z^D—, —Z^D-M^D3-, —Z^D-M^D2-, and -M^D3-Z^D—, independently can have one of the following structures:

[0236]
in which ring A or B independently is cycloalkyl or heterocycloalkyl; R^W is an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety; R^1J is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety; and ring D is heterocycloalkyl.

[0237] For each L^P, each of -M^P2-Z^P—, —Z^P-M^P3-, —Z^P-M^P2-, and -M^P3-Z^P—, independently, can have one of the following structures:

[0238]
in which ring A is cycloalkyl or heterocycloalkyl and R^1J is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

[0239] Each of X^D and X^P, independently can be absent.

[0240] Each of X^D and X^P, independently can be O or NH.

[0241] Each of X^D and X^P, independently can be

[0242]

[0243] Each of Y^D and Y^P independently can be —S—S—, —OCO—, —COO—, —CONH—, or —NHCO—.

[0244] Each of Q^D and Q^P independently can be absent, —S—S—, —OCO—, —COO—, —CONH—, —NHCO—, —OCONHNH— or —NHNHCOO—.

[0245] In particular, this invention features a conjugate of Formula (I):

[0246]
wherein each of n, n₁, n₂, n₃, and n₄, is the molar fraction of the corresponding polymer unit ranging between 0 and 1; n+n₁+n₂+n₃+n₄=1; provided that none of n, n₂, and n₄ is 0.

[0247] In Formula (I) above, the disconnection or gap between 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, W^D, and W^P, can be randomly distributed along the polymer backbone.

[0248] In the protein-polymer-drug conjugate of Formula (I), each D can be the same or different moiety and each PBRM can be the same or different moiety.

[0249] The ratio between n₂ and n₄ can be greater than 1:1, and up to 200:1 (e.g., up to 100:1), e.g., between 2:1 and 40:1; between 5:1 and 20:1; between 10:1 and 50:1, between 25:1 and 50:1, or between 30:1 and 50:1.

[0250] The ratio between n₂ and n₄ can be about 50:1, 40:1, 25:1, 20:1, 10:1, 5:1 or 2:1.

[0251] For example the ratio between D and PBRM can be greater than 1:1, and up to 200:1 (e.g., up to 100:1), e.g., between 2:1 and 40:1; between 5:1 and 20:1; between 10:1 and 50:1, between 25:1 and 50:1, or between 30:1 and 50:1. Examples of PBRM include but are not limited to, full length antibodies such as IgG and IgM, antibody fragments such as Fabs, scFv, camelids, Fab2, and the like, small proteins, and peptides.

[0252] In one embodiment the ratio between D and PBRM can be about 50:1, 40:1, 25:1, 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

[0253] In another embodiment the ratio between D and PBRM can be about 25:1, 20:1, 15:1, 10:1, 5:1 or 2:1.

[0254] In another aspect, the invention provides compositions comprising the conjugates, methods for their preparation, and methods of use thereof in the treatment of various disorders, including, but not limited to cancer.

[0255] The invention also features a drug-polymer conjugate (e.g., therapeutic agent-polymer conjugate) that is similar to the protein-polymer-drug conjugate described above except that drug-polymer conjugate does not contain a PBRM. In this embodiment the polymer-drug conjugate may comprise a plurality of drug moieties in which each D can be the same or different. In this embodiment, n₄ is 0 in the conjugate of Formula (I). The methods of producing the drug-polymer conjugates and methods of treating various disorders (e.g., cancer) are also contemplated and described herein.

[0256] The invention also features a protein-polymer conjugate (e.g., PBRM-polymer conjugate) that is similar to the protein-polymer-drug conjugate described above except that protein-polymer conjugate does not contain a drug. In this embodiment the protein-polymer conjugate may comprise a plurality of protein moieties in which each PBRM can be the same or different. In this embodiment, n₂ is 0 in the conjugate of Formula (I). The methods of producing the drug-polymer conjugates or polymeric scaffolds and methods of treating various disorders (e.g., cancer) are also contemplated and described herein. The target cancer can be anal, astrocytoma, leukemia, lymphoma, head and neck, liver, testicular, cervical, sarcoma, hemangioma, esophageal, eye, laryngeal, mouth, mesothelioma, skin, myeloma, oral, rectal, throat, bladder, breast, uterus, ovary, prostate, lung, colon, pancreas, renal, or gastric cancer.

[0257] The invention further relates to a pharmaceutical composition comprising a polymeric scaffold or conjugate described herein and a pharmaceutically acceptable carrier.

[0258] In yet another aspect, the invention relates to a method of diagnosing a disorder in a subject suspected of having the disorder. The method comprises administering an effective amount of the conjugate described herein to the subject suspected of having the disorder or performing an assay to detect a target antigen/receptor in a sample from the subject so as to determine whether the subject expresses target antigen or receptor.

[0259] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the specification, the singular forms also include the plural unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference. The references cited herein are not admitted to be prior art to the claimed invention. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods and examples are illustrative only and are not intended to be limiting.

[0260] One of the advantages of the present invention is that the protein-polymer-drug conjugates or the polymeric scaffolds described herein greatly enhances the bioavailability of the drugs to be delivered and/or enhances the bioavailability of the protein attached to the polymeric carrier. Another advantage of the present invention is that the efficacy of the protein-polymer-drug conjugates described herein increases or at least remains substantially the same with increases in the drug load of the conjugates. Yet another advantage of the present invention is that the protein-polymer conjugates via thiol conjugation to the cysteine moiety of the protein exhibits substantially improved stability. Other features and advantages of the invention will be apparent from the following detailed description and claims.

[0271] The present invention provides novel protein-polymer-drug conjugates, polymeric scaffolds for making the conjugates, synthetic methods for making the conjugates or polymeric scaffolds, pharmaceutical compositions containing them and various uses of the conjugates.

[0272] The present invention also provides novel polymer-drug conjugates, synthetic methods for making the conjugates, pharmaceutical compositions containing them and various uses of the conjugates.

[0273] The present invention further provides novel drug derivatives, synthetic methods for making the derivatives, pharmaceutical compositions containing them and various uses of the drug derivatives.

Definition/Terminology

[0274] Certain compounds of the present invention, and definitions of specific functional groups are also described in more detail herein. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, the entire contents of which are incorporated herein by reference. Furthermore, it will be appreciated by one of ordinary skill in the art that the synthetic methods, as described herein, utilize a variety of protecting groups.

[0275] The use of the articles “a”, “an”, and “the” in both the following description and claims are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising”, “having”, “including”, and “containing” are to be construed as open terms (i.e., meaning “including but not limited to”) unless otherwise noted. Additionally whenever “comprising” or another open-ended term is used in an embodiment, it is to be understood that the same embodiment can be more narrowly claimed using the intermediate term “consisting essentially of” or the closed term “consisting of:”

[0276] Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. A range used herein, unless otherwise specified, includes the two limits of the range. For example, the expressions “x being an integer between 1 and 6” and “x being an integer of 1 to 6” both mean “x being 1, 2, 3, 4, 5, or 6”.

[0277] “Protecting group”: as used herein, the term protecting group means that a particular functional moiety, e.g., O, S, or N, is temporarily blocked so that a reaction can be carried out selectively at another reactive site in a multifunctional compound. In preferred embodiments, a protecting group reacts selectively in good yield to give a protected substrate that is stable to the projected reactions; the protecting group must be selectively removed in good yield by readily available, preferably nontoxic reagents that do not attack the other functional groups; the protecting group forms an easily separable derivative (more preferably without the generation of new stereogenic centers); and the protecting group has a minimum of additional functionality to avoid further sites of reaction. As detailed herein, oxygen, sulfur, nitrogen and carbon protecting groups may be utilized. For example, in certain embodiments, certain exemplary oxygen protecting groups may be utilized. These oxygen protecting groups include, but are not limited to methyl ethers, substituted methyl ethers (e.g., MOM (methoxymethyl ether), MTM (methylthiomethyl ether), BOM (benzyloxymethyl ether), and PMBM (p-methoxybenzyloxymethyl ether)), substituted ethyl ethers, substituted benzyl ethers, silyl ethers (e.g., TMS (trimethylsilyl ether), TES (triethylsilylether), TIPS (triisopropylsilyl ether), TBDMS (t-butyldimethylsilyl ether), tribenzyl silyl ether, and TBDPS (t-butyldiphenyl silyl ether), esters (e.g., formate, acetate, benzoate (Bz), trifluoroacetate, and dichloroacetate), carbonates, cyclic acetals and ketals. In certain other exemplary embodiments, nitrogen protecting groups are utilized. Nitrogen protecting groups, as well as protection and deprotection methods are known in the art. Nitrogen protecting groups include, but are not limited to, carbamates (including methyl, ethyl and substituted ethyl carbamates (e.g., Troc), amides, cyclic imide derivatives, N-Alkyl and N-Aryl amines, imine derivatives, and enamine derivatives. In yet other embodiments, certain exemplary sulphur protecting groups may be utilized. The sulfur protecting groups include, but are not limited to those oxygen protecting group describe above as well as aliphatic carboxylic acid (e.g., acrylic acid), maleimide, vinyl sulfonyl, and optionally substituted maleic acid. Certain other exemplary protecting groups are detailed herein, however, it will be appreciated that the present invention is not intended to be limited to these protecting groups; rather, a variety of additional equivalent protecting groups can be readily identified using the above criteria and utilized in the present invention. Additionally, a variety of protecting groups are described in “Protective Groups in Organic Synthesis” Third Ed. Greene, T. W. and Wuts, P. G., Eds., John Wiley & Sons, New York: 1999, the entire contents of which are hereby incorporated by reference.

[0278] “Leaving group” refers to a molecular fragment that departs with a pair of electrons in heterolytic bond cleavage. Leaving groups can be anions or neutral molecules. Leaving groups include, but are not limited to halides such as Cl⁻, Br⁻, and I⁻, sulfonate esters, such as para-toluenesulfonate (“tosylate”, TsO⁻), and RC(O)O— in which R is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

[0279] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illustrate the invention and is not to be construed as a limitation on the scope of the claims unless explicitly otherwise claimed. No language in the specification is to be construed as indicating that any non-claimed element is essential to what is claimed.

[0280] “Antibody” refers to an immunoglobulin molecule of the class IgG including but not limited to IgG subclasses (IgG1, 2, 3 and 4) and class IgM which is able to specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, camelized single domain antibodies, intracellular antibodies (“intrabodies”), recombinant antibodies, anti-idiotypic antibodies, domain antibodies, linear antibody, multispecific antibody, antibody fragments, such as, Fv, Fab, Fab′, Fab′-SH, F(ab′)₂, single chain variable fragment antibodies (scFv), Fc, pFc′, scFvFc, disulfide Fv (dsfv), bispecific antibodies (bc-scFv) such as BiTE antibodies; camelid antibodies, resurfaced antibodies, humanized antibodies, fully human antibodies, single-domain antibody (sdAb, also known as NANOBODY®), chimeric antibodies, chimeric antibodies comprising at least one human constant region, dual-affinity antibodies such as, dual-affinity retargeting proteins (DART™), divalent (or bivalent) single-chain variable fragments (di-scFvs, bi-scFvs) including but not limited to minibodies, diabodies, triabodies or tribodies, tetrabodies, and the like, and multivalent antibodies. “Antibody fragment” refers to at least a portion of the variable region of the immunoglobulin molecule that binds to its target, i.e., the antigen-binding region. As used herein, the term “antibody” refers to both the full-length antibody and antibody fragments unless otherwise specified.

[0281] “Protein based recognition-molecule” or “PBRM” refers to a molecule that recognizes and binds to a cell surface marker or receptor such as, a transmembrane protein, surface immobilized protein, or protoglycan. Examples of PBRMs include but are not limited to, antibodies (e.g., Trastuzumab, Cetuximab, Rituximab, Bevacizumab, Epratuzumab, Veltuzumab, Labetuzumab) or peptides (LHRH receptor targeting peptides, EC-1 peptide), lipocalins, such as, for example, anticalins, proteins such as, for example, interferons, lymphokines, growth factors, colony stimulating factors, and the like, peptides or peptide mimics, and the like. The protein based recognition molecule, in addition to targeting the modified polymer conjugate to a specific cell, tissue or location, may also have certain therapeutic effect such as antiproliferative (cytostatic and/or cytotoxic) activity against a target cell or pathway. The protein based recognition molecule comprises or may be engineered to comprise at least one chemically reactive group such as, —COOH, primary amine, secondary amine —NHR, —SH, or a chemically reactive amino acid moiety or side chains such as, for example, tyrosine, histidine, cysteine, or lysine.

[0282] “Biocompatible” as used herein is intended to describe compounds that exert minimal destructive or host response effects while in contact with body fluids or living cells or tissues. Thus a biocompatible group, as used herein, refers to an aliphatic, cycloalkyl, heteroaliphatic, heterocycloalkyl, aryl, or heteroaryl moiety, which falls within the definition of the term biocompatible, as defined above and herein. The term “Biocompatibility” as used herein, is also taken to mean that the compounds exhibit minimal interactions with recognition proteins, e.g., naturally occurring antibodies, cell proteins, cells and other components of biological systems, unless such interactions are specifically desirable. Thus, substances and functional groups specifically intended to cause the above minimal interactions, e.g., drugs and prodrugs, are considered to be biocompatible. Preferably (with exception of compounds intended to be cytotoxic, such as, e.g., antineoplastic agents), compounds are “biocompatible” if their addition to normal cells in vitro, at concentrations similar to the intended systemic in vivo concentrations, results in less than or equal to 1% cell death during the time equivalent to the half-life of the compound in vivo (e.g., the period of time required for 50% of the compound administered in vivo to be eliminated/cleared), and their administration in vivo induces minimal and medically acceptable inflammation, foreign body reaction, immunotoxicity, chemical toxicity and/or other such adverse effects. In the above sentence, the term “normal cells” refers to cells that are not intended to be destroyed or otherwise significantly affected by the compound being tested.

[0283] “Biodegradable”: As used herein, “biodegradable” polymers are polymers that are susceptible to biological processing in vivo. As used herein, “biodegradable” compounds or moieties are those that, when taken up by cells, can be broken down by the lysosomal or other chemical machinery or by hydrolysis into components that the cells can either reuse or dispose of without significant toxic effect on the cells. The term “biocleavable” as used herein has the same meaning of “biodegradable”. The degradation fragments preferably induce little or no organ or cell overload or pathological processes caused by such overload or other adverse effects in vivo. Examples of biodegradation processes include enzymatic and non-enzymatic hydrolysis, oxidation and reduction. Suitable conditions for non-enzymatic hydrolysis of the biodegradable protein-polymer-drug conjugates (or their components, e.g., the biodegradable polymeric carrier and the linkers between the carrier and the antibody or the drug molecule) described herein, for example, include exposure of the biodegradable conjugates to water at a temperature and a pH of lysosomal intracellular compartment. Biodegradation of some protein-polymer-drug conjugates (or their components, e.g., the biodegradable polymeric carrier and the linkers between the carrier and the antibody or the drug molecule), can also be enhanced extracellularly, e.g., in low pH regions of the animal body, e.g., an inflamed area, in the close vicinity of activated macrophages or other cells releasing degradation facilitating factors. In certain preferred embodiments, the effective size of the polymer carrier at pH˜7.5 does not detectably change over 1 to 7 days, and remains within 50% of the original polymer size for at least several weeks. At pH˜5, on the other hand, the polymer carrier preferably detectably degrades over 1 to 5 days, and is completely transformed into low molecular weight fragments within a two-week to several-month time frame. Polymer integrity in such tests can be measured, for example, by size exclusion HPLC. Although faster degradation may be in some cases preferable, in general it may be more desirable that the polymer degrades in cells with the rate that does not exceed the rate of metabolization or excretion of polymer fragments by the cells. In preferred embodiments, the polymers and polymer biodegradation byproducts are biocompatible.

[0284] “Bioavailability”: The term “bioavailability” refers to the systemic availability (i.e., blood/plasma levels) of a given amount of drug or compound administered to a subject. Bioavailability is an absolute term that indicates measurement of both the time (rate) and total amount (extent) of drug or compound that reaches the general circulation from an administered dosage form.

[0285] “Hydrophilic”: The term “hydrophilic” as it relates to substituents on the polymer monomeric units does not essentially differ from the common meaning of this term in the art, and denotes chemical moieties which contain ionizable, polar, or polarizable atoms, or which otherwise may be solvated by water molecules. Thus a hydrophilic group, as used herein, refers to an aliphatic, cycloalkyl, heteroaliphatic, heterocycloalkyl, aryl or heteroaryl moiety, which falls within the definition of the term hydrophilic, as defined above. Examples of particular hydrophilic organic moieties which are suitable include, without limitation, aliphatic or heteroaliphatic groups comprising a chain of atoms in a range of between about one and twelve atoms, hydroxyl, hydroxyalkyl, amine, carboxyl, amide, carboxylic ester, thioester, aldehyde, nitryl, isonitryl, nitroso, hydroxylamine, mercaptoalkyl, heterocycle, carbamates, carboxylic acids and their salts, sulfonic acids and their salts, sulfonic acid esters, phosphoric acids and their salts, phosphate esters, polyglycol ethers, polyamines, polycarboxylates, polyesters and polythioesters. In preferred embodiments of the present invention, at least one of the polymer monomeric units include a carboxyl group (COOH), an aldehyde group (CHO), a methylol (CH₂OH) or a glycol (for example, CHOH—CH₂OH or CH—(CH₂OH)₂).

[0286] The term “hydrophilic” as it relates to the polymers of the invention generally does not differ from usage of this term in the art, and denotes polymers comprising hydrophilic functional groups as defined above. In a preferred embodiment, hydrophilic polymer is a water-soluble polymer. Hydrophilicity of the polymer can be directly measured through determination of hydration energy, or determined through investigation between two liquid phases, or by chromatography on solid phases with known hydrophobicity, such as, for example, C4 or C18.

[0287] “Polymeric Carrier”: The term polymeric carrier, as used herein, refers to a polymer or a modified polymer, which is suitable for covalently attaching to or can be covalently attached to one or more drug molecules with a designated linker and/or one or more PBRMs with a designated linker.

[0288] “Physiological conditions”: The phrase “physiological conditions”, as used herein, relates to the range of chemical (e.g., pH, ionic strength) and biochemical (e.g., enzyme concentrations) conditions likely to be encountered in the extracellular fluids of living tissues. For most normal tissues, the physiological pH ranges from about 7.0 to 7.4. Circulating blood plasma and normal interstitial liquid represent typical examples of normal physiological conditions.

[0289] “Polysaccharide”, “carbohydrate” or “oligosaccharide”: The terms “polysaccharide”, “carbohydrate”, or “oligosaccharide” are known in the art and refer, generally, to substances having chemical formula (CH₂O)_n, where generally n>2, and their derivatives. Carbohydrates are polyhydroxyaldehydes or polyhydroxyketones, or change to such substances on simple chemical transformations, such as hydrolysis, oxidation or reduction. Typically, carbohydrates are present in the form of cyclic acetals or ketals (such as, glucose or fructose). These cyclic units (monosaccharides) may be connected to each other to form molecules with few (oligosaccharides) or several (polysaccharides) monosaccharide units. Often, carbohydrates with well defined number, types and positioning of monosaccharide units are called oligosaccharides, whereas carbohydrates consisting of mixtures of molecules of variable numbers and/or positioning of monosaccharide units are called polysaccharides. The terms “polysaccharide”, “carbohydrate”, and “oligosaccharide”, are used herein interchangeably. A polysaccharide may include natural sugars (e.g., glucose, fructose, galactose, mannose, arabinose, ribose, and xylose) and/or derivatives of naturally occurring sugars (e.g., 2′-fluororibose, 2′-deoxyribose, and hexose).

[0290] “Small molecule”: As used herein, the term “small molecule” refers to molecules, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have a relatively low molecular weight. Preferred small molecules are biologically active in that they produce a local or systemic effect in animals, preferably mammals, more preferably humans. In certain preferred embodiments, the small molecule is a drug and the small molecule is referred to as “drug molecule” or “drug” or “therapeutic agent”. In certain embodiments, the drug molecule has MW less than or equal to about 5 kDa. In other embodiments, the drug molecule has MW less than or equal to about 1.5 kDa. In embodiments, the drug molecule is selected from vinca alkaloids, auristatins, tubulysins, duocarmycins, kinase inhibitors, MEK inhibitors, KSP inhibitors, and analogs thereof. Preferably, though not necessarily, the drug is one that has already been deemed safe and effective for use by an appropriate governmental agency or body, e.g., the FDA. For example, drugs for human use listed by the FDA under 21 C.F.R. §§330.5, 331 through 361, and 440 through 460; drugs for veterinary use listed by the FDA under 21 C.F.R. §§500 through 589, incorporated herein by reference, are all considered suitable for use with the present hydrophilic polymers.

[0291] Classes of drug molecules that can be used in the practice of the present invention include, but are not limited to, anti-cancer substances, radionuclides, vitamins, anti-AIDS substances, antibiotics, immunosuppressants, anti-viral substances, enzyme inhibitors, neurotoxins, opioids, hypnotics, anti-histamines, lubricants, tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinson substances, anti-spasmodics and muscle contractants including channel blockers, miotics and anti-cholinergics, anti-glaucoma compounds, anti-parasite and/or anti-protozoal compounds, modulators of cell-extracellular matrix interactions including cell growth inhibitors and anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNA or protein synthesis, anti-hypertensives, analgesics, anti-pyretics, steroidal and non-steroidal anti-inflammatory agents, anti-angiogenic factors, anti-secretory factors, anticoagulants and/or antithrombotic agents, local anesthetics, ophthalmics, prostaglandins, anti-depressants, anti-psychotic substances, anti-emetics, imaging agents. Many large molecules are also drugs.

[0292] A more complete, although not exhaustive, listing of classes and specific drugs suitable for use in the present invention may be found in “Pharmaceutical Substances: Syntheses, Patents, Applications” by Axel Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999 and the “Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals”, Edited by Susan Budavari et al., CRC Press, 1996, both of which are incorporated herein by reference. In preferred embodiments, the drug used in this invention is a therapeutic agent that has antiproliferative (cytostatic and/or cytotoxic) activity against a target cell or pathway. The drug may have a chemically reactive group such as, for example, —COOH, primary amine, secondary amine —NHR, —OH, —SH, —C(O)H, —C(O)R, —C(O)NHR^2b, C(S)OH, —S(O)₂OR^2b, —P(O)₂OR^2b, —CN, —NC or —ONO, in which R is an aliphatic, heteroaliphatic, carbocyclic or heterocycloalkyl moiety and R^2b is a hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocyclic moiety.

[0293] “Drug derivative” or “modified drug” or the like as used herein, refers to a compound that comprises the drug molecule intended to be delivered by the conjugate of the invention and a functional group capable of attaching the drug molecule to the polymeric carrier.

[0294] “Active form” as used herein refers to a form of a compound that exhibits intended pharmaceutical efficacy in vivo or in vitro. In particular, when a drug molecule intended to be delivered by the conjugate of the invention is released from the conjugate, the active form can be the drug itself or its derivatives, which exhibit the intended therapeutic properties. The release of the drug from the conjugate can be achieved by cleavage of a biodegradable bond of the linker which attaches the drug to the polymeric carrier. The active drug derivatives accordingly can comprise a portion of the linker.

[0295] “Diagnostic label”: As used herein, the term diagnostic label refers to an atom, group of atoms, moiety or functional group, a nanocrystal, or other discrete element of a composition of matter, that can be detected in vivo or ex vivo using analytical methods known in the art. When associated with a conjugate of the present invention, such diagnostic labels permit the monitoring of the conjugate in vivo. Alternatively or additionally, constructs and compositions that include diagnostic labels can be used to monitor biological functions or structures. Examples of diagnostic labels include, without limitation, labels that can be used in medical diagnostic procedures, such as, radioactive isotopes (radionuclides) for gamma scintigraphy and Positron Emission Tomography (PET), contrast agents for Magnetic Resonance Imaging (MRI) (for example paramagnetic atoms and superparamagnetic nanocrystals), contrast agents for computed tomography and other X-ray-based imaging methods, agents for ultrasound-based diagnostic methods (sonography), agents for neutron activation (e.g., boron, gadolinium), fluorophores for various optical procedures, and, in general moieties which can emit, reflect, absorb, scatter or otherwise affect electromagnetic fields or waves (e.g., gamma-rays, X-rays, radiowaves, microwaves, light), particles (e.g., alpha particles, electrons, positrons, neutrons, protons) or other forms of radiation, e.g., ultrasound.

[0296] “Aliphatic”: In general, the term aliphatic, as used herein, includes both saturated and unsaturated, straight chain (i.e., unbranched) or branched aliphatic hydrocarbons, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, “aliphatic” is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl moieties. Thus, as used herein, the term “alkyl” includes straight and branched alkyl groups. An analogous convention applies to other generic terms such as “alkenyl”, “alkynyl” and the like. In certain embodiments, as used herein, “lower alkyl” is used to indicate those alkyl groups (substituted, unsubstituted, branched or unbranched) having about 1-6 carbon atoms. “Substituted alkyl” refers to alkyl groups that are substituted with one or more functional groups. Substituents include, but are not limited to, any of the substituents mentioned below, i.e., the substituents recited below resulting in the formation of a stable compound.

[0297] “Alkenyl”: the term alkenyl denotes a monovalent group derived from a hydrocarbon moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom. “Substituted alkenyl” groups are substituted with one or more functional groups. Substituents include, but are not limited to, any of the substituents mentioned below, i.e., the substituents recited below resulting in the formation of a stable compound. Alkenyl groups include, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.

[0298] “Alkynyl”: the term alkynyl as used herein refers to a monovalent group derived from a hydrocarbon having at least one carbon-carbon triple bond by the removal of a single hydrogen atom. “Substituted alkenyl” groups are substituted with one or more functional groups. Substituents include, but are not limited to, any of the substituents mentioned below, i.e., the substituents recited below resulting in the formation of a stable compound. Representative alkynyl groups include ethynyl, 2-propynyl(propargyl), 1-propynyl, and the like.

[0299] In certain embodiments, the alkyl, alkenyl and alkynyl groups employed in the invention contain about 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain about 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain about 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain about 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain about 1-4 carbon atoms. Illustrative aliphatic groups thus include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, n-hexyl, sec-hexyl, moieties and the like, which again, may bear one or more substituents. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl(propargyl), 1-propynyl and the like.

[0300] “Alkylene” as used herein, the term alkylene by itself or part of another term refers to a saturated, branched or straight chain having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Alkylene radicals include, but are not limited to, methylene, 1,2, ethylene, 1,3-propyl, and the like. Suitable alkylenes include, but are not limited to methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, ocytylene, nonylene, decalene, and the like. The term “cycloalkylene” similarly refers to bivalent cycloalkyl. Cycloalkylene radicals include, but are not limited to, 1,1-cyclopentylene, 1,2-cyclopentylene, 1,1-cyclobutylene, 1,3-cyclobutylene, etc.

[0301] “Heteroaliphatic”: as used herein, the term heteroaliphatic refers to aliphatic moieties in which one or more carbon atoms in the main chain have been substituted with a heteroatom. Thus, a heteroaliphatic group refers to an aliphatic chain which contains one or more oxygen, sulfur, nitrogen, phosphorus or silicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moieties may be branched or linear unbranched. In certain embodiments, heteroaliphatic moieties are substituted (“substituted heteroaliphatic”) by independent replacement of one or more of the hydrogen atoms thereon with one or more moieties including, but not limited to aliphatic; heteroaliphatic; cycloalkyl; heterocycloalkyl; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; —N_O2; —CN; —C_F3; —C_H2C_F3; —CHC₁₂; —C_H2OH; —C_H2C_H2OH; —C_H2N_H2; —C_H2S_O2C_H3; - or -G^RG1 wherein G is —O—, —S—, —N^RG2—, —C(═O)—, —S(═O)—, —S_O2—, —C(═O)O—, —C(═O)N^RG2—, —OC(═O)—, —N^RG2C(═O)—, —OC(═O)O—, —OC(═O)N^RG2—, —N^RG2C(O)O—, —N^RG2C(═O)N^RG2—, —C(═S)—, —C(═S)S—, —SC(═S)—, —SC(═S)S—, —C(═N^RG2)—, —C(═N^RG2)O—, —C(═N^RG2)N^RG3—, —OC(═N^RG2)—, —N^RG2C(═N^RG3)—, —N^RG2S_O2—, —N^RG2S_O2N^RG3—, or —S_O2N^RG2—, wherein each occurrence of ^RG1, ^RG2 and ^RG3 independently includes, but is not limited to, hydrogen, halogen, or an aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkylaryl, or alkylheteroaryl moiety, each of which is optionally substituted. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein.

[0302] “Cycloalkyl”: as used herein, the term cycloalkyl refers to a saturated or unsaturated nonaromatic hydrocarbon mono- or multi-ring system having 3 to 30 carbon atoms (e.g., C₃-C₁₀). Suitable cycloalkyls include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cycloheptynyl, adamantyl, and the like.

[0303] “Heterocycloalkyl” as used herein refers to a saturated or unsaturated nonaromatic 3-8 membered monocyclic, 8-12 membered bicyclic, or 11-19 membered tricyclic ring system having one or more heteroatoms (such as O, N, S, or Se), unless specified otherwise. In certain embodiments, the term “heterocycloalkyl” refers to a non-aromatic 5-, 6-, 7- or 8-membered ring or a polycyclic group, including, but not limited to a bi- or tri-cyclic group comprising fused six-membered rings having between one and three heteroatoms independently selected from oxygen, sulfur and nitrogen, wherein (i) each 5-membered ring has 0 to 2 double bonds and each 6-membered ring has 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms may optionally be oxidized, (iii) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above heterocycloalkyl; rings may be fused to an aryl or heteroaryl ring. Examples of heterocycloalkyl groups include, but are not limited to, piperidinyl, piperazinyl, pyrrolidinyl, dioxanyl, tetrahydrofuranyl, tetrahydrothienyl, isoindolinyl, indolinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl, tetrahyrofuranyl, oxiranyl, azetidinyl, oxetanyl, thietanyl, 1,2,3,6-tetrahydropyridinyl, tetrahydro-2H-pyranyl, 3,6-dihydro-2H-pyranyl, morpholinyl, and the like.

[0304] “Aryl”: as used herein, refers to groups with aromaticity, including “conjugated,” or multicyclic systems with at least one aromatic ring and do not contain any heteroatom in the ring structure. Examples include phenyl, benzyl, 1,2,3,4-tetrahydronaphthalenyl, etc.

[0305] “Heteroaryl”: as used herein, refers to aryl groups, as defined above, except having from one to four heteroatoms in the ring structure, and may also be referred to as “aryl heterocycles” or “heteroaromatics.” As used herein, the term “heteroaryl” is intended to include a stable 5-, 6-, or 7-membered monocyclic or 7-, 8-, 9-, 10-, 11- or 12-membered bicyclic aromatic heterocyclic ring which consists of carbon atoms and one or more heteroatoms, e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or e.g., 1, 2, 3, 4, 5, or 6 heteroatoms, independently selected from the group consisting of nitrogen, oxygen and sulfur. The nitrogen atom may be substituted or unsubstituted (i.e., N or NR wherein R is H or other substituents, as defined). The nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., NO and S(O)_p, where p=1 or 2). It is to be noted that total number of S and O atoms in the aromatic heterocycle is not more than 1. Examples of heteroaryl include pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, tetrazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, tetrazolyl, pyridazinyl, quinazolinyl, dihydroquinazolyl, and tetrahydroquinazolyl and the like.

[0306] Furthermore, the terms “aryl” and “heteroaryl” include multicyclic aryl and heteroaryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, naphthrydine, indole, benzofuran, purine, benzofuran, deazapurine, indolizine.

[0307] In the case of multicyclic aromatic rings, only one of the rings needs to be aromatic (e.g., 2,3-dihydroindole), although all of the rings may be aromatic (e.g., quinoline). The second ring can also be fused or bridged.

[0308] “Carbocycle” or “carbocyclic moiety” as used herein, is intended to include any stable monocyclic, bicyclic or tricyclic ring having the specified number of carbons, any of which may be saturated, unsaturated, or aromatic. Carbocycle includes cycloalkyl and aryl. For example, a C₃-C₁₄ carbocycle is intended to include a monocyclic, bicyclic or tricyclic ring having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms. Examples of carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cycloheptenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclooctadienyl, fluorenyl, phenyl, naphthyl, indanyl, adamantyl and tetrahydronaphthyl. Bridged rings are also included in the definition of carbocycle, including, for example, [3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane and [2.2.2]bicyclooctane. A bridged ring occurs when one or more carbon atoms link two non-adjacent carbon atoms. In one embodiment, bridge rings are one or two carbon atoms. It is noted that a bridge always converts a monocyclic ring into a tricyclic ring. When a ring is bridged, the substituents recited for the ring may also be present on the bridge. Fused (e.g., naphthyl, tetrahydronaphthyl) and spiro rings are also included.

[0309] “Heterocycle” or “heterocyclic moiety” as used herein, includes any ring structure (saturated, unsaturated, or aromatic) which contains at least one ring heteroatom (e.g., N, O or S). Heterocycle includes heterocycloalkyl and heteroaryl. Examples of heterocycles include, but are not limited to, morpholine, pyrrolidine, tetrahydrothiophene, piperidine, piperazine and tetrahydrofuran.

[0310] Examples of heterocyclic groups include, but are not limited to, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazol5(4H)-one, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and xanthenyl. Multiple-ring heterocycle can include fused, bridged or spiro rings.

[0311] The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring (or the carbocyclic or heterocyclic group) can be substituted at one or more ring positions (e.g., the ring-forming carbon or heteroatom such as N) with such substituents as described above, for example, aliphatic; heteroaliphatic; cycloalkyl; heterocycloalkyl; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃; - or -Gr^G1 wherein G is —O—, —S—, —NR^G2—, —C(═O)—, —S(═O)—, —SO₂—, —C(═O)O—, —C(═O)NR^G2—, —OC(═O)—, —NR^G2C(═O)—, —OC(═O)O—, —OC(═O)NR^G2—, —NR^G2C(═O)O—, —NR^G2C(═O)NR^G2—, —C(═S)—, —C(═S)S—, —SC(═S)—, —SC(═S)S—, —C(═NR^G2)—, —C(═NR^G2)O—, —C(═NR^G2)NR^G3—, —OC(═NR^G2)—, —NR—, —NR—, —NR—, or —SO₂NR^G2—, wherein each occurrence of R^G1, R^G2 and R^G3 independently includes, but is not limited to, hydrogen, halogen, or an aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkylaryl, or alkylheteroaryl moiety, each of which is optionally substituted. Aryl and heteroaryl groups can also be fused or bridged with cycloalkyl or heterocyclic rings, which are not aromatic so as to form a multicyclic system (e.g., tetralin, methylenedioxyphenyl).

[0312] “Alkoxy” (or “alkyloxy”): as used herein, the term alkoxy (or alkyloxy) refers to an alkyl group, as previously defined, attached to the parent molecular moiety through an oxygen atom (“alkoxy”). In certain embodiments, the alkyl group contains about 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl group contains about 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl group contains about 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl group contains about 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl group contains about 1-4 aliphatic carbon atoms. Examples of alkoxy groups, include but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy and n-hexoxy.

[0313] “Aryloxy”: as used herein, the term aryloxy refers to an aryl group, as defined herein, attached to the parent molecular moiety through an oxygen atom. Examples of aryloxy groups include but are not limited to phenoxy and napthyloxy.

[0314] “Heteroaryloxy”: as used herein, the term heteroaryloxy refers to a heteroaryl group, as defined herein, attached to the parent molecular moiety through an oxygen atom. Examples of heteroaryloxy groups include but are not limited to, quinolyloxy and isoquinolizinyloxy.

[0315] “Amine”: the term amine refers to a group having the structure —N(R)₂ wherein each occurrence of R is independently hydrogen, or an aliphatic or heteroaliphatic moiety, or the R groups, taken together, may form a heterocyclic moiety. In certain instances, an amine group can be charged (protonized) or quarternized, e.g., —HN⁺(R)₂ or —N⁺(R)₃.

[0316] “Alkylamino”: as used herein, the term alkylamino refers to a group having the structure —NHR′ wherein R′ is alkyl, as defined herein. The term “aminoalkyl” refers to a group having the structure NH₂R′—, wherein R′ is alkyl, as defined herein. In certain embodiments, the alkyl group contains about 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl group contains about 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain about 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl group contains about 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl group contains about 1-4 aliphatic carbon atoms. Examples of alkylamino include, but are not limited to, methylamino, ethylamino, iso-propylamino and the like.

[0317] “Alkylthio” (or “thioalkyl”) means an alkyl group as defined herein with the indicated number of carbon atoms attached through a sulfur atom. C_1-6 alkylthio, is intended to include C₁, C₂, C₃, C₄, C₅, and C₆ alkylthio groups. C_1-8 alkylthio, is intended to include C₁, C₂, C₃, C₄, C₅, C₆, C₇, and C₈ alkylthio groups. The thioalkyl groups can be substituted with groups such as alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, carboxyacid, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl alkylaryl, or an aryl or heteroaryl moieties.

[0318] “Thiocarbonyl” or “thiocarboxy” includes compounds and moieties which contain a carbon connected with a double bond to a sulfur atom.

[0319] “Thioether” includes moieties which contain a sulfur atom bonded to two carbon atoms or heteroatoms. Examples of thioethers include, but are not limited to alkthioalkyls, alkthioalkenyls and alkthioalkynyls. The term “alkthioalkyls” include moieties with an alkyl, alkenyl or alkynyl group bonded to a sulfur atom which is bonded to an alkyl group. Similarly, the term “alkthioalkenyls” refers to moieties wherein an alkyl, alkenyl or alkynyl group is bonded to a sulfur atom which is covalently bonded to an alkenyl group; and alkthioalkynyls” refers to moieties wherein an alkyl, alkenyl or alkynyl group is bonded to a sulfur atom which is covalently bonded to an alkynyl group.

[0320] “Arylthio” (or “thioaryl”) means an aryl group as defined herein with the indicated number of carbon atoms attached through a sulfur atom.

[0321] “Carboxylic acid” as used herein refers to a compound comprising a group of formula —CO₂H.

[0322] “Dicarboxylic acid” refers to a compound comprising two groups of formula —CO₂H.

[0323] “Halo, halide and halogen”: The terms halo, halide and halogen as used herein refer to an atom selected from fluorine, chlorine, bromine, and iodine.

[0324] “Methylol”: The term methylol as used herein refers to an alcohol group of the structure —CH₂OH.

[0325] “Hydroxyalkyl”: As used herein, the term hydroxyalkyl refers to an alkyl group, as defined above, bearing at least one OH group.

[0326] “Mercaptoalkyl”: The term mercaptoalkyl as used therein refers to an alkyl group, as defined above, bearing at least one SH group.

[0327] “Acyl” includes moieties that contain the acyl radical (—C(O)—) or a carbonyl group. “Substituted acyl” includes acyl groups where one or more of the hydrogen atoms are replaced by, for example, alkyl groups, alkynyl groups, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aryl or heteroaryl moiety.

[0328] “Hydrocarbon”: The term hydrocarbon, as used herein, refers to any chemical group comprising hydrogen and carbon. The hydrocarbon may be substituted or unsubstituted. The hydrocarbon may be unsaturated, saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic. Illustrative hydrocarbons include, for example, methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl, methoxy, diethylamino, heterocycloalkyl, aryl, heteroaryl, thioalkyl, and the like. As would be known to one skilled in this art, all valencies must be satisfied in making any substitutions.

[0329] “Alkylaryl” as used herein refers to an aryl group substituted with one or more alkyl groups (e.g., methylphenyl).

[0330] “Alkylarylamino” as used herein refers to —NR^G4R^G5, wherein R^G4 is alkyl, as defined herein, and R^G5 is an aryl, as defined herein, or at least one of R^G4 and R^G5 is an alkylaryl as defined herein.

[0331] “Substituted”: The terms substituted, whether preceded by the term “optionally” or not, and substituent, as used herein, refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. Examples of substituents include, but are not limited to aliphatic; heteroaliphatic; cycloalkyl; heterocycloalkyl; aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃; - or -GR^G1 wherein G is —O—, —S—, —NR^G2—, —C(═O)—, —S(═O)—, —SO₂—, —C(═O)O—, —C(═O)NR^G2—, —OC(═O)—, —NR^G2C(═O)—, —OC(═O)O—, —OC(═O)NR^G2—, —NR^G2C(═O)O—, —NR^G2C(═O)NR^G2—, —C(═S)—, —C(═S)S—, —SC(═S)—, —SC(═S)S—, —C(═NR^G2)—, —C(═NR^G2)O—, —C(═NR^G2)NR^G3—, —OC(═NR^G2)—, —NR^G2C(═NR^G3)—, —NR^G2SO₂—, —NR^G2SO₂NR^G3—, or —SO₂NR^G2—, wherein each occurrence of R^G1, R^G2 and R^G3 independently includes, but is not limited to, hydrogen, halogen, or an aliphatic, heteroaliphatic, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkylaryl, or alkylheteroaryl moiety, each of which is optionally substituted. Additional examples of generally applicable substituents are illustrated by the specific embodiments shown in the Examples that are described herein.

[0332] The following are more general terms used throughout the present application:

[0333] “Animal”: The term animal, as used herein, refers to humans as well as non-human animals, at any stage of development, including, for example, mammals, birds, reptiles, amphibians, fish, worms and single cells. Cell cultures and live tissue samples are considered to be pluralities of animals. Preferably, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig). An animal may be a transgenic animal or a human clone. The term “subject” encompasses animals.

[0334] “Efficient amount”: In general, as it refers to an active agent or drug delivery device, the term “efficient amount” refers to the amount necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the efficient amount of an agent or device may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the encapsulating matrix, the target tissue, etc. For example, the efficient amount of microparticles containing an antigen to be delivered to immunize an individual is the amount that results in an immune response sufficient to prevent infection with an organism having the administered antigen.

[0335] “Natural amino acid” as used herein refers to any one of the common, naturally occurring L-amino acids found in naturally occurring proteins: glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), lysine (Lys), arginine (Arg), histidine (His), proline (Pro), serine (Ser), threonine (Thr), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), glutamine (Gln), cysteine (Cys) and methionine (Met).

[0336] “Unnatural amino acid” as used herein refers to any amino acid which is not a natural amino acid. This includes, for example, amino acids that comprise α-, β-, ω-, D-, L-amino acyl residues. More generally, the unnatural amino acid comprises a residue of the general formula

[0337]
wherein the side chain R is other than the amino acid side chains occurring in nature. Exemplary unnatural amino acids, include, but are not limited to, sarcosine (N-methylglycine), citrulline (cit), homocitrulline, β-ureidoalanine, thiocitrulline, hydroxyproline, allothreonine, pipecolic acid (homoproline), α-aminoisobutyric acid, tert-butylglycine, tert-butylalanine, allo-isoleucine, norleucine, α-methylleucine, cyclohexylglycine, β-cyclohexylalanine, β-cyclopentylalanine, α-methylproline, phenylglycine, α-methylphenylalanine and homophenylalanine.

[0338] “Amino acyl”: More generally, the term amino acyl, as used herein, encompasses natural amino acid and unnatural amino acids.

[0339] “Polyamide”: refers to homo- or hetero-polymers of natural amino acid and unnatural amino acids. Illustrative homo-polymers include, but are not limited to, poly-lysine, poly-arginine, poly-γ-glutaric acid, and the like. Illustrative hetero-polymers include, but are not limited to, polymers comprising peptides fragments selected from peptidases, lysozymes, metalloproteinases, and the like.

[0340] “PHF” refers to poly(1-hydroxymethylethylene hydroxymethyl-formal).

[0341] As used herein, the terms “polymer unit”, “monomeric unit”, “monomer”, “monomer unit”, “unit” all refer to a repeatable structural unit in a polymer.

[0342] As used herein, “molecular weight” or “MW” of a polymer or polymeric carrier/scaffold or polymer conjugates refers to the weight average molecular weight unless otherwise specified.

[0343] The present invention is intended to include all isotopes of atoms occurring in the present compounds. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.

[0344] The present invention is intended to include all isomers of the compound, which refers to and includes, optical isomers, and tautomeric isomers, where optical isomers include enantiomers and diastereomers, chiral isomers and non-chiral isomers, and the optical isomers include isolated optical isomers as well as mixtures of optical isomers including racemic and non-racemic mixtures; where an isomer may be in isolated form or in a mixture with one or more other isomers.

Polymeric Carriers

[0345] In certain exemplary embodiments, the conjugates of the invention find use in biomedical applications, such as drug delivery and tissue engineering, and the carrier is biocompatible and biodegradable. In certain embodiments, the carrier is a soluble polymer, nanoparticle, gel, liposome, micelle, suture, implant, etc. In certain embodiments, the term “soluble polymer” encompasses biodegradable biocompatible polymer such as a polyal (e.g., hydrophilic polyacetal or polyketal). In certain other embodiments, the carrier is a fully synthetic, semi-synthetic or naturally-occurring polymer. In certain other embodiments, the carrier is hydrophilic.

[0346] In certain exemplary embodiments, the carriers used in the present invention are biodegradable biocompatible polyals comprising at least one hydrolysable bond in each monomer unit positioned within the main chain. This ensures that the degradation process (via hydrolysis/cleavage of the monomer units) will result in fragmentation of the polymer conjugate to the monomeric components (i.e., degradation), and confers to the polymer conjugates of the invention their biodegradable properties. The properties (e.g., solubility, bioadhesivity and hydrophilicity) of biodegradable biocompatible polymer conjugates can be modified by subsequent substitution of additional hydrophilic or hydrophobic groups. Examples of biodegradable biocompatible polymers suitable for practicing the invention can be found inter alia in U.S. Pat. Nos. 5,811,510; 5,863,990; 5,958,398; 7,838,619 and 7,790,150; and U.S. Publication No. 2006/0058512; each of the above listed patent documents is incorporated herein by reference in its entirety. Guidance on the significance, preparation, and applications of this type of polymers may be found in the above-cited documents. In certain embodiments, it is anticipated that the present invention will be particularly useful in combination with the above-referenced patent documents, as well as U.S. Pat. Nos. 5,582,172 and 6,822,086, each of the above listed patent documents is incorporated herein by reference in its entirety.

[0347] The conjugates of this invention are hydrophilic, hydrolysable and comprise drug molecules (e.g., vinca alkaloids or derivatives, non-natural camptothecin compounds or derivatives, auristatins, tubulysins, duocarmycins, PI3 kinases, MEK inhibitors, KSP inhibitors, and analogs thereof) and antibodies (e.g., Trastuzumab, Cetuximab, Rituximab, Bevacizumab, Epratuzumab, Veltuzumab, Labetuzumab) or peptides (LHRH receptor targeting peptides, EC-1 peptide) covalently attached to the polymer carrier via linkages that contain one or more biodegradable bonds. Thus, in certain exemplary embodiments, carriers suitable for practicing the present invention are polyals having at least one acetal/ketal oxygen atom in each monomer unit positioned within the main chain. As discussed above, this ensures that the degradation process (via hydrolysiscleavage of the polymer acetal/ketal groups) will result in fragmentation of the polyal conjugate to low molecular weight components (i.e., degradation).

[0348] In certain embodiments, biodegradable biocompatible polymer carriers, used for preparation of polymer conjugates of the invention, are naturally occurring polysaccharides, glycopolysaccharides, and synthetic polymers of polyglycoside, polyacetal, polyamide, polyether, and polyester origin and products of their oxidation, fictionalization, modification, cross-linking, and conjugation.

[0349] In certain other embodiments, the carrier is a hydrophilic biodegradable polymer selected from the group consisting of carbohydrates, glycopolysaccharides, glycolipids, glycoconjugates, polyacetals, polyketals, and derivatives thereof.

[0350] In certain exemplary embodiments, the carrier is a naturally occurring linear and/or branched biodegradable biocompatible homopolysaccharide selected from the group consisting of cellulose, amylose, dextran, levan, fucoidan, carraginan, inulin, pectin, amylopectin, glycogen and lixenan.

[0351] In certain other exemplary embodiments, the carrier is a naturally occurring linear and branched biodegradable biocompatible heteropolysaccharide selected from the group consisting of agarose, hyluronan, chondroitinsulfate, dermatansulfate, keratansulfate, alginic acid and heparin.

[0352] In yet other exemplary embodiments, the polymeric carrier comprises a copolymer of a polyacetal/polyketal and a hydrophilic polymer selected from the group consisting of polyacrylates, polyvinyl polymers, polyesters, polyorthoesters, polyamides, polypeptides, and derivatives thereof.

[0353] In yet another embodiment, the polymeric carrier is dextrin that is produced by the hydrolysis of a starch obtained from various natural products such as, for example, wheat, rice, maize and tapioca. Depending on the structure of the starch starting material each dextrin comprises a unique distribution of α-1,4 linkages and α-1,6 linkages. Since the rate of biodegradability of α-1,6 linkages is typically less than that for α-1,4 linkages, preferably the percentage of α-1,6 linkages is less than 10% and more preferably less than 5%. In one embodiment the molecular weight of the dextrin is in the range of about 1 kDa to about 200 kDa, more preferably from about 2 kDa to about 55 kDa.

[0354] In certain embodiments, the carrier comprises polysaccharides activated by selective oxidation of cyclic vicinal diols of 1,2-, 1,4-, 1,6-, and 2,6-pyranosides, and 1,2-, 1,5-, 1,6-furanosides, or by oxidation of lateral 6-hydroxy and 5,6-diol containing polysaccharides prior to conjugation with drug molecules or PBRMs.

[0355] In still other embodiments, the polymeric carrier comprises a biodegradable biocompatible polyacetal wherein at least a subset of the polyacetal repeat structural units have the following chemical structure:

[0356]

[0357] wherein for each occurrence of the n bracketed structure, one of R₁ and R₂ is hydrogen, and the other is a biocompatible group and includes a carbon atom covalently attached to C¹; R^x is a carbon atom covalently attached to C²; n″ is an integer; each occurrence of R₃, R₄, R₅ and R₆ is a biocompatible group and is independently hydrogen or an organic moiety; and for each occurrence of the bracketed structure n, at least one of R₁, R₂, R₃, R₄, R₅ and R₆ comprises a functional group suitable for coupling. In certain embodiments, the functional group is a hydroxyl moiety.

[0358] In one embodiment, the polymeric carrier comprises activated hydrophilic biodegradable biocompatible polymers comprising from 0.1% to 100% polyacetal moieties whose backbone is represented by the following chemical structure:
(—CH₂—CHR₇—O—CHR₈—O—)_o,
wherein:

[0359] R₇ and R₈ are independently hydrogen, hydroxyl, hydroxy alkyl (e.g., —CH₂OH, —CH(OH)—CH(OH), —CHO, —CH(OH)—CHO or -carbonyl; and

[0360] o is an integer from 20 to 2000.

[0361] In yet other embodiments, the polymeric carrier comprises a biodegradable biocompatible polyketal wherein at least a subset of the polyketal repeatable structural units have the following chemical structure:

[0362]
wherein each occurrence of R₁ and R₂ is a biocompatible group and R^x, R₃, R₄, R₅, R₆ and are as defined herein.

[0363] In certain embodiments, the ketal units are monomers of Formula (IIa) or (IIb):

[0364]

[0365] Biodegradable, biocompatible polyketal polymers and their methods of making have been described in U.S. Pat. Nos. 5,811,510, 7,790,150 and 7,838,619, which are hereby incorporated by reference in their entireties.

[0366] In one embodiment, the polymeric carrier can be obtained from partially oxidized dextran (β1→6)-D-glucose) followed by reduction. In this embodiment, the polymer comprises a random mixture of the unmodified dextran (A), partially oxidized dextran acetal units (B) and exhaustively dextran acetal units (C) of the following structures:

[0367]

[0368] In another embodiment, the polymeric carrier comprises unmodified acetal units, i.e., polyacetal segments. In some embodiments, the polyacetals can be derived from exhaustively oxidized dextran followed by reduction. These polymers have been described in references, see, e.g., U.S. Pat. No. 5,811,510, which is hereby incorporated by reference for its description of polyacetals at column 2, line 65 to column 8, line 55 and their synthesis at column 10, line 45 to column 11, line 14. In one embodiment, the unmodified polyacetal polymer is a poly(hydroxymethylethylene hydroxymethyl formal) polymer (PHF).

[0369] In addition to poly(hydroxymethylethylene hydroxymethyl formal) polymers, the backbone of the polymeric carrier can also comprise co-polymers of poly(hydroxymethylethylene hydroxymethyl formal) blocks and other acetal or non-acetal monomers or polymers. For example, polyethylene glycol polymers are useful as a stealth agent in the polymer backbone because they can decrease interactions between polymer side chains of the appended functional groups. Such groups can also be useful in limiting interactions such as between serum factors and the modified polymer. Other stealth agent monomers for inclusion in the polymer backbone include, for example, ethyleneimine, methacrylic acid, acrylamide, glutamic acid, and combinations thereof.

[0370] The acetal or ketal units are present in the modified polymer in an amount effective to promote biocompatibility. The unmodified acetal or ketal unit can be described as a “stealth agent” that provides biocompatibility and solubility to the modified polymers. In addition, conjugation to a polyacetal or polyketal polymer can modify the susceptibility to metabolism and degradation of the moieties attached to it, and influence biodistribution, clearance and degradation.

[0371] The unmodified acetal units are monomers of Formula (III):

[0372]

[0373] The molar fraction, n, of unmodified polyacetal units is the molar fraction available to promote biocompatibility, solubility and increase half-life, based on the total number of polymer units in the modified polymer. The molar fraction n may be the minimal fraction of unmodified monomer acetal units needed to provide biocompatibility, solubility, stability, or a particular half-life, or can be some larger fraction. The most desirable degree of cytotoxicity is substantially none, i.e., the modified polymer is substantially inert to the subject. However, as is understood by those of ordinary skill in the art, some degree of cytotoxicity can be tolerated depending on the severity of disease or symptom being treated, the efficacy of the treatment, the type and degree of immune response, and like considerations.

[0374] In one embodiment, the modified polymer backbone comprises units of Formula (IV):

[0375]
wherein X′ indicates the substituent for the hydroxyl group of the polymer backbone. As shown in Formula (IV) and the other formulae described herein, each polyacetal unit has a single hydroxyl group attached to the glycerol moiety of the unit and an X′ group (or another substituent such as -L^D-D) attached to the glycolaldehyde moiety of the unit. This is for convenience only and it should be construed that the polymer 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 -L^D-D) attached to the glycolaldehyde moiety of the units and those having a single X′ group (or another substituent such as -L^D-D) attached to the glycerol moiety of the units as well as units having two X′ groups (or other substituents such as -L^D-D) with one attached to the glycolaldehyde moiety and the other attached to the glycerol moiety of the units.

[0376] In one embodiment, biodegradable biocompatible polyals suitable for practicing the present invention have a molecular weight of between about 0.5 and about 300 kDa. In a preferred embodiment of the present invention, the biodegradable biocompatible polyals have a molecular weight of between about 1 and about 300 kDa (e.g., between about 1 and about 200 kDa, between about 2 and about 300 kDa, between about 2 and about 200 kDa, between about 5 and about 100 kDa, between about 10 and about 70 kDa, between about 20 and about 50 kDa, between about 20 and about 300 kDa, between about 40 and about 150 kDa, between about 50 and about 100 kDa, between about 2 and about 40 kDa, between about 6 and about 20 kDa, or between about 8 and about 15 kDa).

[0377] In one embodiment, the biodegradable biocompatible polyals suitable for practicing the present invention are modified before conjugating with a drug or a PBRM. For example, the polyals may contain subunits of linkers L^D or L^P, such as —C(═O)—X—(CH₂)_v—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 derivatives thereof. Unless otherwise specified, reference numbers in Tables A through E below correspond to the Example numbers described herein; the term “ND” means not determined; and X is CH₂, O, or NH.

[0378]

TABLE A
Ref #	Polymer Scaffold
Ex 2
Ex 1
X = CH2 Ex 5 X = NH Ex 74
X = CH2, Ex 12
X = CH2 Ex 71
X = CH2 Ex 68

Therapeutic Agents

[0379] In certain embodiments, the therapeutic agent is a small molecule having a molecular weight preferably ≦about 5 kDa, more preferably ≦about 4 kDa, more preferably ≦about 3 kDa, most preferably ≦about 1.5 kDa or ≦about 1 kDa.

[0380] In certain embodiments, the therapeutic agent has an IC₅₀ of about less than 1 nM.

[0381] In another embodiment, the therapeutic agent has an IC₅₀ of about greater than 1 nM, for example, the therapeutic agent has an IC₅₀ of about 1 to 50 nM.

[0382] Some therapeutic agents having an IC₅₀ of greater than about 1 nM (e.g., “less potent drugs”) are unsuitable for conjugation with a PBRM using art-recognized conjugation techniques. Without wishing to be bound by theory, such therapeutic agents have a potency that is insufficient for use in targeted PBRM-drug conjugates using conventional techniques as sufficient copies of the drug (i.e., more than 8) cannot be conjugated using art-recognized techniques without resulting in diminished pharmacokinetic and physiochemical properties of the conjugate. However sufficiently high loadings of these less potent drugs can be achieved using the conjugation strategies described herein thereby resulting in high loadings of the therapeutic agent while maintaining the desirable pharmacokinetic and physiochemical properties. Thus, the invention also relates to a PBRM-drug conjugate which includes a PBRM, PHF and at least eight therapeutic agent moieties, wherein the therapeutic agent has an IC₅₀ of greater than about 1 nM.

[0383] In certain embodiments, about 0.1 to about 25% monomers comprise a therapeutic agent, more preferably about 0.5 to about 20%, more preferably about 1 to about 15%, and even more preferably about 2 to about 10%.

[0384] The small molecule therapeutic agents used in this invention (e.g., antiproliferative (cytotoxic and cytostatic) agents capable of being linked to a polymer carrier) include cytotoxic compounds (e.g., broad spectrum), angiogenesis inhibitors, cell cycle progression inhibitors, PI3K/m-TOR/AKT pathway inhibitors, MAPK signaling pathway inhibitors, kinase inhibitors, protein chaperones inhibitors, HDAC inhibitors, PARP inhibitors, Wnt/Hedgehog signaling pathway inhibitors and RNA polymerase inhibitors.

[0385] Broad spectrum cytotoxins include, but are not limited to, DNA-binding or alkylating drugs, microtubule stabilizing and destabilizing agents, platinum compounds, and topoisomerase I inhibitors.

[0386] Exemplary DNA-binding or alkylating drugs include, CC-1065 and its analogs, anthracyclines (doxorubicin, epirubicin, idarubicin, daunorubicin) and its analogs, alkylating agents, such as calicheamicins, dactinomycines, mitromycines, pyrrolobenzodiazepines, and the like.

[0387] Exemplary CC-1065 analogs include duocarmycin SA, duocarmycin C1, duocarmycin C2, duocarmycin B2, DU-86, KW-2189, bizelesin, seco-adozelesin, and those described in U.S. Pat. Nos. 5,475,092; 5,595,499; 5,846,545; 6,534,660; 6,586,618; 6,756,397 and 7,049,316. Doxorubicin and its analogs include those described in U.S. Pat. No. 6,630,579. Calicheamicins include those described in U.S. Pat. Nos. 5,714,586 and 5,739,116. Duocarmycins include those described in U.S. Pat. Nos. 5,070,092; 5,101,038; 5,187,186; 6,548,530; 6,660,742; and 7,553,816 B2; and Li et al., Tet Letts., 50:2932-2935 (2009). Pyrrolobenzodiazepines include those described in Denny, Exp. Opin. Ther. Patents., 10(4):459-474 (2000).

[0388] Exemplary microtubule stabilizing and destabilizing agents include taxane compounds, such as paclitaxel, docetaxel; maytansinoids, auristatins and analogs thereof, tubulysin A and B derivatives, vinca alkaloid derivatives, epothilones and cryptophycins.

[0389] Exemplary maytansinoids or maytansinoid analogs include maytansinol and maytansinol analogs, maytansine or DM-1 and DM-4 are those described in U.S. Pat. Nos. 5,208,020; 5,416,064; 6,333,410; 6,441,163; 6,716,821; RE39,151 and 7,276,497. In certain embodiments, the cytotoxic agent is a maytansinoid, another group of anti-tubulin agents (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res. 52:127-131), maytansinoids or maytansinoid analogs. Examples of suitable maytansinoids include maytansinol and maytansinol analogs. Suitable maytansinoids are disclosed in U.S. Pat. Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545.

[0390] Exemplary auristatins include auristatin E (also known as a derivative of dolastatin-10), auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F and dolastatin. Suitable auristatins are also described in U.S. Publication Nos. 2003/0083263, 2011/0020343, and 2011/0070248; PCT Application Publication Nos. WO 09/117,531, WO 2005/081711, WO 04/010957; WO 02/088172 and WO01/24763, and U.S. Pat. Nos. 7,498,298; 6,884,869; 6,323,315; 6,239,104; 6,124,431; 6,034,065; 5,780,588; 5,767,237; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414, the disclosures of which are incorporated herein by reference in their entirety.

[0391] Exemplary tubulysin compounds include compounds described in U.S. Pat. Nos. 7,816,377; 7,776,814; 7,754,885; U.S. Publication Nos. 2011/0021568; 2010/004784; 2010/0048490; 2010/00240701; 2008/0176958; and PCT Application Nos. WO 98/13375; WO 2004/005269; WO 2008/138561; WO 2009/002993; WO 2009/055562; WO 2009/012958; WO 2009/026177; WO 2009/134279; WO 2010/033733; WO 2010/034724; WO 2011/017249; WO 2011/057805; the disclosures of which are incorporated by reference herein in their entirety.

[0392] Exemplary vinca alkaloids include vincristine, vinblastine, vindesine, and navelbine (vinorelbine). Suitable Vinca alkaloids that can be used in the present invention are also disclosed in U.S. Publication Nos. 2002/0103136 and 2010/0305149, and in U.S. Pat. No. 7,303,749 B 1, the disclosures of which are incorporated herein by reference in their entirety.

[0393] Exemplary epothilone compounds include epothilone A, B, C, D, E and F, and derivatives thereof. Suitable epothilone compounds and derivatives thereof are described, for example, in U.S. Pat. Nos. 6,956,036; 6,989,450; 6,121,029; 6,117,659; 6,096,757; 6,043,372; 5,969,145; and 5,886,026; and WO 97/19086; WO 98/08849; WO 98/22461; WO 98/25929; WO 98/38192; WO 99/01124; WO 99/02514; WO 99/03848; WO 99/07692; WO 99/27890; and WO 99/28324; the disclosures of which are incorporated herein by reference in their entirety.

[0394] Exemplary cryptophycin compounds are described in U.S. Pat. Nos. 6,680,311 and 6,747,021.

[0395] Exemplary platinum compounds include cisplatin (PLATINOL®), carboplatin (PARAPLATIN®), oxaliplatin (ELOXATINE®), iproplatin, ormaplatin, and tetraplatin.

[0396] Exemplary topoisomerase I inhibitors include camptothecin, camptothecin, derivatives, camptothecin analogs and non-natural camptothecins, such as, for example, CPT-11 (irinotecan), SN-38, topotecan, 9-aminocamptothecin, rubitecan, gimatecan, karenitecin, silatecan, lurtotecan, exatecan, diflomotecan, belotecan, lurtotecan and 539625. Other camptothecin compounds that can be used in the present invention include those described in, for example, J. Med. Chem., 29:2358-2363 (1986); J. Med. Chem., 23:554 (1980); J. Med. Chem., 30:1774 (1987).

[0397] Angiogenesis inhibitors include, but are not limited, MetAP2 inhibitors, VEGF inhibitors, PIGF inhibitors, VGFR inhibitors, PDGFR inhibitors, MetAP2 inhibitors. Exemplary VGFR and PDGFR inhibitors include sorafenib (Nexavar), sunitinib (Sutent) and vatalanib. Exemplary MetAP2 inhibitors include fumagillol analogs, meaning any compound that includes the fumagillin core structure, including fumagillamine, that inhibits the ability of MetAP-2 to remove NH₂-terminal methionines from proteins as described in Rodeschini et al., J. Org. Chem., 69, 357-373, 2004 and Liu, et al., Science 282, 1324-1327, 1998. Non limiting examples of “fumagillol analogs” are disclosed in J. Org. Chem., 69, 357, 2004; J. Org. Chem., 70, 6870, 2005; European Patent Application 0 354 787; J. Med. Chem., 49, 5645, 2006; Bioorg. Med. Chem., 11, 5051, 2003; Bioorg. Med. Chem., 14, 91, 2004; Tet. Lett. 40, 4797, 1999; WO99/61432; U.S. Pat. Nos. 6,603,812; 5,789,405; 5,767,293; 6,566,541; and 6,207,704.

[0398] Exemplary cell cycle progression inhibitors include CDK inhibitors such as, for example, BMS-387032 and PD0332991; Rho-kinase inhibitors such as, for example GSK429286; checkpoint kinase inhibitors such as, for example, AZD7762; aurora kinase inhibitors such as, for example, AZD1152, MLN8054 and MLN8237; PLK inhibitors such as, for example, BI 2536, BI6727 (Volasertib), GSK461364, ON-01910 (Estybon); and KSP inhibitors such as, for example, SB 743921, SB 715992 (ispinesib), MK-0731, AZD8477, AZ3146 and ARRY-520.

[0399] Exemplary PI3K/m-TOR/AKT signaling pathway inhibitors include phosphoinositide 3-kinase (PI3K) inhibitors, GSK-3 inhibitors, ATM inhibitors, DNA-PK inhibitors and PDK-1 inhibitors.

[0400] Exemplary PI3 kinases are disclosed in U.S. Pat. No. 6,608,053, and include BEZ235, BGT226, BKM120, CAL101, CAL263, demethoxyviridin, GDC-0941, GSK615, IC87114, LY294002, Palomid 529, perifosine, PF-04691502, PX-866, SAR245408, SAR245409, SF1126, Wortmannin, XL147 and XL765.

[0401] Exemplary AKT inhibitors include, but are not limited to AT7867.

[0402] Exemplary MAPK signaling pathway inhibitors include MEK, Ras, JNK, B-Raf and p38 MAPK inhibitors.

[0403] Exemplary MEK inhibitors are disclosed in U.S. Pat. No. 7,517,994 and include GDC-0973, GSK1120212, MSC1936369B, AS703026, R05126766 and R04987655, PD0325901, AZD6244, AZD 8330 and GDC-0973.

[0404] Exemplary B-raf inhibitors include CDC-0879, PLX-4032, and SB590885.

[0405] Exemplary B p38 MAPK inhibitors include BIRB 796, LY2228820 and SB 202190.

[0406] Receptor tyrosine kinases (RTK) are cell surface receptors which are often associated with signaling pathways stimulating uncontrolled proliferation of cancer cells and neoangiogenesis. Many RTKs, which over express or have mutations leading to constitutive activation of the receptor, have been identified, including, but not limited to, VEGFR, EGFR, FGFR, PDGFR, EphR and RET receptor family receptors. Exemplary specific RTK targets include ErbB2, FLT-3, c-Kit, and c-Met.

[0407] Exemplary inhibitors of ErbB2 receptor (EGFR family) include but not limited to AEE788 (NVP-AEE 788), BIBW2992, (Afatinib), Lapatinib, Erlotinib (Tarceva), and Gefitinib (Iressa).

[0408] Exemplary RTK inhibitors targeting more then one signaling pathway (multitargeted kinase inhibitors) include AP24534 (Ponatinib) that targets FGFR, FLT-3, VEGFR-PDGFR and Bcr-Abl receptors; ABT-869 (Linifanib) that 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, and c-Kit receptors; Sunitinib (Sutent) that targets VEGFR, PDGFR, KIT, FLT-3 and CSF-IR; Sorafenib (Nexavar) and Vatalanib that target VEGFR, PDGFR as well as intracellular serine/threonine kinases in the Raf/Mek/Erk pathway.

[0409] Exemplary protein chaperon inhibitors include HSP90 inhibitors. Exemplary HSP90 inhibitors include 17AAG derivatives, BIIB021, BIIB028, SNX-5422, NVP-AUY-922 and KW-2478.

[0410] Exemplary HDAC inhibitors include Belinostat (PXD101), CUDC-101, Droxinostat, ITF2357 (Givinostat, Gavinostat), JNJ-26481585, LAQ824 (NVP-LAQ824, Dacinostat), LBH-589 (Panobinostat), MC1568, MGCD0103 (Mocetinostat), MS-275 (Entinostat), PCI-24781, Pyroxamide (NSC 696085), SB939, Trichostatin A and Vorinostat (SAHA).

[0411] Exemplary PARP inhibitors include iniparib (BSI 201), olaparib (AZD-2281), ABT-888 (Veliparib), AG014699, CEP 9722, MK 4827, KU-0059436 (AZD2281), LT-673, 3-aminobenzamide, A-966492, and AZD2461.

[0412] Exemplary Wnt/Hedgehog signaling pathway inhibitors include vismodegib (RG3616/GDC-0449), cyclopamine (11-deoxojervine) (Hedgehog pathway inhibitors) and XAV-939 (Wnt pathway inhibitor)

[0413] Exemplary RNA polymerase inhibitors include amatoxins. Exemplary amatoxins include α-amanitins, β-amanitins, γ-amanitins, ε-amanitins, amanullin, amanullic acid, amaninamide, amanin, and proamanullin.

[0414] In one embodiment the drug of the invention is a non-natural camptothecin compound, vinca alkaloid, kinase inhibitor (e.g., PI3 kinase inhibitor (GDC-0941 and PI-103)), MEK inhibitor, KSP inhibitor, RNA polymerse inhibitor, PARP inhibitor, docetaxel, paclitaxel, doxorubicin, duocarmycin, tubulysin, auristatin or a platinum compound. In specific embodiments, the drug is a derivative of SN-38, vindesine, vinblastine, PI-103, AZD 8330, auristatin E, auristatin F, a duocarmycin compound, tubulysin compound, or ARRY-520.

[0415] In another embodiment, the drug used in the invention is a combination of two or more drugs, such as, for example, PI3 kinases and MEK inhibitors; broad spectrum cytotoxic compounds and platinum compounds; PARP inhibitors and platinum compounds; broad spectrum cytotoxic compounds and PARP inhibitors.

[0416] In one embodiment, the Vinca alkaloid is a compound of Formula (V):

[0417]
wherein:

[0418] R₁₄ is hydrogen, —C(O)—C_1-3 alkyl or —C(O)-chloro substituted C_1-3 alkyl;

[0419] R₁₅ is hydrogen, —CH₃ or —CHO;

[0420] when R₁₇ and R₁₈ are taken independently, R₁₈ is hydrogen, and either R₁₆ or R₁₇ is ethyl and the other is hydroxyl;

[0421] when R₁₇ and R₁₈ are taken together with the carbon to which they are attached to form an oxiran ring, R₁₆ is ethyl;

[0422] R₁₉ is hydrogen, OH, amino group, alkyl amino or —[C(R₂₀R₂₁)]_a—R₂₂;

[0423] each of R₂₀ and R₂₁ independently is hydrogen, C_1-6 alkyl, C_6-10 aryl, hydroxylated C_6-10 aryl, polyhydroxylated C_6-10 aryl, 5 to 12-membered heterocycle, C_3-8 cycloalkyl, hydroxylated C_3-8 cycloalkyl, polyhydroxylated C_3-8 cycloalkyl or a side chain of a natural or unnatural amino acid;

[0424] R₂₂ is —OH, —NH₂, —COOH, —R₈₂—C(O)(CH₂)_c—C(H)(R₂₃)—N(H)(R₂₃), —R₈₂—C(O)(CH₂)_d—(OCH₂—CH₂)_f—N(H)(R₂₃) or —R₈₂—(C(O)—CH(X²)—NH)_d—R₇₇;

[0425] each R₂₃ independently is hydrogen, C_1-6 alkyl, C_6-10 aryl, C_3-8 cycloalkyl, —COOH, or —COO—C_1-6 alkyl;

[0426] X² is a side chain of a natural or unnatural amino acid;

[0427] R₇₇ is hydrogen or X² and NR₇₇ form a nitrogen containing heterocyclic moiety;

[0428] R₈₂ is —NH or oxygen;

[0429] a is an integer from 1 to 6;

[0430] c is an integer from 0 to 3;

[0431] d is an integer from 1 to 3; and

[0432] f is an integer from 1 to 12.

[0433] Further examples of Vinca alkaloids are described in US 2010/0305149 and US 2002/0103136.

[0434] In one embodiment the Vinca alkaloid of Formula (V) is a compound of Formula (VI):

[0435]
wherein:

[0436] R₄₀ is hydrogen, —OH, —NH₂, or any of the following structures:

[0437]
wherein:

[0438] a is an integer from 1 to 6; and

[0439] c is an integer from 0 to 3.

[0440] In one embodiment, R₄₀ is

[0441]

[0442] In another embodiment, non-natural camptothecin is a compound of Formula (VII):

[0443]
wherein:

[0444] R₂₄ is —H, —Cl, —F, —OH or alkyl; or R₂₄ and R₂₅, may be taken together to form an optionally substituted five- or six-membered ring;

[0445] R₂₅ is —H, —F, —OH, —CH₃, —CH═N—O-t-Butyl, —CH₂CH₂Si(CH₃)₃, —Si((CH₃)₂)-t-butyl, —O—C(O)—R₂₉;

[0446] R₂₉ is —NH₂, —R₂₈—C_1-6 alkyl-R₂₂, 5 to 12-membered heterocycloalkyl, R₂₈—C_5-12 heterocycloalkyl-C_1-6 alkyl-R₂₂ or —R₂₈—C_1-6 alkyl-C_6-12 aryl-C_1-6 alkyl-R₂₂; or R₂₉ is R₄₇ as defined herein;

[0447] R₂₆ is —H, —CH₂—N(CH₃)₂, NH₂, or NO₂;

[0448] R₂₇ is ethyl, N-methyl piperidine, cycloalkyl, —CH₂CH₂NHCH(CH₃)₂, or —N-4-methylcyclohexylamine;

[0449] R₇₉ is —H or —C(O)—R₂₈—[C(R₂₀R₂₁)]_a—R₂₂;

[0450] each of R₂₀ and R₂₁ independently is hydrogen, C_1-6 alkyl, C_6-10 aryl, hydroxylated C_6-10 aryl, polyhydroxylated C_6-10 aryl, 5 to 12-membered heterocycle, C_3-8 cycloalkyl, hydroxylated C_3-8 cycloalkyl, polyhydroxylated C_3-8 cycloalkyl or a side chain of a natural or unnatural amino acid;

[0451] R₂₂ is —OH, —NH₂, —COOH, —R₈₂—C(O)(CH₂)_c—C(H)(R₂₃)—N(H)(R₂₃), —R₈₂—C(O)(CH₂)_d—(OCH₂—CH₂)_f—N(H)(R₂₃), or —R₈₂—(C(O)—CH(X²)—NH)_d—R₇₇;

[0452] each R₂₃ independently is hydrogen, C_1-6 alkyl, C_6-10 aryl, C_3-8 cycloalkyl, —COOH, or —COO—C_1-6 alkyl;

[0453] X² is a side chain of a natural or unnatural amino acid;

[0454] R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cyclic compound;

[0455] R₈₂ is —NH or oxygen;

[0456] or R₂₆ and R₂₇ when taken together with the two carbon atoms to which they attach and the third carbon atom connecting the two carbon atoms form an optionally substituted six-membered ring;

[0457] R₂₈ is absent, NH or oxygen;

[0458] a is an integer from 1 to 6;

[0459] c is an integer from 0 to 3;

[0460] d is an integer from 1 to 3;

[0461] f is an integer from 1 to 12;

[0462] u is an integer 0 or 1;

[0463] w is an integer 0 or 1; and

[0464] with the proviso that the compound of Formula (VII) must contain at least one of R₂₉ and R₇₉.

[0465] In one embodiment the non-natural camptothecin compound of Formula (VII) is a compound of Formula (VIII) or Formula (XXV):

[0466]
wherein R₃₀ is —NH₂, —R₂₈—C_1-6 alkyl-R₂₂, 5 to 12-membered heterocycloalkyl, R₂₈—C_5-12 heterocycloalkyl-C_1-6 alkyl-R₂₂ or —R₂₈—C_1-6 alkyl-C_6-12 aryl-C_1-6 alkyl-R₂₂;

[0467] R₂₈ is absent, NH or oxygen;

[0468] R₂₂ is —OH, —NH₂, —COOH, —R₈₂—C(O)(CH₂)_c—C(H)(R₂₃)—N(H)(R₂₃), —R₈₂—C(O)(CH₂)_d—(OCH₂—CH₂)_f—N(H)(R₂₃) or —R₈₂—(C(O)—CH(X²)—NH)_d—R₇₇;

[0469] each R₂₃ independently is hydrogen, C_1-6 alkyl, C_6-10 aryl, C_3-8 cycloalkyl, —COOH, or —COO—C_1-6 alkyl;

[0470] X² is a side chain of a natural or unnatural amino acid;

[0471] R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cyclic compound;

[0472] R₈₂ is —NH or oxygen;

[0473] c is an integer from 0 to 3;

[0474] d is an integer from 1 to 3; and

[0475] f is an integer from 1 to 12.

[0476] In some embodiments R₃₀ is any one of the following structures:

[0477]
wherein:

[0478] a is an integer from 1 to 6;

[0479] c is an integer from 0 to 3; and

[0480] g is an integer from 2 to 6.

[0481] In another embodiment the PI3 kinase is a compound of Formula (IX):

[0482]
wherein

[0483] R₄₇ is an amino group, —R₉—[C(R₂₀R₂₁)]_a—R₁₀, —R₉—C_5-12 heterocycloalkyl-C_1-6 alkyl-R₁₀, 5 to 12-membered heterocycloalkyl, or —R₉—C_6-10 aryl;

[0484] each of R₂₀ and R₂₁ independently is hydrogen, C_1-6 alkyl, C_6-10 aryl, hydroxylated C_6-10 aryl, polyhydroxylated C_6-10 aryl, 5 to 12-membered heterocycle, C_3-8 cycloalkyl, hydroxylated C_3-8 cycloalkyl, polyhydroxylated C_3-8 cycloalkyl or a side chain of a natural or unnatural amino acid;

[0485] R₁₀ is —OH, —NHR₈₃, —N—(R₈₃)R₁₁, —COOH, —R₈₂—C(O)(CH₂)_c—C(H)(R₂₃)—N(H)(R₂₃), —R₈₂—C(O)(CH₂)_d—(OCH₂—CH₂)_f—N(H)(R₂₃), —R₈₂—(C(O)—CH(X²)—NH)_d—R₇₇ or —R₈₂—C(O)—[C(R₂₀R₂₁)]_a—R₈₂-R₈₃ or

[0486]

[0487] each R₂₃ independently is hydrogen, C_1-6 alkyl, C_6-10 aryl, C_3-8 cycloalkyl, —COOH, or —COO—C_1-6 alkyl;

[0488] X² is a side chain of a natural or unnatural amino acid;

[0489] R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cyclic compound;

[0490] R₈₂ is —NH or oxygen;

[0491] R₉ is absent, N—(R₈₃) or oxygen;

[0492] R₈₃ is hydrogen or CH₃;

[0493] R₁₁ is:

[0494]

[0495] each R₁₂ independently is hydrogen, chloride, —CH₃ or —OCH₃;

[0496] R₁₃ is hydrogen or —C(O)—(CH₂)_d—(O—CH₂—CH₂)_f—NH₂;

[0497] R₈₂ is —NH or oxygen

[0498] X₄ is the side chain of lysine, arginine, citrulline, alanine or glycine;

[0499] X₅ is the side chain of phenylalanine, valine, leucine, isoleucine or tryptophan;

[0500] each of X₆ and X₇ is independently the side chain of glycine, alanine, serine, valine or proline;

[0501] a is an integer from 1 to 6;

[0502] c is an integer from 0 to 3;

[0503] d is an integer from 1 to 3;

[0504] f is an integer from 1 to 12; and

[0505] each u independently is an integer 0 or 1;

[0506] or R₁₁ is —Y_u—W_q—R₈₈,

wherein:

[0507] Y is any one of the following structures:

[0508]

[0509] in each of which the terminal NR₈₃ group of Y is proximal to R₈₈;

[0510] R₈₃ is hydrogen or CH₃;

[0511] each W is an amino acid unit;

[0512] each R₁₂′ independently is halogen, —C_1-8 alkyl, —O—C_1-8 alkyl, nitro or cyano;

[0513] R₈₈ is hydrogen or —C(O)—(CH₂)_ff—(NH—C(O))_aa-E_j(CH₂)_bb—R₈₅

[0514] R₈₅ is NH₂, OH or

[0515]

[0516] E is —CH₂— or —CH₂CH₂O—;

[0517] u is an integer 0 or 1;

[0518] q is an integer from 0 to 12;

[0519] aa is an integer 0 or 1;

[0520] bb is an integer 0 or 2;

[0521] ff is an integer from 0 to 10;

[0522] h is an integer from 0 to 4;

[0523] j is an integer from 0 to 12; and

[0524] when E is —CH₂—, bb is 0 and j is an integer from 0 to 10; and when E is —CH₂CH₂—O—, bb is 2 and j is an integer from 1 to 12;

[0525] or R₁₁ is

[0526]
wherein:

[0527] R₈₃ is hydrogen or CH₃;

[0528] R₈₄ is C_1-6 alkyl or C_6-10 aryl;

[0529] each R₁₂′ independently is halogen, —C_1-8 alkyl, —O—C_1-8 alkyl, nitro or cyano;

[0530] h is an integer from 0 to 4; and

[0531] u is an integer 0 or 1.

[0532] In some embodiments, R₁₁ is:

[0533]
wherein:

[0534] each R₁₂′ independently is chloride, —CH₃ or —OCH₃;

[0535] R₈₈ is hydrogen or —C(O)—(CH₂)_ff—(CH₂—CH₂O)_j—CH₂—CH₂—NH₂;

[0536] R₈₂ is —NH or oxygen

[0537] X₄ is the side chain of lysine, arginine, citrulline, alanine or glycine;

[0538] X₅ is the side chain of phenylalanine, valine, leucine, isoleucine or tryptophan;

[0539] each of X₆ and X₇ is independently the side chain of glycine, alanine, serine, valine or proline;

[0540] ff is an integer from 1 to 3;

[0541] j is an integer from 1 to 12

[0542] h is an integer from 0 to 4; and

[0543] each u independently is an integer 0 or 1.

[0544] In some embodiments,

[0545]
is citrulline-valine; lysine-phenylalanine; citrulline-phenylalanine; citrulline-leucine; citrulline-valine-glycine-glycine; glycine-phenylalanine-glycine-glycine; valine; proline; leucine or isoleucine.

[0546] In another embodiment, R₁₁ is any one of the following structures:

[0547]

[0548] In some embodiments R₄₇ is any one of the following structures:

[0549]
wherein:

[0550] a is an integer from 1 to 6;

[0551] c is an integer from 0 to 3; and

[0552] g is an integer from 2 to 6.

[0553] In another embodiment the auristatin is a compound of Formula (X):

[0554]
wherein:

[0555] each of R₃₁ and R₃₂ independently is hydrogen or C_1-8 alkyl and at most one of R₃₁ and R₃₂ is hydrogen;

[0556] R₃₃ is hydrogen, C_1-8 alkyl, C_3-8 carbocycle, C_6-10 aryl, C_1-8 alkyl-C_6-10 aryl, X¹—(C_3-8 carbocycle), C_3-8 heterocycle or X¹—(C_3-8 heterocycle);

[0557] R₃₄ is hydrogen, C_1-8 alkyl, C_3-8 carbocycle, C_6-10 aryl, X¹—C_6-10 aryl, X¹—(C_3-8 carbocycle), C_3-8 heterocycle or X¹—(C_3-8 heterocycle);

[0558] R₃₅ is hydrogen or methyl;

[0559] or R₃₄ and R₃₅, together with the carbon atom to which they attach form a carbocyclic ring having the formula —(CR₅₅R₄₁)_b— wherein each of R₅₅ and R₄₁ independently is hydrogen or C_1-8 alkyl and b is an integer from 3 to 7;

[0560] R₃₆ is hydrogen or C_1-8 alkyl;

[0561] R₃₇ is hydrogen, C_1-8 alkyl, C_3-8 carbocycle, C_6-10 aryl, —X¹—C_6-10 aryl, —X¹—(C_3-8 carbocycle), C_3-8 heterocycle or —X¹—(C_3-8 heterocycle);

[0562] each R₃₈ independently is hydrogen, OH, C_1-8 alkyl, C_3-8 carbocycle or O—(C_1-8 alkyl);

[0563] R₅₃ is: or R₅₄

[0564]

[0565] R₃₉ is H, C_1-8 alkyl, C_6-10 aryl, —X¹—C_6-10 aryl, C_3-8 carbocycle, C_3-8 heterocycle, —X¹—C_3-8 heterocycle, —C_1-8 alkylene-NH₂, or (CH₂)₂SCH₃

[0566] each X¹ independently is C_1-10 alkylene or C_3-10 cycloalkylene;

[0567] R₄₄ is hydrogen or C_1-8 alkyl;

[0568] R₄₅ is X³—R₄₂ or NH—R₁₉;

[0569] X³ is O or S;

[0570] R₁₉ is hydrogen, OH, amino group, alkyl amino or —[C(R₂₀R₂₁)]_a—R₂₂;

[0571] R₄₂ is an amino group, C_1-6 alkyl amino or —[C(R₂₀R₂₁)]_a—R₂₂;

[0572] each of R₂₀ and R₂₁ independently is hydrogen, C_1-6 alkyl, C_6-10 aryl, hydroxylated C_6-10 aryl, polyhydroxylated C_6-10 aryl, 5 to 12-membered heterocycle, C_3-8 cycloalkyl, hydroxylated C_3-8 cycloalkyl, polyhydroxylated C_3-8 cycloalkyl or a side chain of a natural or unnatural amino acid;

[0573] R₂₂ is —OH, —NHR₂₃, —COOH, —R₈₂—C(O)(CH₂)_c—C(H)(R₂₃)—N(H)(R₂₃), —R₈₂—C(O)(CH₂)_d—(OCH₂—CH₂)_f—N(H)(R₂₃) or —R₈₂—(C(O)—CH(X²)—NH)_d—R₇₇;

[0574] each R₂₃ independently is hydrogen, C_1-6 alkyl, C_6-10 aryl, C_3-8 cycloalkyl, —COOH, or —COO—C_1-6 alkyl;

[0575] X² is a side chain of a natural or unnatural amino acid;

[0576] R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cyclic compound;

[0577] R₈₂ is —NH or oxygen;

[0578] R₅₄ is —C(R₅₆)₂—C(R₅₆)₂—C_6-10 aryl, —C(R₅₆)₂—C(R₅₆)₂—C_3-8 heterocycle or —C(R₅₆)₂—C(R₅₆)₂—C_3-8 carbocycle;

[0579] R₅₆ is independently selected from H, OH, C_1-8 alkyl, C_3-8 carbocycle, —O—C_1-8 alkyl, —O—C(O)—R₂₉ and —O—R₂₃—O—C_1-6 alkyl-NH₂;

[0580] R₂₉ is an amino group, 5 to 12-membered heterocycloalkyl, —R₂₈—C_1-6 alkyl-R₂₂, R₂₈—C_5-12 heterocycloalkyl-C_1-6 alkyl-R₂₂, —[C(R₂₀R₂₁)]_a—R₂₂, or —R₂₈—C_1-6 alkyl-C_6-12 aryl-C_1-6 alkyl-R₂₂; or R₂₉ is R₄₇ as defined herein;

[0581] R₂₈ is absent, NH or oxygen;

[0582] a is an integer from 1 to 6;

[0583] c is an integer from 0 to 3;

[0584] d is an integer from 1 to 3; and

[0585] f is an integer from 1 to 12.

[0586] In some embodiments, in the auristatin compound of Formula (X):

[0587] R₃₉ is benzyl or

[0588]
and

[0589] R₄₄ is hydrogen.

[0590] In another embodiment the auristatin is a compound of Formula (Xa):

[0591]
wherein:

[0592] R₃₃ through R₃₈, and R₄₄ are as defined herein,

[0593] one of R₃₁ and R₃₂ is hydrogen or C_1-8 alkyl and the other is:

[0594]

[0595] wherein:

[0596] R₈₃ is hydrogen or CH₃;

[0597] R₈₄ is C_1-6 alkyl or C_6-10 aryl;

[0598] each R₁₂′ independently is halogen, —C_1-8 alkyl, —O—C_1-8 alkyl, nitro or cyano;

[0599] h is an integer from 0 to 4; and

[0600] u is an integer 0 or 1;

[0601] R₅₃ is:

[0602]
or R₅₄

[0603] R₃₉ is H, C_1-8 alkyl, C_6-10 aryl, —X¹—C_6-10 aryl, C_3-8 carbocycle, C_3-8 heterocycle, —X¹—C_3-8 heterocycle, —C_1-8 alkylene-NH₂, or (CH₂)₂SCH₃,

[0604] each X¹ independently is C_1-10 alkylene or C_3-10 cycloalkylene;

[0605] R₄₅ is X³—R₄₂ or NH—R₁₉;

[0606] X³ is O or S;

[0607] R₁₉ is hydrogen, OH, amino group, alkyl amino or —[C(R₂₀R₂₁)]_a—R₂₂;

[0608] R₄₂ is H, an amino group, C_1-6 alkyl amino or —[C(R₂₀R₂₁)]_a—R₂₂;

[0609] each of R₂₀ and R₂₁ independently is hydrogen, C_1-6 alkyl, C_6-10 aryl, hydroxylated C_6-10 aryl, polyhydroxylated C_6-10 aryl, 5 to 12-membered heterocycle, C_3-8 cycloalkyl, hydroxylated C_3-8 cycloalkyl, polyhydroxylated C_3-8 cycloalkyl or a side chain of a natural or unnatural amino acid;

[0610] R₂₂ is —OH, —NHR₂₃, —COOH, —R₈₂—C(O)(CH₂)_c—C(H)(R₂₃)—N(H)(R₂₃), —R₈₂—C(O)(CH₂)_d—(OCH₂—CH₂)_f—N(H)(R₂₃) or —R₈₂—(C(O)—CH(X²)—NH)_d—R₇₇;

[0611] each R₂₃ independently is hydrogen, C_1-6 alkyl, C_6-10 aryl, C_3-8 cycloalkyl, —COOH, or —COO—C_1-6 alkyl;

[0612] X² is a side chain of a natural or unnatural amino acid;

[0613] R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cyclic compound;

[0614] R₈₂ is —NH or oxygen;

[0615] R₅₄ is —C(R₅₆)₂—C(R₅₆)₂—C_6-10 aryl, —C(R₅₆)₂—C(R₅₆)₂—C_3-8 heterocycle or —C(R₅₆)₂—C(R₅₆)₂—C_3-8 carbocycle;

[0616] R₅₆ is independently selected from H, OH, C_1-8 alkyl, C_3-8 carbocycle, —O—C_1-8 alkyl, —O—C(O)—R₂₉ and —O—R₂₃—O—C_1-6 alkyl-NH₂;

[0617] R₂₉ is an amino group, 5 to 12-membered heterocycloalkyl, —R₂₈—C_1-6 alkyl-R₂₂, R₂₈—C_5-12 heterocycloalkyl-C_1-6 alkyl-R₂₂, —[C(R₂₀R₂₁)]_a—R₂₂, or —R₂₈—C_1-6 alkyl-C_6-12 aryl-C_1-6 alkyl-R₂₂; or R₂₉ is R₄₇ as defined herein;

[0618] R₂₈ is absent, NH or oxygen;

[0619] a is an integer from 1 to 6;

[0620] c is an integer from 0 to 3;

[0621] d is an integer from 1 to 3; and

[0622] f is an integer from 1 to 12.

[0623] In one embodiment, the auristatin compound of Formula (Xa) is a compound of Formula (XIa) or Formula (XIb):

[0624]
wherein:

[0625] R₉₂ is:

[0626]
and

[0627] R₈₃ is hydrogen or CH₃.

[0628] In one embodiment the auristatin of Formula (X) is a compound of Formula (XI), Formula (XII) or Formula (XIII):

[0629] wherein the compound of Formula (XI) is:

[0630]

[0631] wherein R₄₂ is —CH₃ or any one of the following structures:

[0632]
wherein:

[0633] a is an integer from 1 to 6; and

[0634] c is an integer from 0 to 3;

[0635] wherein the compound of Formula (XII) is:

[0636]

[0637] wherein R₄₀ is hydrogen, —OH, —NH₂, or any of the following structures:

[0638]
wherein:

[0639] a is an integer from 1 to 6; and

[0640] c is an integer from 0 to 3;

[0641] wherein the compound of Formula (XIII) is:

[0642]

[0643] wherein R₂₉ is an amino group, 5 to 12-membered heterocycloalkyl, —R₂₈—C_1-6 alkyl-R₂₂, R₂₈—C_5-12 heterocycloalkyl-C_1-6 alkyl-R₂₂, —R₂₈—[C(R₂₀R₂₁)]_a—R₂₂, or —R₂₈—C_1-6 alkyl-C_6-12 aryl-C_1-6 alkyl-R₂₂; or R₂₉ is R₄₇ as defined herein;

[0644] each of R₂₀ and R₂₁ independently is hydrogen, C_1-6 alkyl, C_6-10 aryl, hydroxylated C_6-10 aryl, polyhydroxylated C_6-10 aryl, 5 to 12-membered heterocycle, C_3-8 cycloalkyl, hydroxylated C_3-8 cycloalkyl, polyhydroxylated C_3-8 cycloalkyl or a side chain of a natural or unnatural amino acid;

[0645] R₂₂ is —OH, —NHR₂₃, —COOH, —R₈₂—C(O)(CH₂)_c—C(H)(R₂₃)—N(H)(R₂₃), —R₈₂—C(O)(CH₂)_d—(OCH₂—CH₂)_f—N(H)(R₂₃) or —R₈₂—(C(O)—CH(X²)—NH)_d—R₇₇;

[0646] each R₂₃ independently is hydrogen, C_1-6 alkyl, C_6-10 aryl, C_3-8 cycloalkyl, —COOH, or —COO—C_1-6 alkyl;

[0647] X² is a side chain of a natural or unnatural amino acid;

[0648] R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cyclic compound;

[0649] R₈₂ is —NH or oxygen;

[0650] R₂₈ is absent, NH or oxygen;

[0651] a is an integer from 1 to 6;

[0652] c is an integer from 0 to 3;

[0653] d is an integer from 1 to 3; and

[0654] f is an integer from 1 to 12.

[0655] In one embodiment, in Formula (XII), R₄₀ is

[0656]

[0657] In one embodiment in the compound of Formula (XIII), R₂₉ is —NH₂, 5 membered heterocycloalkyl, —R₂₈—C_1-6 alkyl-R₂₂, R₂₈—C_5-12 heterocycloalkyl-C_1-6 alkyl-R₂₂ or —R₂₈—C_1-6 alkyl-C_6-12 aryl-C_1-6 alkyl-R₂₂; or R₂₉ is R₄₇ as defined herein;

[0658] R₂₈ is absent, NH or oxygen;

[0659] R₂₂ is —OH, —NHR₂₃, —COOH, —R₈₂—C(O)(CH₂)_c—C(H)(R₂₃)—N(H)(R₂₃), —R₈₂—C(O)(CH₂)_d—(OCH₂—CH₂)_f—N(H)(R₂₃) or —R₈₂—(C(O)—CH(X²)—NH)_d—R₇₇;

[0660] each R₂₃ independently is hydrogen, C_1-6 alkyl, C_6-10 aryl, C_3-8 cycloalkyl, —COOH, or —COO—C_1-6 alkyl;

[0661] X² is a side chain of a natural or unnatural amino acid;

[0662] R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cyclic compound;

[0663] R₈₂ is —NH or oxygen;

[0664] c is an integer from 0 to 3;

[0665] d is an integer from 1 to 3; and

[0666] f is an integer from 1 to 12.

[0667] In yet another embodiment, R₂₉ is any one of the following structures:

[0668]
wherein:

[0669] a is an integer from 1 to 6;

[0670] c is an integer from 0 to 3; and

[0671] g is an integer from 2 to 6.

[0672] In one embodiment, the MEK inhibitor is a compound of Formula (XIV):

[0673]

[0674] wherein R₄₃ is H or —R₄₆-R₄₇;

[0675] each of R₂₀ and R₂₁ independently is hydrogen, C_1-6 alkyl, C_6-10 aryl, hydroxylated C_6-10 aryl, polyhydroxylated C_6-10 aryl, 5 to 12-membered heterocycle, C_3-8 cycloalkyl, hydroxylated C_3-8 cycloalkyl, polyhydroxylated C_3-8 cycloalkyl or a side chain of a natural or unnatural amino acid;

[0676] R₂₂ is —OH, —NH₂, —COOH, —R₈₂—C(O)(CH₂), C(H)(R₂₃)—N(H)(R₂₃), —R₈₂—C(O)(CH₂)_d—(OCH₂—CH₂)_f—N(H)(R₂₃) or —R₈₂—(C(O)—CH(X²)—NH)_d—R₇₇;

[0677] each R₂₃ independently is hydrogen, C_1-6 alkyl, C_6-10 aryl, C_3-8 cycloalkyl, —COOH, or —COO—C_1-6 alkyl;

[0678] X² is a side chain of a natural or unnatural amino acid;

[0679] R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cyclic compound;

[0680] R₈₂ is —NH or oxygen;

[0681] R₄₆ is —C(O)—; —C(O)—O—, —C(O)—NH—, or absent;

[0682] R₄₇ is as defined herein;

[0683] a is an integer from 1 to 6;

[0684] c is an integer from 0 to 3;

[0685] d is an integer from 1 to 3; and

[0686] f is an integer from 1 to 12.

[0687] Further examples of the MEK inhibitor are disclosed in U.S. Pat. No. 7,517,994 B2.

[0688] In some embodiments R₄₃ is —C(O)—(CH₂)_a—NH₂, or —C(O)—C(H)(CH₃)—(CH₂)_c—NH₂; in which a is an integer from 1 to 6; and c is an integer from 0 to 3.

[0689] In another embodiment, the duocarmycin compound is a compound of Formula (XV):

[0690]
wherein:

[0691] R₄₇ is as defined herein;

[0692] R₄₈ is hydrogen, —COOC_1-6 alkyl, —COOH, —NH₂ or —CH₃;

[0693] R₄₉ is Cl, Br or —OH;

[0694] R₅₀ is hydrogen, —OCH₃,

[0695]

[0696] each of R₅₁ and R₅₂ independently is hydrogen or —OCH₃; and

[0697] ring AA is either a phenyl or pyrrolyl ring.

[0698] Further examples of duocarmycin compounds are disclosed in U.S. Pat. No. 7,553,816.

[0699] In one embodiment the duocarmycin compound of Formula (XV) is a compound of Formula (XVI), (XVII), (XVIII) or (XIX):

[0700]

[0701] wherein:

[0702] R₄₉ is Cl, Br or —OH; and

[0703] R₄₇ is as defined herein.

[0704] In another embodiment, the duocarmycin compound is a duocarmycin SA compound of Formula (XX): U.S. Pat. No. 5,101,038; or (XXI):

[0705]
wherein:

[0706] R₄₂ is C_1-6 alkyl amino or —[C(R₂₀R₂₁)]_a—R₂₂;

[0707] each of R₂₀ and R₂₁ independently is hydrogen, C_1-6 alkyl, C_6-10 aryl, hydroxylated C_6-10 aryl, polyhydroxylated C_6-10 aryl, 5 to 12-membered heterocycle, C_3-8 cycloalkyl, hydroxylated C_3-8 cycloalkyl, polyhydroxylated C_3-8 cycloalkyl or a side chain of a natural or unnatural amino acid;

[0708] R₂₂ is —OH, —NH₂, —COOH, —R₈₂—C(O)(CH₂)_c—C(H)(R₂₃)—N(H)(R₂₃), —R₈₂—C(O)(CH₂)_d—(OCH₂—CH₂)_f—N(H)(R₂₃), or —R₈₂—(C(O)—CH(X²)—NH)_d—R₇₇;

[0709] each R₂₃ independently is hydrogen, C_1-6 alkyl, C_6-10 aryl, C_3-8 cycloalkyl, —COOH, or —COO—C_1-6 alkyl;

[0710] X² is a side chain of a natural or unnatural amino acid;

[0711] R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cyclic compound;

[0712] R₈₂ is —NH or oxygen;

[0713] a is an integer from 1 to 6;

[0714] c is an integer from 0 to 3;

[0715] d is an integer from 1 to 3; and

[0716] f is an integer from 1 to 12.

[0717] In some embodiments, R₄₂ is any one of the following structures:

[0718]
wherein:

[0719] a is an integer from 1 to 6; and

[0720] c is an integer from 0 to 3.

[0721] In another embodiment the tubulysin is a compound of Formula (XXII):

[0722]

[0723] wherein:

[0724] R₅₇ is C_1-4 alkyl or —C(O)R₅₈;

[0725] R₅₈ is C_1-6 alkyl, CF₃ or C_6-10 aryl;

[0726] R₅₉ is C_1-6 alkyl;

[0727] R₆₀ is hydrogen, C_1-6 alkyl, C_2-7 alkenyl, —CH₂-phenyl, CH₂OR₆₅ or CH₂OCOR₆₆;

[0728] R₆₅ is hydrogen, C_1-6 alkyl, C_2-7 alkenyl, C_6-10 aryl or C(O)R₆₇;

[0729] R₆₇ is C_1-6 alkyl, C_2-6 alkenyl, C_6-10 aryl or heteroaryl;

[0730] R₆₆ is C_1-6 alkyl, —C₆H₅ or —CH₂-phenyl;

[0731] R₆₁ is C_1-6 alkyl;

[0732] R₆₂ is hydrogen, OH, O—C_1-4 alkyl or O—C(O)—C_1-4 alkyl;

[0733] R₆₃ is hydrogen, OH, O—C_1-4 alkyl, O—C(O)—C_1-4 alkyl, halogen or C_1-6 alkyl;

[0734] e is an integer from 1 to 3;

[0735] R₆₄ is:

[0736]
wherein:

[0737] R₆₈ is hydrogen or C₁-C₆ alkyl;

[0738] R₆₉ is CO₂R₇₀, C(O)—R₇₈, CONHNH₂, OH, NH₂, SH, or an optionally substituted alkyl, an optionally substituted cycloalkyl, an optionally substituted heteroalkyl or an optionally substituted heterocycloalkyl group;

[0739] R₇₀ is an optionally substituted alkyl (e.g., amino C_1-6 alkyl), an optionally substituted heteroalkyl or an optionally substituted heterocycloalkyl group;

[0740] each of R₇₁ and R₇₃ independently is hydrogen, halo, —NO₂, —CN, —NHR₇₄, C_1-6 alkyl, haloalkyl, alkoxy, and haloalkoxy;

[0741] R₇₂ is hydrogen, OR₄₃, alkoxy, halogen, —NHR₇₄, —O—C(O)—R₄₇, NO₂, —CN, C_6-10 aryl, C_1-6 alkyl, amino or dialkylamino;

[0742] R₇₄ is hydrogen, —CHO, —C(O)—C_1-4 alkyl, OH, amino group, alkyl amino or —[C(R₂₀R₂₁)]_a—R₂₂;

[0743] R₄₃ is H or —R₄₆-R₄₇;

[0744] R₄₆ is —C(O)—; —C(O)—O—, —C(O)—NH—, or absent;

[0745] R₄₇ is as defined herein;

[0746] R₇₈ is X³—R₇₅ or NH—R₁₉;

[0747] X³ is O or S;

[0748] R₁₉ is hydrogen, OH, amino group, alkyl amino or —[C(R₂₀R₂₁)]_a—R₂₂;

[0749] R₇₅ is a hydrogen, an amino group, C_1-6 alkyl amino or —[C(R₂₀R₂₁)]_a—R₂₂;

[0750] each of R₂₀ and R₂₁ independently is hydrogen, C_1-6 alkyl, C_6-10 aryl, hydroxylated C_6-10 aryl, polyhydroxylated C_6-10 aryl, 5 to 12-membered heterocycle, C_3-8 cycloalkyl, hydroxylated C_3-8 cycloalkyl, polyhydroxylated C_3-8 cycloalkyl or a side chain of a natural or unnatural amino acid;

[0751] R₂₂ is —OH, —NH₂, —COOH, —R₈₂—C(O)(CH₂)_c—C(H)(R₂₃)—N(H)(R₂₃), —R₈₂—C(O)(CH₂)_d—(OCH₂—CH₂)_f—N(H)(R₂₃), or —R₈₂—(C(O)—CH(X²)—NH)_d—R₇₇;

[0752] each R₂₃ independently is hydrogen, C_1-6 alkyl, C_6-10 aryl, C_3-8 cycloalkyl, —COOH, or —COO—C_1-6 alkyl;

[0753] X² is a side chain of a natural or unnatural amino acid;

[0754] R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cyclic compound;

[0755] R₈₂ is —NH or oxygen;

[0756] R₄₇ is as defined herein;

[0757] a is an integer from 1 to 6;

[0758] c is an integer from 0 to 3;

[0759] d is an integer from 1 to 3;

[0760] f is an integer from 1 to 12; and

[0761] with the proviso that when R₆₉ is C(O)—X³—R₇₅ or C(O)—NH—R₁₉, one or both of R₇₁ and R₇₃ are —NHR₇₄, and R₇₂ is OR₄₃, —NHR₇₄ or —O—C(O)—R₄₇, at least one of R₁₉, R₄₃, R₇₄ and R₇₅ cannot be hydrogen.

[0762] In some embodiments in the compound of Formula (XXII):

[0763] R₅₇ is —CH₃;

[0764] R₅₉ is sec-butyl;

[0765] R₆₀ is hydrogen, methyl, ethyl, propyl, iso-propyl or iso-butyl;

[0766] R₆₁ is iso-propyl,

[0767] R₆₂ is hydrogen;

[0768] R₆₃ is hydrogen, OH, —O—C₃H₇, O—C(O)—CH₃;

[0769] R₆₈ is hydrogen or —CH₃;

[0770] R₆₉ is CO₂H, CO₂R₇₀ or C(O)—R₇₈;

[0771] R₇₀ is C_1-6 alkyl amine;

[0772] each of R₇₁ and R₇₃ independently is hydrogen;

[0773] R₇₂ is hydrogen, —OR₄₃, OH, F, —CH₃ or —OCH₃;

[0774] R₇₈ is OH, —OR₇₅ or —NHR₄₀;

[0775] e is the integer 2;

[0776] R₄₀ is hydrogen, —OH, —NH₂, or any of the following structures:

[0777]
wherein:

[0778] a is an integer from 1 to 6;

[0779] c is an integer from 0 to 3;

[0780] R₇₅ is any one of the following structures:

[0781]
wherein:

[0782] a is an integer from 1 to 6; and

[0783] c is an integer from 0 to 3;

[0784] R₄₃ is hydrogen, —C(O)—(CH₂)_a—NH₂, or —C(O)—C(H)(CH₃)—(CH₂)_c—NH₂;

wherein:

[0785] a is an integer from 1 to 6;

[0786] c is an integer from 0 to 3; and

[0787] R₄₇ is any one of the following structures:

[0788]
wherein:

[0789] a is an integer from 1 to 6;

[0790] c is an integer from 0 to 3; and

[0791] g is an integer from 2 to 6;

[0792] with the proviso that if R₇₂ is —OH, then R₇₅ cannot be hydrogen; if R₆₉ is COOH then R₇₂ must be —OR₄₃ or —O—C(O)—R₄₇.

[0793] In some embodiments, the tubulysin of Formula (XXII) is a compound of Formula (XXIII) or (XXIV):

[0794]
wherein:

[0795] R₇₆ is hydrogen, OH, OCH₃, F, —OR₄₃ or —O—C(O)—R₄₇;

[0796] wherein R₇₈, R₇₅, R₁₉, R₄₇ and R₄₃ are as defined herein; and with the proviso that if R₇₆ is —OH, OCH₃ or F, then R₇₅ and R₁₉ cannot be hydrogen.

[0797] In one embodiment, R₄₇ is

[0798]

[0799] In another embodiment, R₄₇ is

[0800]

[0801] In yet another embodiment, R₄₇ is

[0802]

[0803] In another embodiment, the KSP inhibitor compound is a compound of Formula (XXVI):

[0804]
wherein R₃₀ is as defined herein.

[0805] In some embodiments R₃₀ is:

[0806]
wherein:

[0807] a is an integer from 1 to 6;

[0808] c is an integer from 0 to 3; and

[0809] g is an integer from 2 to 6.

[0810] In another embodiment the KSP inhibitor compound is a compound of Formula (XXVII), (XXVIII) or (XXIX):

[0811]
wherein:

[0812] R₁₁ is as defined herein.

[0813] 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 therapeutic agents described herein. Thus, the therapeutic agents of the present invention include analogues and derivatives of the compounds described herein.

[0814] Table B below provides more examples of the therapeutic agents and derivatives thereof suitable for conjugation to form the polymer-drug-protein conjugates or polymer-drug scaffolds of the invention. Spectral data of certain compounds are also provided (ND in the table means “not determined”). These examples may also be the active form of the drug when it is released from the conjugates in vitro or in vivo.

[0815]

TABLE B
	(VI)
Ref #	R40
Ex 6
Ex 22
Ex 23
	(IX)
Ref #	R47	m/z
Ex 24		ND
Ex 25		ND
Ex 30		ND
Ex 33		ND
	(XI)
Ref #	R42	m/z
	H
	—CH3	760
Ex 39		802.6
		790
Ex 64		804
	(XII)
Ref #	R40	m/z
	—H
Ex 48		803.5
		789.1
Ex 49		974.2
Ex 50		874.5
		902.2
		ND
		ND
	—OH	788
Ex 61		803.4
Ex 62		803.4
		874.4
		874.4
		874.4
		874.4
		900.2
		900.2
		900.5
		900.5
	(XIII)
—C(O)—R29	m/z
	903.2
	803.1
	790
	832.6
	829.1
	802
	(XIV)
Ref #	R43	m/z
Ex 36		ND
		644.9
	(XVII)
R47	m/z
	553.1
	538.1
	564.1
	566.1
	568.1
	ND
	ND
	667.2
	622.2
	632.02
	986.2
	ND
	ND
	(XXIII)
Ref #	R76	—R78	m/z
	OH	—OH	772.1
	OH	—OCH3	786.4
	OH	—NH2	771.4
	OH		829.4
	OH		ND
		OH	ND
		—OCH3	900.4
		—OH	ND
		—OCH3	ND
		—OCH3	ND
		—OCH3	1000.5
Ex 73		—OH	986.5
Ex 63		—OH	869.4
		—OH	927.3
Ex 76		—OH	871.4
Ex 42	F		ND
Ex 43	F		ND
			1027.2
	—OH
Ex 73	—OH		862.5
Ex 73			1076.4
Ex 73		—OH	886.3
Ex 79		—OH	886.4
Ex 79		—OH	1291.7
Ex 82		—OH	1316.7
		—OH	ND
	(XXX)
Ref #	—R89
Ex 45
Ex 46
	(XXVII)
	(XXVIII)
	(XXIX)
Ref #	R11	m/z (XXVII)
Ex 84		922.3
Ex 87		732.2
		1101.7
		ND
		ND
		ND
		ND
		ND
		ND

Protein-Based Recognition Molecules (PBRMs)

[0816] The protein-based recognition molecule directs the drug-polymer carrier conjugates to specific tissues, cells, or locations in a cell. The protein-based recognition molecule can direct the modified polymer in culture or in a whole organism, or both. In each case, the protein-based recognition molecule has a ligand that is present on the cell surface of the targeted cell(s) to which it binds with an effective specificity, affinity and avidity. In some embodiments, the protein-based recognition molecule targets the modified polymer to tissues other than the liver. In other embodiments the protein-based recognition molecule targets the modified polymer to a specific tissue such as the liver, kidney, lung or pancreas. The protein-based recognition molecule can target the modified polymer to a target cell such as a cancer cell, such as a receptor expressed on a cell such as a cancer cell, a matrix tissue, or a protein associated with cancer such as tumor antigen. Alternatively, cells comprising the tumor vasculature may be targeted. Protein-based recognition molecules can direct the polymer to specific types of cells such as specific targeting to hepatocytes in the liver as opposed to Kupffer cells. In other cases, protein-based recognition molecules can direct the polymer to cells of the reticular endothelial or lymphatic system, or to professional phagocytic cells such as macrophages or eosinophils. (In such cases the polymer itself might also be an effective delivery system, without the need for specific targeting).

[0817] In still other embodiments, the protein based recognition molecule can target the modified polymer to a location within the cell, such as the nucleus, the cytoplasm, or the endosome, for example. In specific embodiments, the protein based recognition molecule can enhance cellular binding to receptors, or cytoplasmic transport to the nucleus and nuclear entry or release from endosomes or other intracellular vesicles.

[0818] In specific embodiments the protein based recognition molecules include antibodies, proteins and peptides or peptide mimics.

[0819] Exemplary antibodies or antibodies derived from Fab, Fab2, scFv or camel antibody heavy-chain fragments specific to the cell surface markers, include, but are not limited to, 5T4, AOC3, C242, CA-125, CCL11, CCR 5, CD2, CD3, CD4, CD5, CD15, CD18, CD19, CD20, CD22, CD23, CD25, CD28, CD30, CD31, CD33, CD37, CD38, CD40, CD41, CD44, CD51, CD52, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD74, CD80, CD125, CD138, CD141, CD147, CD152, CD 154, CD326, CEA, clumping factor, CTLA-4, EGFR, ErbB2, ErbB3, EpCAM, folate receptor, FAP, GD2, GD3, GPNMB, HGF, HER2, ICAM, IGF-1 receptor, VEGFR1, EphA2, TRPV1, CFTR, gpNMB, CA9, Cripto, ACE, APP, adrenergic receptor-beta2, Claudine 3, Mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor, integrins (including α₄, α_vβ₃, α_vβ₅, α_vβ₆, α₁β₄, α₄β₁, α₄β₇, α₅β₁, α₆β₄, α_IIbβ₃ intergins), IFN-α, IFN-γ, IgE, IgE, IGF-1 receptor, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6, interferon receptor, ITGB2 (CD18), LFA-1 (CD11a), L-selectin (CD62L), mucin, MUC1, myostatin, NCA-90, NGF, PDGFRα, phosphatidylserine, prostatic carcinoma cell, Pseudomonas aeruginosa, rabies, RANKL, respiratory syncytial virus, Rhesus factor, SLAMF7, sphingosine-1-phosphate, TAG-72, T-cell receptor, tenascin C, TGF-1, TGF-β2, TGF-β, TNF-α, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR2, vimentin, and the like.

[0820] In one embodiment the antibodies or antibody derived from Fab, Fab2, scFv or camel antibody heavy-chain fragments specific to the cell surface markers include CA-125, C242, CD3, CD19, CD22, CD25, CD30, CD31, CD33, CD37, CD40, CD44, CD51, CD54, CD56, CD62E, CD62P, CD62L, CD70, CD138, CD141, CD326, CEA, CTLA-4, EGFR, ErbB2, ErbB3, FAP, folate receptor, IGF-1 receptor, GD3, GPNMB, HGF, HER2, VEGF-A, VEGFR2, VEGFR1, EphA2, EpCAM, 5T4, TAG-72, tenascin C, TRPV1, CFTR, gpNMB, CA9, Cripto, ACE, APP, PDGFR α, phosphatidylserine, prostatic carcinoma cells, adrenergic receptor-beta2, Claudine 3, mucin, MUC1, Mesothelin, IL-2 receptor, IL-4 receptor, IL-13 receptor and integrins (including α_vβ₃, α_vβ₅, α_vβ₆, α₁β₄, α₄β₁, α₅β₁, α₆β₄ intergins), tenascin C, TRAIL-R2 and vimentin.

[0821] Exemplary antibodies include 3F8, abagovomab, abciximab (REOPRO), adalimumab (HUMIRA), adecatumumab, afelimomab, afutuzumab, alacizumab, ALD518, alemtuzumab (CAMPATH), altumomab, amatuximab, anatumomab, anrukinzumab, apolizumab, arcitumomab (CEA-SCAN), aselizumab, atlizumab (tocilizumab, Actemra, RoActemra), atorolimumab, bapineuzumab, basiliximab (Simulect), bavituximab, bectumomab (LYMPHOSCAN), belimumab (BENLYSTA), benralizumab, bertilimumab, besilesomab (SCINITIMUN), bevacizumab (AVASTIN), biciromab (FIBRISCINT), bivatuzumab, blinatumomab, brentuximab, briakinumab, canakinumab (ILARIS), cantuzumab, capromab, catumaxomab (REMOVAB), CC49, cedelizumab, certolizumab, cetuximab (ERBITUX), citatuzumab, cixutumumab, clenoliximab, clivatuzumab, conatumumab, CR6261, dacetuzumab, daclizumab (ZENAPAX), daratumumab, denosumab (PROLIA), detumomab, dorlimomab, dorlixizumab, ecromeximab, eculizumab (SOLIRIS), edobacomab, edrecolomab (PANOREX), efalizumab (RAPTIVA), efungumab (MYCOGRAB), elotuzumab, elsilimomab, enlimomab, epitumomab, epratuzumab, erlizumab, ertumaxomab (REXOMUN), etaracizumab (ABEGRIN), exbivirumab, fanolesomab (NEUTROSPEC), faralimomab, farletuzumab, felvizumab, fezakinumab, figitumumab, fontolizumab (HuZAF), foravirumab, fresolimumab, galiximab, gantenerumab, gavilimomab, gemtuzumab girentuximab, glembatumumab, golimumab (SIMPONI), gomiliximab, ibalizumab, ibritumomab, igovomab (INDIMACIS-125), imciromab (MYOSCINT), infliximab (REMICADE), intetumumab, inolimomab, inotuzumab, ipilimumab, iratumumab, keliximab, labetuzumab (CEA-CIDE), lebrikizumab, lemalesomab, lerdelimumab, lexatumumab, libivirumab, lintuzumab, lucatumumab, lumiliximab, mapatumumab, maslimomab, matuzumab, mepolizumab (BOSATRIA), metelimumab, milatuzumab, minretumomab, mitumomab, morolimumab, motavizumab (NUMAX), muromonab-CD3 (ORTHOCLONE OKT3), nacolomab, naptumomab, natalizumab (TYSABRI), nebacumab, necitumumab, nerelimomab, nimotuzumab (THERACIM), nofetumomab, ocrelizumab, odulimomab, ofatumumab (ARZERRA), olaratumab, omalizumab (XOLAIR), ontecizumab, oportuzumab, oregovomab (OVAREX), otelixizumab, pagibaximab, palivizumab (SYNAGIS), panitumumab (VECTIBIX), panobacumab, pascolizumab, pemtumomab (THERAGYN), pertuzumab (OMNITARG), pexelizumab, pintumomab, priliximab, pritumumab, PRO 140, rafivirumab, ramucirumab, ranibizumab (LUCENTIS), raxibacumab, regavirumab, reslizumab, rilotumumab, rituximab (RITUXAN), robatumumab, rontalizumab, rovelizumab (LEUKARREST), ruplizumab (ANTOVA), satumomab pendetide, sevirumab, sibrotuzumab, sifalimumab, siltuximab, siplizumab, solanezumab, sonepcizumab, sontuzumab, stamulumab, sulesomab (LEUKOSCAN), tacatuzumab (AFP-CIDE), tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tefibazumab (AUREXIS), telimomab, tenatumomab, teneliximab, teplizumab, TGN1412, ticilimumab (tremelimumab), tigatuzumab, TNX-650, tocilizumab (atlizumab, ACTEMRA), toralizumab, tositumomab (BEXXAR), trastuzumab (HERCEPTIN), tremelimumab, tucotuzumab, tuvirumab, urtoxazumab, ustekinumab (STELERA), vapaliximab, vedolizumab, veltuzumab, vepalimomab, visilizumab (NUVION), volociximab (HUMASPECT), votumumab, zalutumumab (HuMEX-EGFr), zanolimumab (HuMAX-CD4), ziralimumab and zolimomab.

[0822] In some embodiments the antibodies are directed to cell surface markers for 5T4, CA-125, CEA, CD3, CD19, CD20, CD22, CD30, CD33, CD40, CD44, CD51, CTLA-4, EpCAM, HER2, EGFR, FAP, folate receptor, HGF, integrin α_vβ₃, integrin α₅β₁, IGF-1 receptor, GD3, GPNMB, mucin, MUC1, phosphatidylserine, prostatic carcinoma cells, PDGFR α, TAG-72, tenascin C, TRAIL-R2, VEGF-A and VEGFR2. In this embodiment the antibodies are abagovomab, adecatumumab, alacizumab, altumomab, anatumomab, arcitumomab, bavituximab, bevacizumab (AVASTIN), bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, capromab, cetuximab, citatuzumab, clivatuzumab, conatumumab, dacetuzumab, edrecolomab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, figitumumab, gemtuzumab, glembatumumab, ibritumomab, igovomab, intetumumab, inotuzumab, labetuzumab, lexatumumab, lintuzumab, lucatumumab, matuzumab, mitumomab, naptumomab estafenatox, necitumumab, oportuzumab, oregovomab, panitumumab, pemtumomab, pertuzumab, pritumumab, rituximab (RITUXAN), rilotumumab, robatumumab, satumomab, sibrotuzumab, taplitumomab, tenatumomab, tenatumomab, ticilimumab (tremelimumab), tigatuzumab, trastuzumab (HERCEPTIN), tositumomab, tremelimumab, tucotuzumab celmoleukin, volociximab and zalutumumab.

[0823] In specific embodiments the antibodies directed to cell surface markers for HER2 are pertuzumab or trastuzumab and for EGFR the antibody is cetuximab and for CD20 the antibody is rituximab and for VEGF-A is bevacizumab and for CD-22 the antibody is epratuzumab or veltuzumab and for CEA the antibody is labetuzumab.

[0824] Exemplary peptides or peptide mimics include integrin targeting peptides (RGD peptides), LHRH receptor targeting peptides, ErbB2 (HER2) receptor targeting peptides, prostate specific membrane bound antigen (PSMA) targeting peptides, lipoprotein receptor LRP1 targeting, ApoE protein derived peptides, ApoA protein peptides, somatostatin receptor targeting peptides, chlorotoxin derived peptides, and bombesin.

[0825] In specific embodiments the peptides or peptide mimics are LHRH receptor targeting peptides and ErbB2 (HER2) receptor targeting peptides

[0826] Exemplary proteins comprise insulin, transferrin, fibrinogen-gamma fragment, thrombospondin, claudin, apolipoprotein E, Affibody molecules such as, for example, ABY-025, Ankyrin repeat proteins, ankyrin-like repeats proteins and synthetic peptides.

[0827] In some embodiments of the invention the protein drug polymer conjugates comprise broad spectrum cytotoxins in combination with cell surface markers for HER2 such as pertuzumab or trastuzumab; for EGFR such as cetuximab; for CEA such as labetuzumab; for CD20 such as rituximab; for VEGF-A such as bevacizumab; or for CD-22 such as epratuzumab or veltuzumab.

[0828] In other embodiments of the invention the protein-drug-polymer conjugates or protein-polymer conjugates used in the invention comprise combinations of two or more protein based recognition molecules, such as, for example, combination of bispecific antibodies directed to the EGF receptor (EGFR) on tumor cells and to CD3 and CD28 on T cells; combination of antibodies or antibody derived from Fab, Fab2, scFv or camel antibody heavy-chain fragments and peptides or peptide mimetics; combination of antibodies or antibody derived from Fab, Fab2, scFv or camel antibody heavy-chain fragments and proteins; combination of two bispecific antibodies such as CD3×CD19 plus CD28×CD22 bispecific antibodies.

[0829] Table C below provides more examples of the PBRM described hereof, which are suitable for conjugation to form the polymer-drug-protein conjugates or polymer-PBRM scaffolds of the invention.

[0830]

TABLE C
Ref #	PBRM
Ex 3
Ex 4
Ex 53
Ex 60	TRASTUZUMAB-Fab'-SH
Ex 10
Ex 14
Ex 16
Ex 91	TRASTUZUMAB-Fab-SH

Linkers (L^D and L^P)

[0831] As described above, the drug or PBRM is connected to the polymeric carrier via a linker L^D or L^P. In some embodiments, the linker is biocleavable/biodegradable under intracellular conditions, such that the cleavage of the linker releases the drug or PBRM from the polymer unit in the intracellular environment.

[0832] A linker is any chemical moiety that is capable of linking a drug or a PBRM to a polymer backbone through chemical bonds such that the drug or PBRM and the polymer are chemically coupled (e.g., covalently bonded) to each other. In some embodiments, the linker comprises a biodegradable linker moiety (e.g., a biodegradable bond such as an ester or amide bond).

[0833] In other embodiments, the linker L^D or L^P is biodegradable under mild conditions, i.e., conditions within a cell under which the activity of the drug is not affected. Examples of suitable biodegradable linker moiety include disulfide linkers, acid labile linkers, photolabile linkers, peptidase labile linkers, and esterase labile linkers.

[0834] In some embodiments, the linker L^D or L^P is biocleavable under reducing conditions (e.g., a disulfide linker). In this embodiment the drug or PBRM moiety is linked to the polymer through a disulfide bond. The linker molecule comprises a reactive chemical group that can react with the drug. Preferred reactive chemical groups for reaction with the drug or PBRM moiety are N-succinimidyl esters and N-sulfosuccinimidyl esters. Additionally the linker molecule comprises a reactive chemical group, preferably a dithiopyridyl group that can react with the drug to form a disulfide bond. In some embodiments the linker molecules include, for example, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-5-acetylthioacetate (SATA) and N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene or 2,5-dioxopyrrolidin-1-yl 4-(1-(pyridin-2-yldisulfanyl)ethyl)benzoate (SMPT).

[0835] In other embodiments, the biocleavable linker L^D or L^P is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, the pH-sensitive linker is hydrolysable under acidic conditions. For example, an acid-labile linker that is hydrolysable in the lysosome or endosome (e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like) can be used. Such linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5.0, the approximate pH of the lysosome. In certain embodiments, the hydrolysable linker is a thioether linker (such as, e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond.

[0836] In other embodiments the linker L^D or L^P is photo-labile and is useful at the body surface and in many body cavities that are accessible to light. Furthermore, L^D or L^P is biocleavable by infrared light which can penetrate tissue. Accordingly, L^D or L^P is useful for both applications on the body surface and in the tissue.

[0837] In some embodiments, the linker L^D or L^P is biocleavable by a cleaving agent that is present in the intracellular environment (e.g., within a lysosome or endosome or caveolea). The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease.

[0838] In some embodiments the linker L^D or L^P is cleaved by esterases. Only certain esters can be cleaved by esterases present inside or outside cells. Esters are formed by the condensation of a carboxylic acid and an alcohol. Simple esters are esters produced with simple alcohols, such as aliphatic alcohols, and small cyclic and small aromatic alcohols.

[0839] In yet other embodiments, the linker L^D or L^P is not biocleavable and the drug is released by antibody degradation. See, for example, U.S. Pat. No. 7,498,298, which is incorporated by reference herein in its entirety and for all purposes.

[0840] Typically, the linker L^D or L^P is not substantially sensitive to the extracellular environment. As used herein, “not substantially sensitive to the extracellular environment,” in the context of a linker, means that no more than about 20%, typically no more than about 15%, more typically no more than about 10%, and even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the linkers, in a sample of Polymer Drug Conjugate, are cleaved when the Polymer Drug Conjugate presents in an extracellular environment (e.g., in plasma) for 24 hours. Whether a linker is not substantially sensitive to the extracellular environment can be determined, for example, by incubating the Polymer Drug Conjugate with plasma for a predetermined time period (e.g., 2, 4, 8, 16, or 24 hours) and then quantitating the amount of free drug present in the plasma.

[0841] In embodiments, the linker L^D has the structure: —R^L1—C(═O)—X^D-M^D1-Y^D-M^D2-Z^D-M^D3-Q^D-M^D4-, with R^L1 connected to an oxygen atom of the polymeric carrier and M^D4 connected to the drug molecule to be delivered.

[0842] In embodiments, the linker L^P has the structure: —R^L2—C(═O)—X^P-M^P1-Y^P-M^P2-Z^P-M^P3-Q^P-M^P4-, with R^L2 connected to an oxygen atom of the polymeric carrier and M^P4 connected to the PBRM.

[0843] For example, each of R^L1 and R^L2 independently is absent, alkyl, alkenyl, alkynyl, cycloalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, aryl, or heteroaryl.

[0844] For example, each of R^L1 and R^L2 independently is absent, alkyl, cycloalkyl, heteroalkyl, or heterocycloalkyl.

[0845] For example, R^L1 is absent.

[0846] For example, R^L2 is absent.

[0847] For example, each of X^D and X^P, independently is —O—, —S—, —N(R¹)—, or absent, in which R¹ is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety, —C(═O)R^1B, —C(═O)OR^1B, —SO₂R^1B or —N(R¹)— is a heterocycloalkyl moiety, wherein R^IB is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

[0848] For example, each of Y^D, Y^P, Z^D, Z^P, Q^D, and Q^P, independently, is absent or a biodegradable linker moiety selected from the group consisting of —S—S—, —C(═O)O—, —C(═O)NR²—, —OC(═O)—, —NR²C(═O)—, —OC(═O)O—, —OC(═O)NR²—, —NR²C(═O)O—, —NR²C(═O)NR³—, —C(OR²)O—, —C(OR²)S—, —C(OR²)NR³—, —C(SR²)O—, —C(SR²)S—, C(SR²)NR³—, —C(NR²R³)O—, —C(NR²R³)S—, —C(NR²R³)NR⁴—, —C(═O)S—, —SC(═O)—, —SC(═O)S—, —OC(═O)S—, —SC(═O)O—, —C(═S)S—, —SC(═S)—, —OC(═S)—, —C(═S)O—, —SC(═S)O—, —OC(═S)S—, —OC(═S)O—, —SC(═S)S—, —C(═NR²)O—, —C(═NR²)S—, —C(═NR²)NR³—, —OC(═NR²)—, —SC(═NR²)—, —NR³C(═NR²)—, —NR²SO₂—, —NR²NR³—, —C(═O)NR²NR³—, —NR²NR³C(═O)—, —OC(═O)NR²NR³—, —NR²NR³C(═O)O—, —C(═S)NR²NR³—, —NR²NR³C(═S)—, —C(═NR⁴)NR²NR³—, —NR²NR³C(═NR⁴)—, —O(N═CR³)—, —(CR³═N)O—, —C(═O)NR²—(N═CR³)—, —(CR³═N)—NR²C(═O)—, —SO₃—, —NR²SO₂NR³—, —SO₂NR²—, and polyamide, wherein each occurrence of R², R³, and R⁴ independently is hydrogen or an aliphatic, heteroaliphatic, carbocyclic, or heterocyclic moiety, or each occurrence of —NR²— or —NR²NR³— is a heterocycloalkyl moiety.

[0849] For example, each of M^D1, M^D2, M^D3, M^D4, M^P1, M^P2, M^P3 and M^P4, independently, is absent or a non-biodegradable linker moiety selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, aryl, heteroaryl, and a combination thereof and each of M^D1, M^D2, M^D3, M^P1, M^P2, and M^P3 optionally contains one or more —(C═O)— but does not contain any of the biodegradable linker moieties mentioned above.

[0850] For example, each of M^D1, M^D2, M^D3, M^D4, M^P1, M^P2, M^P3 And M^P4, Independently is C_1-6 alkyl, C_1-6 alkyl-C(O)—C_0-6 alkyl, C_1-6 alkyl-NH—C_0-6 alkyl, C_1-6 alkyl-O—C_0-6 alkyl, C_1-6 alkyl-S—C_0-6 alkyl, C_1-6 alkyl-C(O)—C_1-6 alkyl-NH, C_1-6 alkyl-C(O)—C_1-6 alkyl-O, C_1-6 alkyl-C(O)—C_1-6 alkyl-S, C_3-10 cycloalkyl-C(O)—C_0-6 alkyl, 3-19 membered heterocycloalkyl-C(O)—C_0-6 alkyl, aryl-C(O)—C_0-6 alkyl, (CH₂CH₂O)_1-12, and the like.

[0851] For example, for each L^D, M^D1 is not absent when X^D is absent.

[0852] For example, for each L^P, M^P1 is not absent when X^P is absent.

[0853] For example, for each L^D, at least one of X^D, Y^D, Z^D, and Q^D is not absent.

[0854] For example, for each L^P, at least one of X^P, Y^P, Z^P, and Q^P is not absent.

[0855] For example, each of M^D1 and M^P1 independently is C_1-6 alkyl or C_1-6 heteroalkyl.

[0856] For example, each of M^D2, M^D3, M^D4, M^P2, M^P3, and M^P4, independently is absent, C_1-6 alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, or a combination thereof.

[0857] For example, for each L^D, at most two of M^D2, M^D3, and M^D4 are absent.

[0858] For example, for each L^P, at most two of M^P2, M^P3, and M^P4 are absent.

[0859] For example, for each L^D, one of M^D2 and M^D3 has one of the following structures:

[0860]
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 of M^D2 or M^D3 is either absent or a moiety different from the above, such as C_1-6 alkyl.

[0861] For example, for each L^P, one of M^P2 and M^P3 has one of the following structures:

[0862]
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 of M^P2 or M^P3 is either absent or a moiety different from the above, such as C_1-6 alkyl.

[0863] For example, p is 2.

[0864] For example, q is 0 or 12.

[0865] For example, t is 0 or 1.

[0866] For example, each of -M^D2-Z^D—, —Z^D-M^D3-, —Z^D-M^D2, or M^D3-Z^D—, independently has one of the following structures:

[0867]
in which ring A or B independently is cycloalkyl or heterocycloalkyl; R^W is an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety; R^1J is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety; and ring D is heterocycloalkyl.

[0868] For example, each of -M^P2-Z^P—, —Z^P-M^P3-, —Z^P-M^P2-, and -M^P3-Z^P— independently, has one of the following structures:

[0869]
in which ring A is cycloalkyl or heterocycloalkyl and R^1J is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

[0870] For example, ring A is 5-19 membered heterocycloalkyl, e.g.,

[0871]

[0872] For example, ring A is C_3-8 cycloalkyl.

[0873] For example, ring D is piperazinyl or piperidinyl.

[0874] For example, R^W is C_1-6 alkyl.

[0875] For example, R^1J is hydrogen or C_1-6 alkyl.

[0876] For example, Z^D is

[0877]

[0878] For example, Z^P is

[0879]

[0880] For example, X^D is absent, O or NH.

[0881] For example, X^P is absent, O or NH.

[0882] For example, each of X^D and X^P, independently is

[0883]

[0884] For example, each of Y^D and Y^P independently is —S—S—, —OCO—, —COO—, —CONH— or —NHCO—.

[0885] For example, each of Q^D and Q^P independently is absent, —S—S—, —OCO—, —COO—, —CONH—, —NHCO—, —OCONHNH—, or —NHNHCOO—.

[0886] For example, -L^D-D can have one of the following 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:

[0887]
wherein R₈₀ is CH₂, —NH, or oxygen; and
R₈₂ is —NH or oxygen.

[0888] For example, polymeric carrier-L^P-PBRM can have one of the following structures below:

[0889]
wherein:

[0890] R₈₀ is CH₂, NH or oxygen; and

[0891] R₈₁ is

[0892]

[0893] While biocleavable linkers preferably are used in the invention, a non-biocleavable linker also can be used to generate the above-described conjugate. A non-biocleavable linker is any chemical moiety that is capable of linking a drug or PBRM, to a polymer in a stable, covalent manner. Thus, non-biocleavable linkers are substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and/or disulfide bond cleavage, at conditions under which the drug or polymer remains active.

[0894] In one embodiment, a substantial amount of the drug moiety is not cleaved from the conjugate until the protein-polymer-drug conjugate enters a cell with a cell-surface receptor specific for the PBRM of the protein-polymer-drug conjugate, and the drug moiety is cleaved from the protein-polymer-drug conjugate when the protein-polymer-drug conjugate does enter the cell.

[0895] In another embodiment, the bioavailability of the protein-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 comprising the drug moiety of the protein-polymer-drug conjugate, or when compared to an analog of the compound not having the drug moiety.

[0896] In another embodiment, the drug moiety is intracellularly cleaved in a subject from the protein-polymer-drug conjugate, or an intracellular metabolite of the protein-polymer-drug conjugate.

Conjugates or Polymeric Scaffolds

[0897] Conjugates of the invention comprise one or more occurrences of D, where D is a therapeutic agent, e.g., a drug, wherein the one or more occurrences of D may be the same or different.

[0898] In certain other embodiments, one or more occurrences of PBRM is attached to the polymeric carrier, wherein the one or more occurrences of PBRM may be the same or different. In certain other embodiments, one or more polymer carriers that contains one or more occurrences of D are connected to a PBRM (e.g., an antibody).

[0899] As discussed more generally above, in certain embodiments, each polymeric carrier independently, has about 0.1 to about 25% monomers comprising a D, more preferably about 0.5 to about 20%, more preferably about 1 to about 15%, and even more preferably about 2 to about 10%.

[0900] In certain embodiments, the conjugate of this invention is of Formula (I):

[0901]
wherein:
each of n, n₁, n₂, n₃, and n₄, is the molar fraction of the corresponding polymer unit ranging between 0 and 1; n+n₁+n₂+n₃+n₄=1; provided that none of n, n₂, and n₄ is 0.

[0902] For example, the ratio between n₂ and n₄ is greater than 1:1 and ≦200:1.

[0903] For example, the ratio between n₂ and n₄ is between 10:1 and 50:1.

[0904] For example, the ratio between n₂ and n₄ is between 30:1 and 50:1.

[0905] For example, the ratio between n₂ and n₄ is about 50:1, 25:1, 10:1, 5:1 or 2:1.

[0906] In certain embodiments, 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) reacting the 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 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 modified polymer produced by step (1) contains a functional group that can react with a functional group of the PBRM or its derivative.

[0907] In another embodiment the conjugates are formed in several steps: (1) modifying a polymer so that it contains a functional group that can react with a functional group of a first drug or its derivative; (2) reacting the modified polymer with the first drug or its derivative so that the first drug is linked to the polymer; (3) modifying the polymer-drug 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 with the second drug or its derivative so that the second drug is linked to the polymer-drug conjugate; (5) modifying the 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) reacting the modified polymer-drug conjugate of step (5) with the PBRM or its derivative to form the conjugate of this invention. Steps (5) and (6) may be repeated if 2 different PBRM or its derivatives are to be conjugated to form a polymer-drug conjugate comprising two different drugs and two different PBRMs.

[0908] 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 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 polymer; and (4) reacting the modified polymer-drug conjugate 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 of the drug or its derivatives and a functional group of the PBRM or its derivative.

[0909] In another embodiment the conjugates are formed in several steps: (1) modifying a polymer so that it contains a functional group that can react with a functional group of a 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 the modified polymer with the first drug or its derivative so that the first drug is linked to the polymer; (4) modifying the polymer-drug conjugate so that it contains a different functional group that can react with a functional group of a second drug or its derivative (5) reacting the modified polymer-drug conjugate with the second drug or its derivative so that the second drug is linked to the polymer-drug conjugate; (6) reacting the modified polymer-drug conjugate containing 2 different drugs so that the polymer with the PBRM or its derivative to form the conjugate of this invention. Step (6) may be repeated if 2 different PBRM or its derivatives are to be conjugated to form a polymer-drug conjugate comprising two different drugs and two different PBRMs. Step (4) may be carried out after step (1) so that the modified polymer contains two different functional groups that can react with two different drugs or their derivatives. In this embodiment, the modified polymer containing two different functional group that can react with two different drugs or their derivatives can be further modified so that it contains a functional group that can react with a functional group of the PBRM or its derivative; prior to the reaction of the modified polymer with either the two different drugs (step (3) and step (5) or PBRM (step (6).

[0910] The biodegradable biocompatible conjugates of the invention can be prepared to meet desired requirements of biodegradability and hydrophilicity. For example, under physiological conditions, a balance between biodegradability and stability can be reached. For instance, it is known that molecules with molecular weights beyond a certain threshold (generally, above 40-100 kDa, depending on the physical shape of the molecule) are not excreted through kidneys, as small molecules are, and can be cleared from the body only through uptake by cells and degradation in intracellular compartments, most notably lysosomes. This observation exemplifies how functionally stable yet biodegradable materials may be designed by modulating their stability under general physiological conditions (pH=7.5±0.5) and at lysosomal 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 environment than, for example, in blood plasma. One can design a test to compare polymer degradation profile at, for 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” lysosomal compartment after uptake by cells. Polymer 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.

[0911] In many cases, it will be preferable that at pH=7.5 the effective size of the polymer will not detectably change over 1 to 7 days, and remain within 50% from the original for at least several weeks. At pH=5, on the other hand, the polymer should preferably detectably degrade over 1 to 5 days, and be completely transformed into low molecular weight fragments within a two-week to several-month time frame. Although faster degradation may be in some cases preferable, in general it may be more desirable that the polymer degrades in cells with the rate that does not exceed the rate of metabolization or excretion of polymer fragments by the cells. Accordingly, in certain embodiments, the conjugates of the present invention are expected to be biodegradable, in particular upon uptake by cells, and relatively “inert” in relation to biological systems. The products of carrier degradation are preferably uncharged and do not significantly shift the pH of the environment. It is proposed that the abundance of alcohol groups may provide low rate of polymer recognition by cell receptors, particularly of phagocytes. The polymer backbones of the present invention generally contain few, if any, antigenic determinants (characteristic, for example, for some polysaccharides and polypeptides) and generally do not comprise rigid structures capable of engaging in “key-and-lock” type interactions in vivo unless the latter are desirable. Thus, the soluble, crosslinked and solid conjugates of this invention are predicted to have low toxicity and bioadhesivity, which makes them suitable for several biomedical applications.

[0912] In certain embodiments of the present invention, the biodegradable biocompatible conjugates can form linear or branched structures. For example, the biodegradable biocompatible polyal conjugates of the present invention can be chiral (optically active). Optionally, the biodegradable biocompatible polyal conjugates of the present invention can be scalemic.

[0913] In certain embodiments, the conjugates of the invention are water-soluble. In certain embodiments, the conjugates of the invention are water-insoluble. In certain embodiments, the inventive conjugate is in a solid form. In certain embodiments, the conjugates of the invention are colloids. In certain embodiments, the conjugates of the invention are in particle form. In certain embodiments, the conjugates of the invention are in gel form.

[0914] This invention also features a polymeric scaffold useful for conjugating with a PBRM to form a polymer-drug-PBRM conjugate described herein. The scaffold comprises a polymeric carrier, one or more -L^D-D connected to the polymeric carrier, and one or more L^P connected to the polymeric carrier which is suitable for connecting a PBRM to the polymeric carrier, wherein:

[0915] each occurrence of D is independently a therapeutic agent having a molecular weight ≦5 kDa;

[0916] the polymeric carrier is a polyacetal or polyketal,

[0917] L^D is a linker having the structure:

[0918]
with R^L1 connected to an oxygen atom of the polymeric carrier and L^D1 connected to D, and

[0919]
denotes direct or indirect attachment of D to L^D1, and L^D contains a biodegradable bond so that when the bond is broken, D is released from the polymeric carrier in an active form for its intended therapeutic effect;

[0920] L^D1 is a carbonyl-containing moiety;

[0921] L^P is a linker different from L^D and having the structure: —R^L2—C(═O)-L^P1 with R^L2 connected to an oxygen atom of the polymeric carrier and L^P1 suitable for connecting directly or indirectly to a PBRM;

[0922] each of R^L1 and R^L2 independently is absent, alkyl, heteroalkyl, cycloalkyl, or heterocycloalkyl; and

[0923] L^P1 is a moiety containing a functional group that is capable of forming a covalent bond with a functional group of a PBRM.

[0924] For example, L^P is a linker having the structure:

[0925]
in which L^P2 is a moiety containing a functional group that is capable of forming a covalent bond with a functional group of a PBRM, and

[0926]
denotes direct or indirect attachment of L^P2 to L^D1.

[0927] For example, the functional group of L^P1 or L^P2 is selected from —SR^p, —S—S-LG, maleimido, and halo, in which LG is a leaving group and R^p is H or a sulfur protecting group.

[0928] For example, L^D1 comprises —X—(CH₂)_v—C(═O)— with X directly connected to the carbonyl group of R^L1—C(═O), in which X is CH₂, O, or NH, and v is an integer from 1 to 6.

[0929] For example, L^P1 or L^P2 contains a biodegradable bond.

[0930] For example, each of R^L1 and R^L2 is absent.

[0931] For example, the polymeric carrier of the scaffold of the invention is a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF) ranging from about 2 kDa to about 300 kDa. The selection of a polymeric carrier with a specific MW range may depend on the size of the PBRM to be conjugated with.

[0932] For example, for conjugating a PBRM having a molecular weight of 40 kDa or greater (e.g., 60 kDa or greater, 80 kDa or greater, 100 kDa or greater, 120 kDa or greater, 140 kDa or greater, 160 kDa or greater or 180 kDa or greater), the polymeric carrier of the scaffold of the invention is a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF) ranging from about 2 kDa to about 40 kDa (e.g., about 6-20 kDa or about 8-15 kDa).

[0933] For example, for conjugating a PBRM having a molecular weight of 40 kDa to 200 kDa, the polymeric carrier of the scaffold of the invention is a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF) ranging from about 2 kDa to about 40 kDa (e.g., about 6-20 kDa or about 8-15 kDa).

[0934] For example, for conjugating a PBRM having a molecular weight of 60 kDa to 120 kDa, the polymeric carrier of the scaffold of the invention is a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF) ranging from about 8 kDa to about 40 kDa (e.g., about 8-30 kDa, about 8-20 kDa or about 8-15 kDa). For example the PHF has a molecular weight of about 10 kDa, 20 kDa, 30 kDa or 40 kDa.

[0935] PBRMs in this molecular weight range, include but are not limited to, for example, camelids, Fab2, and the like.

[0936] For example, for conjugating a PBRM having a molecular weight of 140 kDa to 180 kDa, the polymeric carrier of the scaffold of the invention is a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF) ranging from about 2 kDa to about 40 kDa (e.g., about 6-20 kDa or about 8-15 kDa). For example the PHF has a molecular weight of about 8 kDa, 10 kDa or 15 kDa.

[0937] PBRMs in this molecular weight range, include but are not limited to, for example, full length antibodies, such as, IgG, IgM.

[0938] For example, for conjugating a PBRM having a molecular weight of 200 kDa or less (e.g., 120 kDa or less, 80 kDa or less, 60 kDa or less, 40 kDa or less, 20 kDa or less or 10 kDa or less), the polymeric carrier of the scaffold of the invention is a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF) ranging from about 20 kDa to about 300 kDa (e.g., about 20-150 kDa, about 30-150 kDa, about 50-150 kDa, about 30-100 kDa, or about 50-100 kDa).

[0939] For example, for conjugating a PBRM having a molecular weight of 4 kDa to 80 kDa (e.g., 4-20 kDa, 20-30 kDa, or 30-70 kDa), the polymeric carrier of the scaffold of the invention is a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF) ranging from about 20 kDa to about 300 kDa (e.g., about 20-150 kDa, about 30-150 kDa, about 50-150 kDa, about 30-100 kDa, or about 50-100 kDa).

[0940] For example, for conjugating a PBRM having a molecular weight of 80 kDa or less (e.g., 70 kDa or less, 60 kDa or less, 50 kDa or less or 40 kDa or less), the polymeric carrier of the scaffold of the invention is a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF) ranging from about 20 kDa to about 300 kDa (e.g., about 20-150 kDa, about 30-150 kDa, about 50-150 kDa, about 30-100 kDa, or about 50-100 kDa). For example the PHF has a molecular weight of about 50 kDa, 70 kDa or 100 kDa.

[0941] PBRMs in this molecular weight range, include but are not limited to, for example, antibody fragments such as, for example Fabs.

[0942] For example, for conjugating a PBRM having a molecular weight of 30 kDa or less (e.g., about 20 kDa or less), the polymeric carrier of the scaffold of the invention is a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF) ranging from about 20 kDa to about 300 kDa (e.g., about 20-150 kDa, about 30-150 kDa, about 50-150 kDa, about 30-100 kDa, or about 50-100 kDa). For example the PHF has a molecular weight of about 30 kDa, 40 kDa, 50 kDa, 70 kDa, 100 kDa, 120 kDa or 150 kDa.

[0943] PBRMs in this molecular weight range, include but are not limited to, for example, antibody fragments, such as, scFv.

[0944] For example, for conjugating a PBRM having a molecular weight of 20 kDa or less (e.g., 10 kDa or less), the polymeric carrier of the scaffold of the invention is a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF) ranging from about 20 kDa to about 300 kDa (e.g., about 20-150 kDa, about 30-150 kDa, about 50-150 kDa, about 30-100 kDa, or about 50-100 kDa). For example the PHF has a molecular weight of about 30 kDa, 40 kDa, 50 kDa, 70 kDa, 100 kDa, 120 kDa or 150 kDa.

[0945] PBRMs in this molecular weight range, include but are not limited to, for example, small proteins and peptides.

[0946] For example, the scaffold is of Formula (Ia):

[0947]

[0948] wherein:

• • [0949] m is an integer from 1 to about 2200,
  • [0950] m₁ is an integer from 1 to about 660,
  • [0951] m₂ is an integer from 1 to about 300,
  • [0952] m₃ is an integer from 1 to about 110, and
  • [0953] the sum of m, m₁, m₂ and m₃ ranges from about 15 to about 2200.

[0954] For example, when the PHF in Formula (Ia) has a molecular weight ranging from about 2 kDa to about 40 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging from about 15 to about 300), m₂ is an integer from 1 to about 40, m₃ is an integer from 1 to about 18, and/or m₁ is an integer from 1 to about 140 (e.g., m₁ being about 1-90).

[0955] For example, when the PHF in Formula (Ia) has a molecular weight ranging from about 6 kDa to about 20 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging from about 45 to about 150), m₂ is an integer from 2 to about 20, m₃ is an integer from 1 to about 9, and/or m₁ is an integer from 1 to about 75 (e.g, m₁ being about 4-45).

[0956] For example, when the PHF in Formula (Ia) has a molecular weight ranging from about 8 kDa to about 15 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging from about 60 to about 110), m₂ is an integer from 2 to about 15, m₃ is an integer from 1 to about 7, and/or m₁ is an integer from 1 to about 55 (e.g, m₁ being about 4-30).

[0957] For example, when the PHF in Formula (Ia) has a molecular weight ranging from 20 kDa to 300 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging from about 150 to about 2200), m₂ is an integer from 3 to about 300, m₃ is an integer from 1 to about 110, and/or m₁ is an integer from 1 to about 660 (e.g, m₁ being about 10-250).

[0958] For example, when the PHF in Formula (Ia) has a molecular weight ranging from 20 kDa to 150 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging from about 150 to about 1100), m₂ is an integer from 3 to about 150, m₃ is an integer from 1 to about 75 (e.g., from 1 to about 55), and/or m₁ is an integer from 1 to about 330 (e.g., m₁ being about 10-330 or about 15-100). This scaffold can be used, for example, for conjugating a PBRM having a molecular weight of about 4 kDa to about 80 kDa.

[0959] For example, when the PHF in Formula (Ia) has a molecular weight ranging from 40 kDa to 150 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging from about 300 to about 1100), m₂ is an integer from 4 to about 150, m₃ is an integer from 1 to about 75 (e.g., from 1 to about 55), and/or m₁ is an integer from 1 to about 330 (e.g, m₁ being about 15-100).

[0960] For example, when the PHF in Formula (Ia) has a molecular weight ranging from 30 kDa to 100 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging from about 220 to about 740), m₂ is an integer from 3 to 100 (e.g., 5-100), m₃ is an integer from 1 to about 40, and/or m₁ is an integer from 1 to about 220 (e.g., m₁ being about 15-80).

[0961] For example, when the PHF in Formula (Ia) has a molecular weight ranging from about 50 kDa to about 100 kDa (i.e., the sum of m, m₁, m₂, and m₃ ranging from about 370 to about 740), m₂ is an integer from 5 to about 100, m₃ is an integer from 1 to about 40, and/or m₁ is an integer from 1 to about 220 (e.g, m₁ being about 15-80).

[0962] For example, the scaffold further comprises a PBRM connected to the polymeric carrier via L^P.

[0963] For example, when the PHF has a molecular weight ranging from 20 kDa to 300 kDa, (e.g., about 20-150 kDa, about 30-150 kDa, about 50-150 kDa, about 30-100 kDa, or about 50-100 kDa), the number of drugs per PHF (e.g., m₂) is an integer from about 3 to about 300, (e.g, about 3 to about 150 or about 3 to about 100). This scaffold can be used, for example, for conjugating a PBRM having a molecular weight of 200 kDa or less (e.g., 80 kDa or less, 60 kDa or less, 40 kDa or less, 20 kDa or less or 10 kDa or less). In this embodiment the ratio of PBRM per PHF is between about 1:1 and about 60:1, for example, between about 1:1 and about 30:1; between about 1:1 and about 20:1, between about 1:1 and about 10:1, between about 1:1 and about 9:1, between about 1:1 and about 8:1, between about 1:1 and about 7:1, between about 1:1 and about 6:1, between about 1:1 and about 5:1, between about 1:1 and about 4:1, between about 1:1 and about 3:1, or between about 1:1 and about 2:1. See, for example, Formula (Ib).

[0964] For example, the scaffold further comprises a PBRM connected to the polymeric carrier via L^P. For example, one or more PBRMs are connected to one drug-carrying polymeric carrier.

[0965] For example, the scaffold (e.g., a PBRM-polymer-drug conjugate) is of Formula (Ib):

[0966]
wherein:

[0967]
between L^P2 and PBRM denotes direct or indirect attachment of PBRM to L^P2,

[0968] each occurrence of PBRM independently has a molecular weight of less than 200 kDa,

[0969] m is an integer from 1 to about 2200,

[0970] m₁ is an integer from 1 to about 660,

[0971] m₂ is an integer from 3 to about 300,

[0972] m₃ is an integer from 0 to about 110,

[0973] m₄ is an integer from 1 to about 60; and

[0974] the sum of m, m₁, m₂, m₃ and m₄ ranges from about 150 to about 2200.

[0975] For example, in Formula (Ib), m₁ is an integer from about 10 to about 660 (e.g., about 10-250).

[0976] For example, when the PHF in Formula (Ib) has a molecular weight ranging from 20 kDa to 150 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ ranging from about 150 to about 1100), m₂ is an integer from 3 to about 150, m₃ is an integer from 1 to about 75 (e.g., from 1 to about 55), m₄ is an integer from 1 to about 30, and/or m₁ is an integer from 1 to about 330 (e.g., m₁ being about 10-330 or about 15-100). The PBRM in Formula (Ib), can have, for example, a molecular weight of about 4 kDa to about 70 kDa.

[0977] For example, when the PHF in Formula (Ib) has a molecular weight ranging from 40 kDa to 150 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ ranging from about 300 to about 1100), m₂ is an integer from 4 to about 150, m₃ is an integer from 1 to about 75 (e.g., from 1 to about 55), m₄ is an integer from 1 to about 30, and/or m₁ is an integer from 1 to about 330 (e.g., m₁ being about 10-330 or about 15-100).

[0978] For example, when the PHF in Formula (Ib) has a molecular weight ranging from 50 kDa to 150 kDa, the number of drugs per PHF (e.g., m₂) is an integer from 3 to about 150). The PBRM in Formula (Ib), can have, for example, a molecular weight of about 4 kDa to about 20 kDa. In this embodiment the ratio of PBRM per PHF is between about 1:1 to 10:1, between about 1:1 and about 9:1, between about 1:1 and about 8:1, between about 1:1 and about 7:1, between about 1:1 and about 6:1, between about 1:1 and about 5:1, between about 1:1 and about 4:1, between about 1:1 and about 3:1, or between about 1:1 and about 2:1.

[0979] PBRMs in this molecular weight range, include but are not limited to, for example, small proteins and peptides.

[0980] For example, when the PHF in Formula (Ib) has a molecular weight ranging from about 30 kDa to about 100 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ ranging from about 225 to about 740), m₂ is an integer from 5 to about 100, m₃ is an integer from 1 to about 40, m₄ is an integer from 1 to about 20, and/or m₁ is an integer from 1 to about 220 (e.g., m₁ being about 15-80).

[0981] For example, when the PHF has a molecular weight ranging from 30 kDa to 150 kDa, (e.g., 50-100 kDa), the number of drugs per PHF (e.g., m₂) is an integer from about 3 to about 150 (e.g., about 3 to about 100). This scaffold can be used, for example, for conjugating a PBRM having a molecular weight of about 30 kDa to about 70 kDa In this embodiment the ratio of PBRM per PHF is between about 1:1 and about 30:1, between about 1:1 and about 10:1, between about 1:1 and about 9:1, between about 1:1 and about 8:1, between about 1:1 and about 7:1, between about 1:1 and about 6:1, between about 1:1 and about 5:1, between about 1:1 and about 4:1, between about 1:1 and about 3:1, or between about 1:1 and about 2:1.

[0982] PBRMs in this molecular weight range, include but are not limited to, for example, antibody fragments such as, for example Fab.

[0983] Alternatively or additionally, one or more drug-carrying polymeric carriers are connected to one PBRM. For example, the scaffold (e.g., a PBRM-polymer-drug conjugate) comprises a PBRM with a molecular weight of greater 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):

[0984]
wherein:

[0985] terminal

[0986]
attached to L^P2 denotes direct or indirect attachment of L^P2 to PBRM such that the D-carrying polymeric carrier is connected to the PBRM,

[0987] m is an integer from 1 to 300,

[0988] m₁ is an integer from 1 to 140,

[0989] m₂ is an integer from 1 to 40,

[0990] m₃ is an integer from 0 to 18,

[0991] m₄ is an integer from 1 to 10; and

[0992] the sum of m, m₁, m₂, m₃, and m₄ ranges from 15 to 300; provided that the total number of L^P2 attached to the PBRM is 10 or less.

[0993] For example, in Formula (Ic), m₁ is an integer from 1 to about 120 (e.g., about 1-90) and/or m₃ is an integer from 1 to about 10 (e.g., about 1-8).

[0994] For example, when the PHF in Formula (Ic) has a molecular weight ranging from about 6 kDa to about 20 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ ranging from about 45 to about 150), m₂ is an integer from 2 to about 20, m₃ is an integer from 1 to about 9, and/or m₁ is an integer from 1 to about 75 (e.g., m₁ being about 4-45).

[0995] For example, when the PHF in Formula (Ic) has a molecular weight ranging from about 8 kDa to about 15 kDa (i.e., the sum of m, m₁, m₂, m₃, and m₄ ranging from about 60 to about 110), m₂ is an integer from 2 to about 15, m₃ is an integer from 1 to about 7, and/or m₁ is an integer from 1 to about 55 (e.g., m₁ being about 4-30).

[0996] For example, when the PHF has a molecular weight ranging from 2 kDa to 40 kDa, (e.g., about 6-20 kDa or about 8-15 kDa), the number of drugs per PHF (e.g., m₂) is an integer from 1 to about 40, (e.g., about 2-20 or about 2-15). This scaffold can be used, for example, for conjugating a PBRM having a molecular weight of 40 kDa or greater (e.g., 60 kDa or greater; 80 kDa or greater; or 100 kDa or greater; 120 kDa or greater; 140 kDa or greater; 160 kDa or greater or 180 kDa or greater). In this embodiment the ratio of PBRM per PHF is between about 1:1 and about 1:10, between about 1:1 and about 1:9, between about 1:1 and about 1:8, between about 1:1 and about 1:7, between about 1:1 and about 1:6, between about 1:1 and about 1:5, between about 1:1 and about 1:4, between about 1:1 and about 1:3, or between about 1:1 and about 1:2.

[0997] For example, when the PHF has a molecular weight ranging from 2 kDa to 40 kDa, (e.g., about 6-20 kDa or about 8-15 kDa), the number of drugs per PHF (e.g., m₂) is an integer from 1 to about 40, (e.g., about 1:10 or about 1-15). This scaffold can be used, for example, for conjugating a PBRM having a molecular weight of 140 kDa to 180 kDa. In this embodiment the ratio of PBRM per PHF is between about 1:1 and about 1:10, between about 1:1 and about 1:9, between about 1:1 and about 1:8, between about 1:1 and about 1:7, between about 1:1 and about 1:6, between about 1:1 and about 1:5, between about 1:1 and about 1:4, between about 1:1 and about 1:3, or between about 1:1 and about 1:2.

[0998] PBRMs in this molecular weight range, include but are not limited to, for example, full length antibodies, such as, IgG, IgM.

[0999] For example, when the PHF has a molecular weight ranging from 2 kDa to 40 kDa, the number of drugs per PHF (e.g., m₂) is an integer from 1 to about 40, (e.g., about 1:20 or about 1:15). This scaffold can be used, for example, for conjugating a PBRM having a molecular weight of 60 kDa to 120 kDa. In this embodiment the ratio of PBRM per PHF is between about 1:1 and about 1:10, between about 1:1 and about 1:9, between about 1:1 and about 1:8, between about 1:1 and about 1:7, between about 1:1 and about 1:6, between about 1:1 and about 1:5, between about 1:1 and about 1:4, between about 1:1 and about 1:3, or between about 1:1 and about 1:2.

[1000] PBRMs in this molecular weight range, include but are not limited to, for example, antibody fragments such as, for example Fab2 and camelids.

[1001] In another aspect, the invention features a polymeric scaffold useful to conjugate with both a protein based recognition-molecule (PBRM) and a therapeutic agent (D). The D-free scaffold comprises a polymeric carrier, one or more L^P connected to the polymeric carrier which is suitable for connecting a PBRM to the polymeric carrier, and one or more —R^L1—C(═O)-L^D1 connected to the polymeric carrier via R^L1, wherein:

[1002] the polymeric carrier is a polyacetal or polyketal,

[1003] R^L1 is connected to an oxygen atom of the polymeric carrier,

[1004] L^D1 is a linker suitable for connecting a D molecule to the polymeric carrier, in which each occurrence of D is independently a therapeutic agent having a molecular weight ≦5 kDa;

[1005] L^P is a linker different from —R^L1—C(═O)-L^D1, and having the structure: —R^L2—C(═O)-L^P1 with R^L2 connected to an oxygen atom of the polymeric carrier and L^P1 suitable for connecting to a PBRM;

[1006] each of R^L1 and R^L2 independently is absent, alkyl, heteroalkyl, cycloalkyl, or heterocycloalkyl;

[1007] L^D1 is a moiety containing a functional group that is capable of forming a covalent bond with a functional group of D, and

[1008] L^P1 is a moiety containing a functional group that is capable of forming a covalent bond with a functional group of a PBRM.

[1009] For example, the D-free scaffold useful to conjugate with a PBRM and a D can have one or more of the following features.

[1010] For example, L^P is a linker having the structure:

[1011]
in which L^P2 is a moiety containing a functional group that is capable of forming a covalent bond with a functional group of a PBRM, and

[1012]
denotes direct or indirect attachment of L^P2 to L^D1.

[1013] For example, the functional group of L^P1 or L^P2 is selected from —SR^p, —S—S-LG, maleimido, and halo, in which LG is a leaving group and R^p is H or a sulfur protecting group.

[1014] For example, L^D1 comprises —X—(CH₂)_v—C(═O)— with X directly connected to the carbonyl group of R^L1—C(═O), in which X is CH₂, O, or NH, and v is an integer from 1 to 6.

[1015] For example, L^P1 or L^P2 contains a biodegradable bond.

[1016] For example, each of R^L1 and R^L2 is absent.

[1017] For example, the polymeric carrier of the D-free scaffold is a polyacetal, e.g., a PHF having a molecular weight (i.e., MW of the unmodified PHF) ranging from about 2 kDa to about 300 kDa.

[1018] The D-free scaffold is of Formula (Id):

[1019]

[1020] wherein:

• • [1021] m is an integer from 1 to about 2200,
  • [1022] m₁ is an integer from 1 to about 660,
  • [1023] m₃ is an integer from 1 to about 110, and
  • [1024] the sum of m, m₁, and m₃ ranges from about 15 to about 2200.

[1025] For example, when the PHF in Formula (Id) has a molecular weight ranging from about 2 kDa to about 40 kDa (i.e., the sum of m, m₁, and m₃ ranging from about 15 to about 300), m₃ is an integer from 1 to about 18, and/or m₁ is an integer from 1 to about 140 (e.g., m₁ being about 2-120).

[1026] For example, when the PHF in Formula (Id) has a molecular weight ranging from about 6 kDa to about 20 kDa (i.e., the sum of m, m₁, and m₃ ranging from about 45 to about 150), m₃ is an integer from 1 to about 9, and/or m₁ is an integer from 1 to about 75 (e.g., m₁ being about 6-60).

[1027] For example, when the PHF in Formula (Id) has a molecular weight ranging from about 8 kDa to about 15 kDa (i.e., the sum of m, m₁, and m₃ ranging from about 60 to about 110), m₃ is an integer from 1 to about 7, and/or m₁ is an integer from 1 to about 55 (e.g., m₁ being about 6-45).

[1028] 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, m₁, and m₃ ranging from about 150 to about 2200), m₃ is an integer from 1 to about 110, and/or m₁ is an integer from 1 to about 660 (e.g., m₁ being about 13-550).

[1029] For example, when the PHF in Formula (Id) has a molecular weight ranging from 40 kDa to 150 kDa (i.e., the sum of m, m₁, and m₃ ranging from about 300 to about 1100), m₃ is an integer from 1 to about 75, and/or m₁ is an integer from 1 to about 330 (e.g., m₁ being about 20-250).

[1030] For example, when the PHF in Formula (Id) has a molecular weight ranging from about 50 kDa to about 100 kDa (i.e., the sum of m, m₁, and m₃ ranging from about 370 to about 740), m₃ is an integer from 1 to about 40, and/or m₁ is an integer from 1 to about 220 (e.g., m₁ being about 20-180).

[1031] For example, the D-free scaffold further comprises a PBRM connected to the polymeric carrier via L^P.

[1032] For example, one or more PBRMs are connected to one D-free polymeric carrier.

[1033] For example, the D-free scaffold is of Formula (Ie):

[1034]
wherein:

[1035]
between L^P2 and PBRM denotes direct or indirect attachment of PBRM to L^P2,

[1036] PBRM has a molecular weight of less than 200 kDa,

[1037] m is an integer from 1 to 2200,

[1038] m₁ is an integer from 1 to 660,

[1039] m₃ is an integer from 0 to 110,

[1040] m₄ is an integer from 1 to about 60; and

[1041] the sum of m, m₁, m₂, m₃ and m₄ ranges from about 150 to about 2200.

[1042] For example, in Formula (Ie), m₁ is an integer from about 10 to about 660 (e.g., about 14-550).

[1043] For example, when the PHF in Formula (Ie) has a molecular weight ranging from 40 kDa to 150 kDa (i.e., the sum of m, m₁, m₃, and m₄ ranging from about 300 to about 1100), m₃ is an integer from 1 to about 75, m₄ is an integer from 1 to about 30, and/or m₁ is an integer from 1 to about 330 (e.g., m₁ being about 20-250).

[1044] For example, when the PHF in Formula (Ie) has a molecular weight ranging from about 50 kDa to about 100 kDa (i.e., the sum of m, m₁, m₃, and m₄ ranging from about 370 to about 740), m₃ is an integer from 1 to about 40, m₄ is an integer from 1 to about 20, and/or m₁ is an integer from 1 to about 220 (e.g., m₁ being about 20-180).

[1045] Alternatively or additionally, one or more D-free polymeric carriers are connected to one PBRM. For example, the scaffold comprises a PBRM with a molecular weight of greater than 40 kDa and one or more polymeric carriers connected to the PBRM, in which each of the polymeric carrier independently is of Formula (Ih):

[1046]
wherein:

[1047] terminal

[1048]
attached to L^P2 denotes direct or indirect attachment of L^P2 to PBRM such that the D-carrying polymeric carrier is connected to the PBRM,

[1049] m is an integer from 1 to 300,

[1050] m₁ is an integer from 1 to 140,

[1051] m₃ is an integer from 0 to 18,

[1052] m₄ is an integer from 1 to 10; and

[1053] the sum of m, m₁, m₃, and m₄ ranges from 15 to 300; provided that the total number of L^P2 attached to the PBRM is 10 or less

[1054] For example, in Formula (Ih), m₁ is an integer from 2 to about 130 (e.g., about 3-120) and/or m₃ is an integer from 1 to about 10 (e.g., about 1-8).

[1055] 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, m₁, m₃, and m₄ ranging from about 45 to about 150), m₃ is an integer from 1 to about 9, and/or m₁ is an integer from 6 to about 75 (e.g., m₁ being about 7-60).

[1056] 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, m₁, m₃, and m₄ ranging from about 60 to about 110), m₃ is an integer from 1 to about 7, and/or m₁ is an integer from 6 to about 55 (e.g., m₁ being about 7-45).

[1057] In one embodiment the protein-polymer drug conjugate comprises a PBRM having a molecular weight of about 140 kDa to about 180 kDa (e.g., an antibody), the PHF has a molecular weight of about 8 to 15 kDa, and a load range of about 1 to about 15 of a therapeutic agent.

[1058] In one embodiment the protein-polymer drug conjugate comprises a PBRM having a molecular weight of about 60 kDa to about 120 kDa (e.g., Fab₂, camelids), the PHF has a molecular weight of about 8 to 40 kDa, and a load range of about 1 to about 20 of a therapeutic agent.

[1059] In one embodiment the protein-polymer drug conjugate comprises a PBRM having a molecular weight of about 30 kDa to about 70 kDa (e.g., Fab), the PHF has a molecular weight of about 50 to 100 kDa, and a load range of about 5 to about 100 of a therapeutic agent.

[1060] In one embodiment the protein-polymer drug conjugate comprises a PBRM having a molecular weight of about 20 kDa to about 30 kDa (e.g., scFv), the PHF has a molecular weight of about 50 to 150 kDa, and a load range of about 5 to about 150 of a therapeutic agent.

[1061] In one embodiment the protein-polymer drug conjugate comprises a PBRM having a molecular weight of about 4 kDa to about 20 kDa (e.g., a small protein), the PHF has a molecular weight of about 50 to 150 kDa, and a load range of about 5 to about 150 of a therapeutic agent.

[1062] In some embodiments, the protein-polymer drug conjugate is one of those characterized by Table 1 of FIG. 10.

[1063] In some embodiment, the protein-polymer drug conjugate is one of those characterized by Table 2 of FIG. 10.

[1064] In some embodiment, the protein-polymer drug conjugate is one of those characterized by Table 3 of FIG. 10.

[1065] In some embodiments, the protein-polymer drug conjugate includes PHF having a MW of up to 60 kDa (e.g., up to 50 kDa) and a drug to PHF ratio of up to 50:1 (e.g., about 45:1, 40:1, or 35:1).

[1066] In some embodiments, the polymeric scaffold (e.g., a polyacetal polymer such as PHF) is conjugated with PBRMs by utilizing random lysine modification. In other embodiments, the polymeric scaffold (e.g., a polyacetal polymer such as PHF) is conjugated with PBRMs by utilizing cysteine-based bioconjugation strategy. See, e.g., WO2010100430 and U.S. Pat. No. 7,595,292, the contents of which are hereby incorporated by reference in their entireties. In one embodiment, the polymeric scaffold (e.g., a polyacetal polymer such as PHF) conjugates with a PBRM (e.g., an antibody) via cysteines in the antibody hinge region. Without wishing to be bound by the theory, the resulting conjugate is stabilized through the formation of inter-chain bridge structures.

[1067] Accordingly, the invention also relates to a polymeric scaffold comprising at least two -G^X moieties connected to the polymeric scaffold, in which each -G^X is capable of conjugation to a thiol group from an amino acid (e.g., cysteine) in a PBRM so as to form a protein-polymer conjugate. In embodiments, -G^X is a maleimide group, a disulfide group, a thiol group, a triflate group, a tosylate group, an aziridine group, a 5-pydriyl functional group, a vinylsulfone group, a vinyl pyridine group, an alkyl halide group, an acrylate group or a methacrylate group.

[1068] In embodiments, one or more free thiol groups of a PBRM are produced by reducing a protein. The one or more free thiol groups of the PBRM then react with the at least two -G^X moieties contained in the polymer scaffold so as to conjugate the PBRM with the polymer scaffold.

[1069] In embodiments, the free thiol groups of the PBRM that are used for the conjugation are derived from a disulfide bridge of a native protein or a disulfide bridge of a protein complex consisting of two or more protein chains connected by the disulfide bridge. A disulfide bridge may be intrachain or interchain bridge. Alternatively, the free thiol groups of the PBRM are from cysteines or the unpaired thiol groups of the native protein that are not involved in inter or intra disulfide bridge formation.

[1070] Disulfide bonds can be reduced, for example, with dithiothreitol, mercaptoethanol, tris-carboxyethylphosphine, dehydroascorbic acid, copper sulfate, using conventional methods. A protein can contain one or more disulfide bridges. Reduction to give free thiol groups can be controlled to reduce one or more specific disulfide bridges in a protein. Depending on the extent of disulfide reduction and the stoichiometry of the -G^X moieties on the polymeric scaffold polymeric, it is possible to conjugate one or more polymer scaffolds to the protein. Immobilized reducing agents may be used if it is desired to reduce less than the total number of disulfides, as can partial reduction using different reaction conditions or the addition of denaturants.

[1071] Advantages of conjugating a polymer to a protein via a thiol include, but are not limited to optimized efficacy, improved dose to dose consistency and homogeneity (as the number of conjugated polymer molecules per protein is the substantially the same for each protein molecule), specific conjugation directed to a specific residue or residues on each protein, and easier purification. Also, the protein-polymer conjugates via the thiol conjugation exhibits substantially improved half-life, mean residence time, and/or clearance rate in circulation as compared to the unconjugated protein.

[1072] In one embodiment, the scaffold for conjugating to thiol groups in a PBRM is of Formula (IIIa):

[1073]

[1074] The wavy line in Formula (IIIa) above denotes direct or indirect attachment of -G^X to the backbone of PHF. m and m₃ are as defined herein. For example, -G^X is connected to the polymeric scaffold by a linker -L^S having the structure:

[1075]
with R^L1 and L^D1 defined as herein and

[1076]
denoting direct or indirect attachment of L^D1 to G^X.

[1077] For example, m is an integer from 1 to 2200.

[1078] For example, m₃ is an integer from 2 to 20 (e.g., an integer from 2 to 10, or an integer from 2 to 6).

[1079] In another embodiment, the scaffold for conjugating to thiol groups in a PBRM is of Formula (IIIb):

[1080]

[1081] The wavy line in Formula (IIIb) above denotes direct or indirect attachment of -G^X to the backbone of PHF. For example, -G^X is connected to the polymeric scaffold by a linker -L^S having the structure:

[1082]
with R^L1 and L^D1 defined as herein and

[1083]
denoting direct or indirect attachment of L^D1 to G^X. m, m₁, and m₃ are as defined herein.

[1084] For example, m is an integer from 1 to 2200.

[1085] For example, m₃ is an integer from 2 to 20 (e.g., an integer from 2 to 10, or an integer from 2 to 6).

[1086] For example, m₁ is an integer from 1 to 660.

[1087] In yet another embodiment, the scaffold for conjugating to thiol groups in a PBRM is of Formula (IIIc):

[1088]

[1089] The wavy line in Formula (IIIc) above denotes direct or indirect attachment of -G^X to the backbone of PHF. For example, -G^X is connected to the polymeric scaffold by a linker -L^S having the structure:

[1090]
with R^L1 and L^D1 defined as herein and

[1091]
denoting direct or indirect attachment of L^D1 to G^X. D, m, m₁, m₂, and m₃ are as defined herein.

[1092] For example, m is an integer from 1 to 2200.

[1093] For example, m₃ is an integer from 2 to 20 (e.g., an integer from 2 to 10, or an integer from 2 to 6).

[1094] For example, m₁ is an integer from 1 to 660.

[1095] For example, m₂ is an integer from 1 to 300.

[1096] In some embodiments, the drug-polymer-PBRM conjugates, drug-polymer conjugates, drug carrying-polymeric scaffolds, or PBRM-carrying polymer scaffolds described herein each have a polydispersity index (PDI) of less than 2.

[1097] PBRM-drug-polymer conjugates, drug carrying-polymeric scaffolds, or PBRM-carrying polymer scaffolds can be purified (i.e., removal of residual unreacted drug, PBRM, or polymeric starting materials) by extensive diafiltration. If necessary, additional purification by size exclusion chromatography 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 determined by SEC and <2% unconjugated PBRM as determined by RP HPLC.

[1098] Tables D and E below provide examples of the drug-carrying polymeric scaffolds and the polymer-drug-protein conjugates of the invention respectively.

[1099]

TABLE D
Ref #	Drug: PHF Ratio	Structure
Ex 9
Ex 9
	7:1 to 11:1
	11:1 to 15:1
Ex 6
Ex 18
Ex 13	24:1 to 28.1
Ex 59	11:1 to 15.1
Ex 20
	4:1 to 8:1
	11:1 to 15.1
	1:1 to 5:1
	1:1 to 5:1
Ex 26
Ex 28
Ex 31
Ex 34
Ex 37
Ex 47
Ex 44
	11:1 to 15:1
Ex 74
Ex 77
Ex 80
Ex 40
	X = CH2  1:1 to 4:1
Ex 51
	1:1 to 4:1
Ex 65	3:1 to 7:1
	1:1 to 5:1
Ex 69	1:1 to 5:1
Ex 71
	1:1 to 6:1
	1:1 to 6:1
	8:1 to 12:1
	1:1 to 5:1
Ex 90 Conjugate A	8:1 to 12:1
	1:1 to 5:1
	1:1 to 5:1
	5:1 to 10:1
	1:1 to 5:1
	12:1 to 16:1
	5:1 to 9:1
	2:1 to 6:1
	X = CH2 or —N  1:1 to 5:1
Ex 85
Ex 88

[1100]

TABLE E
	Drug:
Ref	PBRM
#	Ratio	Structure
Ex 11
Ex 7	14.1 to 17:1 19.1 to 22:1
Ex 19	10:1 to 12:1
Ex 21	47:1 to 50:1
Ex 8	16.1 to 18.1
Ex 15
Ex 17
Ex 54	12.1 to 15:1
Ex 55	~5:1
Ex 56	~10:1
Ex 57	~20:1
Ex 60	10:1 to 14:1
	9:1 to 13:1
Ex 27
Ex 29
Ex 32
Ex 35
Ex 38	2:1 to 6:1
	2:1 to 6:1
	14:1 to 18:1
Ex 41
Ex 52	19:1 to 23:1 24:1- 28:1
Ex 105 Con- ju- gate 1	8:1 to 12:1
Ex 66	9:1 to 13:1 21:1 to 25:1
Ex 70	9:1 to 13:1
Ex 72	11:1 to 15:1
Ex 105 Con- ju- gate 2	X = NH 7:1 to 11:1
Ex 94 X = NH2	40:1 to 45:1 (PHF 22 kDa) 44:1 to 48:1 (PHF 47 kDa)
Ex 94 X = NH2	37:1 to 41:1 (PHF 22 kDa) 47:1 to 51:1 (PHF 47 kDa)
Ex 92	16:1 to 20:1
Ex 93	6:1 to 8:1 (PHF 47 kDa) 6:1 to 10:1 (PHF 105 kDa)
Ex 96	6:1 to 10:1 (PHF 105 kDa or 156 kDa)
	2:1 to 6:1
	12:1 to 16:1
Ex 105 Con- ju- gate 3
	11:1 to 15:1
	X = CH2 or —NH
Ex 75	12:1 to 15:1
Ex 78	6:1 to 10:1
Ex 81	6:1 to 10:1
Ex 86	6:1 to 10:1
Ex 89	10:1 to 20:1

Synthetic Methods