WO2012037687A9 - Therapeutic polypeptides and uses thereof - Google Patents

Abstract

The present invention relates to therapeutic polypeptides having therapeutic activity. In particular, these polypeptides are useful in the treatment of cancer or diseases associated with particular cell types, organs, or tissues, including brain cancer.

Description

THERAPEUTIC POLYPEPTIDES AND USES THEREOF

Cross-Reference to Related Application

This application claims the benefit of U.S. Provisional Application No.

61/385,803, filed September 23, 2010, which is hereby incorporated by reference. Background of the Invention

This invention relates to a therapeutic polypeptide capable of both targeting and treating a tissue, and uses thereof. The invention also relates to methods of treating a subject by administering the therapeutic polypeptide with or without a therapeutic conjugate.

Peptides, such as peptide hormones, have found a variety of therapeutic uses, including the treatment of cancer. One of the challenges in treatment of patients using peptides is to ensure delivery of the polypeptide to the desired tissue. For example, delivery to brain tissues is often reduced or prevented by the blood-brain barrier (BBB).

Cancer is a disease marked by the uncontrolled growth of abnormal cells in any number of tissue types. Cancer cells have overcome the barriers imposed in normal cells, which have a finite lifespan, to grow indefinitely. As the growth of cancer cells continue, genetic alterations may persist until the cancerous cell manifests an even more aggressive growth phenotype. If left untreated, metastasis, the spread of cancer cells to distant areas of the body by way of the lymph system or bloodstream, may ensue, destroying healthy tissue. Cancer metastasis requires that the cancer cells leave the original tumor site, usually by entering the blood or lymphatic system, and spread to other regions of the body. Metastatic cells therefore must become free from the tissues in which they originally developed.

Treating and targeting of cancer and cancer metastasis involving brain tissues can be difficult due to the presence of two barrier systems: the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). The BBB is considered to be the major route for the uptake of serum ligands since its surface area is approximately 5000-fold greater than that of BCSFB. The brain endothelium, which constitutes the BBB, represents the major obstacle for the use of potential drugs against many disorders of the central nervous system (CNS). As a general rule, only small lipophilic molecules may pass across the BBB, i.e., from circulating systemic blood to brain. Many drugs that have a larger size or higher hydrophobicity show promising results in animal studies for treating CNS disorders. Thus, peptide and protein therapeutics are generally excluded from transport from blood to brain, owing to the negligible permeability of the brain capillary endothelial wall to these drugs.

Targeting of cancer cells, including metastatic cancer cells, can be difficult and is an ongoing area of research. In particular, therapy of brain diseases, including brain cancer, can be impaired by the inability of otherwise effective therapeutic agents to cross the BBB. Thus, new agents having both therapeutic activity and targeted delivery into particular tissues, including the brain, are desired.

Summary of the Invention

We have made the surprising discovery that a polypeptide capable of crossing the

BBB and targeting particular cell types as exemplified by Angiopep-2, itself has therapeutic activity, based on the discovery that these polypeptides reduce expression or activity of matrix metalloproteinase (MMP) or plasmin and reduce phosphorylation of IKB, which is an inhibitor of NF-κΒ. Because increased expression or activity of MMP, plasmin, and/or NF-κΒ is associated with cancer and/or cancer metastasis, these polypeptides can be used to treat cancer. MMP, in particular, has been associated with cancer cell migration, cancer cell invasion, and angiogenesis. The invention therefore features methods of treating a subject having cancer or at risk of developing cancer by administering the therapeutic polypeptide. The invention also features a composition including the combination of the therapeutic polypeptide and a therapeutic conjugate, where both the therapeutic polypeptide and the therapeutic conjugate are together present in an effective amount.

In a first aspect, the invention features a method of treating (e.g., prophylactically) a subject (e.g., a human) having cancer, having an increased risk of cancer (e.g., at least a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% increased risk of cancer), or in remission from cancer (e.g., in remission for 6 months, 1 year, 5 years, 10 years, or 20 years), the method including administering to the subject an effective amount of a therapeutic polypeptide, the therapeutic polypeptide including an amino acid sequence substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical) to a sequence selected from the group consisting of SEQ ID NOS: l-93, 97-105, and 107-122, or a fragment thereof (e.g., a fragment comprising any of SEQ ID NOS: 117-122), where the therapeutic polypeptide is not conjugated to a second therapeutic agent. In certain embodiments, the therapeutic polypeptide is Angiopep-2 or a fragment thereof.

In some embodiments, the cancer (e.g., metastatic cancer) is a brain cancer (e.g., glioma, mixed glioma, glioblastoma multiforme, astrocytoma, pilocytic astrocytoma, dysembryoplastic neuroepithelial tumor, oligodendroglioma, ependymoma,

oligoastrocytoma, hemangioma, medulloblastoma, retinoblastoma, neuroblastoma, germinoma, and teratoma). In other embodiments, the cancer (e.g., metastatic) is any cancer described herein (e.g., endothelial-related cancer, such as hemangioma).

In particular embodiments, the therapeutic polypeptide reduces matrix

metalloproteinase (e.g., MMP-2 or MMP-9) activity, reduces plasmin activity, reduces phosphorylation of ΙκΒ (e.g., αΙκΒ), or reduces activation of NF-κΒ. In other embodiments, the therapeutic polypeptide reduces migration or invasion of cancer cells, or reduces angiogenesis.

In any of the embodiments above, the therapeutic polypeptide is present in a composition, and the composition does not include ANG1005 or includes less than 5% (e.g., less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) of ANG1005 in the composition. In other embodiments, the therapeutic polypeptide is present in a composition, and the

composition does not include detectable levels of ANG1005 (e.g., as detected by any method known in the art, such as mass spectrometry such as MALDI-TOF, gel electrophoresis such as SDS-PAGE, fluorescence, or immunological techniques).

In a second aspect, the invention features a composition including:

(a) a therapeutic polypeptide described in the first aspect of the invention (e.g, Angiopep-2 or a fragment thereof) not conjugated to a therapeutic agent and

(b) a therapeutic conjugate (e.g., ANG1005) including a second polypeptide conjugated to a second therapeutic agent or a transport vector, where the therapeutic polypeptide and the therapeutic conjugate are together present in an effective amount, and the therapeutic polypeptide and the second polypeptide independently have an amino acid sequence substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical) to a sequence selected from the group consisting of SEQ ID NOS: 1-93, 97-105, and 107-122, or a fragment thereof (e.g., a fragment comprising any of SEQ ID

NOS: 117-122).

In some embodiments of the second aspect, the second polypeptide is conjugated to the second therapeutic agent or the transport vector by a covalent bond (e.g., a peptide bond) or a linker (e.g., at least one amino acid or an ester linker). In other embodiments, the second polypeptide is conjugated to the second therapeutic agent and the second therapeutic agent is selected from the group consisting of an anticancer agent (e.g., paclitaxel, etoposide, and doxorubicin, or an analog thereof), a therapeutic nucleic acid agent, a small molecule drug, a label, and a therapeutic peptidic agent. In some embodiments, the therapeutic conjugate is a fusion protein. In other embodiments, the therapeutic peptidic agent is a GLP-1 agonist, leptin or a leptin analog, neurotensin or a neurotensin analog, a neurotensin receptor agonist, glial-derived neurotrophic factor (GDNF) or a GDNF analog, or brain-derived neurotrophic factor (BDNF) or a BDNF analog.

In some embodiments of the second aspect, the second polypeptide is conjugated to the transport vector and the transport vector is selected from the group consisting of a lipid vector, a polyplex, a dendrimer, and a nanoparticle. In other embodiments, the transport vector is bound to or contains a third therapeutic agent (e.g., an anticancer agent, a therapeutic nucleic acid agent, a small molecule drug, a label, and a therapeutic peptidic agent).

In any of the above embodiments of the second aspect, the ratio of the therapeutic polypeptide and the therapeutic conjugate is from 1:9 to 9: 1 (e.g., any ranges between the following ratios 1:8, 1 :7, 1 :6, 1:5, 1 :4, 1 :3, 1:2, 1: 1, 2: 1, 2:3, 2:5, 2:7, 2:9, 3: 1, 3:2, 3:4, 3:5, 3:7, 3:8, 4: 1, 4:3, 4:5, 4:7, 4:9, 5: 1, 5:2, 5:3, 5:4, 5:6, 5:7, 5:8, 5:9, 6: 1, 6:5, 6:7,

7: 1,7:2, 7:3, 7:4, 7:5, 7:6, 7:8, 7:9, 8: 1, 8:3, 8:5, 8:7, 8:9, 9:2, 9:4, 9:5, 9:7, and 9:8). In other embodiments, the ratio or range of ratios may include 1: 1000, 1 :500, 1:250, 1: 100, 1 :50, 1 :25, 1:20, 1: 15, 15: 1, 20: 1, 25: 1, 50: 1, 100: 1, 250: 1, 500: 1, and 1000: 1.

In any of the above aspects, the composition further includes a pharmaceutically acceptable carrier.

In a third aspect, the invention features a method of treating (e.g.,

prophylactically) treating a subject (e.g., a human) having cancer, having an increased risk of cancer (e.g., at least a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% increased risk), or in remission from cancer (e.g., in remission for 6 months, 1 year, 5 years, 10 years, or 20 years), the method including administering to the subject any composition described herein in an effective amount. The subject having an increased risk of cancer may have been diagnosed by a physician as having an increased risk. In some embodiments, the cancer (e.g., metastatic cancer) is a brain cancer (e.g., glioma, mixed glioma, glioblastoma multiforme, astrocytoma, pilocytic astrocytoma, dysembryoplastic neuroepithelial tumor, oligodendroglioma, ependymoma, oligoastrocytoma, hemangioma, medulloblastoma, retinoblastoma, neuroblastoma, germinoma, and teratoma). In other embodiments, the cancer (e.g., metastatic) is any cancer described herein (e.g., endothelial-related cancer, such as hemangioma).

In a fourth aspect, the invention features a kit including:

(a) a therapeutic polypeptide (e.g., Angiopep-2 or a fragment thereof) not conjugated to a therapeutic agent and

(b) a therapeutic conjugate (e.g., ANG1005) including a second polypeptide conjugated to a second therapeutic agent or a transport vector, where the therapeutic polypeptide and the therapeutic conjugate are together present in an effective amount, and the therapeutic polypeptide and the second polypeptide independently have an amino acid sequence substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical) to a sequence selected from the group consisting of SEQ ID NOS: 1-93, 97-105, and 107-122, or a fragment thereof.

In any of the above aspects, therapeutic polypeptide, therapeutic conjugate, or polypeptide may include a sequence substantially identical to any of the sequences in Table 1, or a fragment thereof. In certain embodiments, the therapeutic polypeptide, therapeutic conjugate, or polypeptide includes a sequence of Angiopep-1 (SEQ ID NO:67), Angiopep-2 (SEQ ID NO:97), Angiopep-3 (SEQ ID NO: 107), Angiopep-4a (SEQ ID NO: 108), Angiopep-4b (SEQ ID NO: 109), Angiopep-5 (SEQ ID NO: 110), Angiopep-6 (SEQ ID NO: 111), or Angiopep-7 (SEQ ID NO: 112)). The therapeutic polypeptide, therapeutic conjugate, or polypeptide may be efficiently transported into a particular cell type (e.g., any one, two, three, four, or five of liver, lung, kidney, spleen, and muscle) or may cross the mammalian BBB efficiently (e.g., Angiopep-1, -2, -3, -4a, - 4b, -5, and -6). In another embodiment, the therapeutic polypeptide, therapeutic conjugate, or polypeptide is able to enter a particular cell type (e.g., any one, two, three, four, or five of liver, lung, kidney, spleen, and muscle) but does not cross the BBB efficiently (e.g., a conjugate including Angiopep-7). The therapeutic polypeptide, therapeutic conjugate, or polypeptide may be of any length, for example, at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25, 35, 50, 75, 100, 200, or 500 amino acids, or any range between these numbers. In certain embodiments, the therapeutic polypeptide, therapeutic conjugate, or polypeptide is 5 to 50 amino acids in length (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 amino acids in length, or ranges therebetween). The therapeutic polypeptide, therapeutic conjugate, or polypeptide may be produced by recombinant genetic technology or chemical synthesis.

Table 1

SEQ ID NO:

1 T F V Y G G C R A K R N N F K s A E D

2 T F Q Y G G C M G N G N N F V T E K E

3 P F F Y G G C G G N R N N F D T E E Y

4 s F Y Y G G C L G N K N N Y L R E E E

5 T F F Y G G C R A K R N N F K R A K Y

6 T F F Y G G C R G K R N N F K R A K Y

7 T F F Y G G C R A K K N N Y K R A K Y

8 T F F Y G G C R G K K N N F K R A K Y

9 T F Q Y G G C R A K R N N F K R A K Y

10 T F Q Y G G C R G K K N N F K R A K Y

11 T F F Y G G C L G K R N N F K R A K Y

12 T F F Y G G s L G K R N N F K R A K Y

13 P F F Y G G c G G K K N N F K R A K Y

14 T F F Y G G c R G K G N N Y K R A K Y

15 P F F Y G G c R G K R N N F L R A K Y

16 T F F Y G G c R G K R N N F K R E K Y

17 P F F Y G G c R A K K N N F K R A K E

18 T F F Y G G c R G K R N N F K R A K D

19 T F F Y G G c R A K R N N F D R A K Y

20 T F F Y G G c R G K K N N F K R A E Y

21 P F F Y G G c G A N R N N F K R A K Y

22 T F F Y G G c G G K K N N F K T A K Y

23 T F F Y G G c R G N R N N F L R A K Y

24 T F F Y G G c R G N R N N F K T A K Y

25 T F F Y G G s R G N R N N F K T A K Y

26 T F F Y G G c L G N G N N F K R A K Y

27 T F F Y G G c L G N R N N F L R A K Y

28 T F F Y G G c L G N R N N F K T A K Y

29 T F F Y G G c R G N G N N F K S A K Y

30 T F F Y G G c R G K K N N F D R E K Y T F F Y G G C R G K R N N F L R E K E T F F Y G G C R G K G N N F D R A K Y T F F Y G G S R G K G N N F D R A K Y T F F Y G G C R G N G N N F V T A K Y P F F Y G G C G G K G N N Y V T A K Y T F F Y G G C L G K G N N F L T A K Y S F F Y G G C L G N K N N F L T A K Y T F F Y G G C G G N K N N F V R E K Y T F F Y G G C M G N K N N F V R E K Y T F F Y G G S M G N K N N F V R E K Y P F F Y G G C L G N R N N Y V R E K Y T F F Y G G C L G N R N N F V R E K Y T F F Y G G C L G N K N N Y V R E K Y T F F Y G G C G G N G N N F L T A K Y T F F Y G G C R G N R N N F L T A E Y T F F Y G G C R G N G N N F K S A E Y P F F Y G G C L G N K N N F K T A E Y T F F Y G G C R G N R N N F K T E E Y T F F Y G G C R G K R N N F K T E E D P F F Y G G C G G N G N N F V R E K Y S F F Y G G C M G N G N N F V R E K Y P F F Y G G C G G N G N N F L R E K Y T F F Y G G C L G N G N N F V R E K Y S F F Y G G C L G N G N N Y L R E K Y T F F Y G G S L G N G N N F V R E K Y T F F Y G G C R G N G N N F V T A E Y T F F Y G G C L G K G N N F V S A E Y T F F Y G G C L G N R N N F D R A E Y T F F Y G G C L G N R N N F L R E E Y T F F Y G G C L G N K N N Y L R E E Y P F F Y G G C G G N R N N Y L R E E Y P F F Y G G S G G N R N N Y L R E E Y M R P D F C L E P P Y T G P C V A R I A R I I R Y F Y N A K A G L C Q T F V Y G Y G G C R A K R N N Y K S A E D C M R T C G P D F C L E P P Y T G P C V A R I I R Y F Y T F F Y G G C R G K R N N F K T E E Y K F F Y G G C R G K R N N F K T E E Y T F Y Y G G C R G K R N N Y K T E E Y T F F Y G G S R G K R N N F K T E E Y C T F F Y G C C R G K R N N F K T E E Y T F F Y G G C R G K R N N F K T E E Y C C T F F Y G S C R G K R N N F K T E E Y T F F Y G G S R G K R N N F K T E E Y C P F F Y G G C R G K R N N F K T E E Y T F F Y G G C R G K R N N F K T K E Y T F F Y G G K R G K R N N F K T E E Y T F F Y G G C R G K R N N F K T K R Y T F F Y G G K R G K R N N F K T A E Y 80 T F F Y G G K R G K R N N F K T A G Y

81 T F F Y G G K R G K R N N F K R E K Y

82 T F F Y G G K R G K R N N F K R A K Y

83 T F F Y G G C L G N R N N F K T E E Y

84 T F F Y G C G R G K R N N F K T E E Y

85 T F F Y G G R C G K R N N F K T E E Y

86 T F F Y G G C L G N G N N F D T E E E

87 T F Q Y G G C R G K R N N F K T E E Y

88 Y N K E F G T F N T K G C E R G Y R F

89 R F K Y G G C L G N M N N F E T L E E

90 R F K Y G G C L G N K N N F L R L K Y

91 R F K Y G G C L G N K N N Y L R L K Y

92 K T K R K R K K Q R V K I A Y E E I F K N Y

93 K T K R K R K K Q R V K I A Y

97 T F F Y G G S R G K R N N F K T E E Y

98 M R P D F C L E P P Y T G P C V A R I

I R Y F Y N A K A G L C Q T F V Y G G C R A K R N N F K S A E D C M R T C G G A

99 T F F Y G G C R G K R N N F K T K E Y

100 R F K Y G G C L G N K N N Y L R L K Y

101 T F F Y G G C R A K R N N F K R A K Y

102 N A K A G L C Q T F V Y G G C L A K R N N F

E S A E D C M R T C G G A

103 Y G G C R A K R N N F K S A E D C M R T C G

G A

104 G L C Q T F V Y G G C R A K R N N F K S A E

105 L C Q T F V Y G G C E A K R N N F K S A

107 T F F Y G G S R G K R N N F K T E E Y

108 R F F Y G G S R G K R N N F K T E E Y

109 R F F Y G G S R G K R N N F K T E E Y

110 R F F Y G G S R G K R N N F R T E E Y

111 T F F Y G G S R G K R N N F R T E E Y

112 T F F Y G G S R G R R N N F R T E E Y

113 C T F F Y G G S R G K R N N F K T E E Y

114 T F F Y G G S R G K R N N F K T E E Y C

115 C T F F Y G G S R G R R N N F R T E E Y

116 T F F Y G G S R G R R N N F R T E E Y C

117 F Y G G S R G K R N N F K T E E Y C

118 G G S R G K R N N F K T E E Y C

119 S R G K R N N F K T E E Y C

120 G K R N N F K T E E Y C

121 K R N N F K T E E Y C

122 K R N N F K Y C

Polypeptides Nos.5, 67, 76, and 91, include the sequences of SEQ ID NO:5, 67, 76, and 91, respectively, and are ami dated at the C-terminus. Polypeptides Nos. 107, 109, and 110 include the sequences of SEQ ID NO:97, 109, and 110, respectively, and are acetylated at the N-terminus.

In any of the above aspects, the therapeutic polypeptide, therapeutic conjugate, or polypeptide may include an amino acid sequence having the formula:

X1 -X2-X3-X4-X5-X6-X7-X8-X9-X10-X11 -X12-X13-X14-X15-X16-X17-X18-X19 where each of X1-X19 (e.g., X1-X6, X8, X9, X11-X14, and X16-X19) is, independently, any amino acid (e.g., a naturally occurring amino acid such as Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) or absent and at least one (e.g., 2 or 3) of XI, X10, and X15 is Arg. In some embodiments, X7 is Ser or Cys; or X10 and X15 each are independently Arg or Lys. In some embodiments, the residues from XI through XI 9, inclusive, are substantially identical to any of the amino acid sequences of any one of SEQ ID NOS: 1-93, 97-105, and 107-122 (e.g., Angiopep-1, Angiopep-2, Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, Angiopep-6, and Angiopep-7). In some embodiments, at least one (e.g., 2, 3, 4, or 5) of the amino acids XI -XI 9 is Arg. In some embodiments, the therapeutic polypeptide or therapeutic conjugate has one or more additional cysteine residues at the N-terminal of the sequence, the C-terminal of the sequence, or both.

In any of the above embodiments, the therapeutic polypeptide, therapeutic conjugate, or polypeptide includes the amino acid sequence Lys-Arg-X3-X4-X5-Lys (SEQ ID NO: 147), where X3 is Asn or Gin; X4 is Asn or Gin; and X5 is Phe, Tyr, or Trp; where the polypeptide is less than 200 amino acids in length (e.g., less than 150, 100, 75, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 12, 10, 11, 8, or 6 amino acids, or any range between these numbers), or a pharmaceutically acceptable salt thereof. In any of the therapeutic polypeptide, therapeutic conjugate, or polypeptide above, additions or deletions of 1, 2, 3, 4, or 5 amino acids (e.g., from 1 to 3 amino acids) may be made from the amino acid sequence Lys-Arg-X3-X4-X5-Lys. In particular embodiments, the amino acid sequence is Lys-Arg-Asn-Asn-Phe-Lys (SEQ ID NO: 123). In other embodiments, the amino acid sequence is Lys-Arg-Asn-Asn-Phe-Lys-Tyr-Cys (SEQ ID NO: 148). In yet other embodiments, the polypeptide is less than 15 amino acids in length (e.g., less than 10 amino acids in length). In some embodiments, the fragment is a deletion of 1 to 7 amino acids from the N-terminus of any sequence described herein (e.g., a sequence selected from the group consisting of SEQ ID NOS: 1-93, 97-105, and 107-122), a deletion of 1 to 5 amino acids from the C-terminus of any sequence described herein (e.g. a sequence selected from the group consisting of SEQ ID NOS: 1-93, 97-105, and 107- 122), or a combination of deletions of 1 to 7 amino acids from the N-terminus and 1 to 5 amino acids from the C-terminus of any sequence described herein (e.g., a sequence selected from the group consisting of SEQ ID NOS: 1-93, 97-105, and 107-122).

In any of the above aspects, therapeutic polypeptide, therapeutic conjugate, or polypeptide may have a C-terminus that is amidated. In other embodiments, the therapeutic polypeptide, therapeutic conjugate, or polypeptide is efficiently transported across the blood-brain barrier (e.g., the polypeptide is transported across the blood-brain barrier more efficiently than or as efficiently as Angiopep-2).

In any of the above aspects, the therapeutic conjugate has the formula A-X-B, where A is a polypeptide of any of the polypeptides above; X is a linker; and B is a therapeutic agent or a transport vector. In some embodiments, X is a covalent bond (e.g., a peptide bond), at least one amino acid (e.g., 1, 2, 3, 4, 5, 8, or 10 or more amino acids), a chemical linking agent (e.g., as described herein), or an ester linker. In certain embodiments, X is an ester linker having the formula:

where n is an integer between 2 and 15 (e.g., n is 3, 6, or 11); and either Y is a thiol on A and Z is a primary amine on B or Y is a thiol on B and Z is a primary amine on A.

In certain embodiments of any of the above aspects, the linkers can be monovalent or polyvalent (e.g., homomultifunctional, heteromultifunctional, bifunctional, or trifunctional agents). In other embodiments, the linkers can include a flexible arm (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms; or a polyethylene glycol spacer, such as (PEG)_n, where n is 1-20).

In certain embodiments of a therapeutic conjugate having the formula A-X-B, B is the therapeutic agent, such as an anticancer agent (e.g., paclitaxel, etoposide, doxorubicin, or an analog thereof), a therapeutic nucleic acid agent (e.g., an RNAi agent, such as siRNA, dsRNA, miRNA, shRNA or ptgsRNA), a small molecule drug (e.g., an antibiotic, an antiproliferative agent, or a growth factor inhibitor), a label (e.g., an isotope, a radioimaging label, a fluorescent label, or a reporter molecule), or a therapeutic peptidic agent (e.g., as described herein).

In other embodiments of a therapeutic conjugate having the formula A-X-B, B is the transport vector, such as a lipid vector (e.g., a liposome, a micelle, or a lipoplex), a polyplex, a dendrimer, or a nanoparticle (e.g., a polymeric nanoparticle, a solid lipid nanoparticle, or a nanometer-sized micelle). In other embodiments, the transport vector is bound to or contains a therapeutic agent (e.g., an anticancer agent, a therapeutic nucleic acid agent, a small molecule drug, a label, and a therapeutic peptidic agent).

In any of the above aspects, the therapeutic polypeptide, therapeutic conjugate, or polypeptide is efficiently transported across the blood-brain barrier, as defined herein.

In certain embodiments of any of the above aspects, the therapeutic polypeptide, therapeutic conjugate, polypeptide, therapeutic peptidic agent, or therapeutic nucleic acid agent described herein is modified (e.g., as described herein). The therapeutic polypeptide, therapeutic conjugate, polypeptide, or therapeutic peptidic agent may be ami dated, acetylated, or both. Such modifications may be at the amino or carboxy terminus of the polypeptide. The therapeutic polypeptide, therapeutic conjugate, polypeptide, or therapeutic peptidic agent may also include peptidomimetics (e.g., those described herein) of any of the peptides described herein. The therapeutic polypeptide, therapeutic conjugate, polypeptide, therapeutic peptidic agent, or therapeutic nucleic acid agent may also include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) substitutions, deletions, or additions of amino acids or nucleic acids relative or one of the sequences described herein. In particular for the therapeutic polypeptide, therapeutic conjugate, polypeptide, or therapeutic peptidic agent, these substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids (e.g., from 1 to 3 amino acids) may be made from the amino acid sequence Lys-Arg-X3-X4-X5-Lys (SEQ ID NO: 147). The amino acid substitution(s) may be conservative or non-conservative. For example, the therapeutic polypeptide or therapeutic conjugate may have an arginine at one, two, or three of the positions corresponding to positions 1, 10, and 15 of the amino acid sequence of any of SEQ ID NO: l, Angiopep-1, Angiopep-2, Angiopep-3, Angiopep-4a, Angiopep-4b, Angiopep-5, Angiopep-6, and Angiopep-7. In certain embodiments, the therapeutic peptidic agent may have a cysteine or lysine substitution or addition at any position (e.g., a lysine substitution at the N- or C-terminal position). Other modifications include posttranslational processing or by chemical modification, including ubiquitination, pegylation, acetylation, acylation, cyclization, amidation, oxidation, sulfation, formation of cysteine, or covalent attachment of one or more therapeutic agents. In particular, cyclization may be a preferred modification.

In certain embodiments of any of the above aspects, the therapeutic polypeptide, therapeutic conjugate, or polypeptide described herein is multimeric (e.g., dimeric, trimeric, or higher order multimeric, or as described herein). The therapeutic polypeptide or polypeptide may be a multimeric polypeptide (e.g., as described herein). The therapeutic conjugate may include multimeric polypeptides and include one or more therapeutic agents or one or more transport vectors (e.g., as described herein). In some embodiments, multimeric polypeptides and conjugates include any of modifications or further conjugations described herein for polypeptides (e.g., posttranslational processing or by chemical modification, including ubiquitination, pegylation, acetylation, acylation, cyclization, amidation, oxidation, sulfation, formation of cysteine, or covalent attachment of one or more therapeutic agents).

Definitions

By "effective amount" is meant an amount of a compound sufficient to substantially improve at least one symptom or decrease some risk by at least 10%, as compared to no treatment (e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) associated with a disease or a medical condition.

By "fragment" is meant a portion of a full-length amino acid or nucleic acid sequence (e.g., any sequence described herein). Fragments may include at least 4, 5, 6, 8, 10, 11, 12, 14, 15, 16, 17, 18, 20, 25, 30, 35, 40, 45, or 50 amino acids or nucleic acids of the full length sequence. A fragment may retain at least one of the biological activities of the full length polypeptide.

By the term "having an increased risk of cancer" is meant a subject or patient population that has been identified as having at least a 10% (e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) increase in the likelihood of having cancer, as compared to the general population. The likelihood of having cancer may be determined by reviewing the medical history of a subject, including family history of disease and subject's behavior and diet, by determining the presence of a genetic mutation (e.g., in the BRCA1 or BRCA2 gene) or an increase in a tumor marker indicative of increased susceptibility, by conducting a physical examination, or by conducting a radiologic study.

By a polypeptide "linked to" a therapeutic agent or a transport vector is meant a covalent or non-covalent interaction between the polypeptide and the therapeutic agent or the transport vector. Non-covalent interactions include, but are not limited to, hydrogen bonding, ionic interactions among charged groups, electrostatic binding, van der Waals interactions, hydrophobic interactions among non-polar groups, lipophobic interactions, and LogP-based attractions.

By "ANG1005" is meant an Angiopep-2 (SEQ ID NO: 97) polypeptide conjugated to three paclitaxel molecules having the following structure:

By a "multimeric polypeptide" is meant a polypeptide including more than one sequence capable of crossing the BBB or targeting a particular cell type (e.g., as described herein), where each sequence can be the same or different.

By a "multimeric therapeutic polypeptide" is meant a polypeptide including more than one sequence capable of crossing the BBB or targeting a particular cell type (e.g., as described herein), where each sequence can be the same or different, and is a therapeutic polypeptide that is not conjugated to a second therapeutic agent.

By "RNAi agent" is meant any agent or compound that exerts a gene silencing effect by way of an RNA interference pathway. RNAi agents include any nucleic acid molecules that are capable of mediating sequence-specific RNAi, for example, a short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, and post- transcriptional gene silencing RNA (ptgsRNA). By "substantially identical" is meant a polypeptide or nucleic acid exhibiting at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 99% identity to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 4 (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, or 100) amino acids. For nucleic acids, the length of comparison sequences will generally be at least 60 nucleotides, preferably at least 90 nucleotides, and more preferably at least 120 nucleotides, or full length. It is to be understood herein that gaps may be found between the amino acids of sequences that are identical or similar to amino acids of the original polypeptide. The gaps may include no amino acids or one or more amino acids that are not identical or similar to the original polypeptide. Percent identity may be determined, for example, with an algorithm GAP, BESTFIT, or FASTA in the Wisconsin Genetics Software Package Release 7.0, using default gap weights.

By "subject" is meant a human or non-human animal (e.g., a mammal).

By "subject in remission from cancer" is meant a subject previously diagnosed within cancer and currently in a period of time when the cancer is responding to treatment or is under control. Remission can be a complete remission, where the symptoms of cancer are not present and cancer cells cannot be detected by a standard test for detecting that cancer (e.g., by a physical examination, a radiologic study, or a measurement of tumor marker levels from a sample, such as a blood, tissue, or urine sample). Remission also can be a partial remission, where the tumor decreases in size but does not completely disappear. Remission can also be for any period of time (e.g., 6 months, 1 year, 5 years, 10 years, or 20 years) before recurrence.

By "therapeutic agent" is meant an agent that is capable of being used in the treatment or prophylactic treatment of a disease or condition or in the diagnosis of a disease or a condition.

By "therapeutic conjugate" is meant a compound having a polypeptide and a therapeutic agent or a transport vector (e.g., any described herein) linked to the polypeptide.

By "therapeutic nucleic acid agent" is meant a RNA-based or DNA-based therapeutic agent.

By "therapeutic peptidic agent" is meant a protein-based or peptide-based therapeutic agent. By a therapeutic polypeptide, polypeptide, or therapeutic conjugate which is "transported across the blood-brain barrier" is meant a therapeutic polypeptide, polypeptide, or therapeutic conjugate that is able to cross the BBB (e.g., 25%, 50%, 100%, 200%, 500%, 1,000%, 5,000%, or 10,000%) greater extent than either a control substance, or, in the case of a conjugate, as compared to the unconjugated agent, or that is able to cross the BBB at least 10% as efficiently as Angiopep-2 (e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 99% as efficiently) or more efficiently than Angiopep-2 (e.g., 25%, 50%, 100%, 200%, 500%, 1,000%, 5,000%, or 10,000% more efficiently). Ability to cross the BBB may be determined using any method known in the art (e.g., an in vitro model of the BBB or in situ brain perfusion as described in U.S. Patent No. 7,557,182).

By a therapeutic polypeptide, polypeptide, or therapeutic conjugate which is "efficiently transported to a particular cell type" is meant that the therapeutic polypeptide, polypeptide, or therapeutic conjugate is able to accumulate (e.g., either due to increased transport into the cell, decreased efflux from the cell, or a combination thereof) in that cell type to at least a 10% (e.g., 25%, 50%, 100%, 200%, 500%, 1,000%, 5,000%, or 10,000%) greater extent than either a control substance, or, in the case of a conjugate, as compared to the unconjugated agent. Such activities are described in detail in

International Application Publication No. WO 2007/009229, hereby incorporated by reference.

By "transport vector" is meant any compound or composition (e.g., lipid, carbohydrate, polymer, or surfactant) capable of binding or containing a therapeutic agent. The transport vector may be capable of transporting the agent, e.g., such as a small molecule drug or therapeutic peptidic agent. Exemplary transport vectors include lipid micelles, liposomes, lipoplexes, dendrimers, and nanoparticles.

By "treating" a disease, disorder, or condition in a subject is meant reducing at least one symptom of the disease, disorder, or condition by administrating a therapeutic polypeptide or a therapeutic conjugate to the subject.

By "prophylactically treating" a disease, disorder, or condition in a subject is meant reducing the frequency of occurrence or severity of (e.g., ameliorating) a disease, disorder or condition by administering to the subject a therapeutic polypeptide or a therapeutic conjugate to the subject prior to the appearance of a disease symptom or symptoms. Other features and advantages of the invention will be apparent from the following Detailed Description and the claims.

Brief Description of the Drawings

Figures 1A-1B show the inhibition of phorbol 12-myristate 13 -acetate (PMA)- mediated secretion of MMP-9 in DAOY medulloblastoma cells by Angiopep-2. Figure 1 A is a photograph of a gel showing vehicle (control), PMA, or TNF-mediated secretion of proMMP-9 or proMMP-2 with or without treatment with Angiopep-2 (Angiopep). Figure IB is a graph showing inhibition of PMA-mediated secretion of proMMP-2 and proMMP-9 by Angiopep-2.

Figures 2A-2B show minimal phosphorylation of αΙκΒ in U87 glioblastoma cells by Angiopep-2. Figure 2A is a photograph of a gel showing presence of phosphorylated IKB (phospho-ΙκΒ) after treatment with Angiopep-2 (Angiopep), PMA, or TNF. Figure 2B is a graph showing phospho-ΙκΒ expression after treatment as a function of time.

Figures 3A-3B show prevention of PMA-mediated phosphorylation of αΙκΒ in

U87 glioblastoma cells pre-incubated with Angiopep-2 for 30 minutes. Figure 3A is a photograph of a gel showing presence of phosphorylated IKB (phospho-ΙκΒ) after preincubation with Angiopep-2 (Ang) and later treatment with PMA or TNF. Figure 3B is a graph showing phospho-ΙκΒ expression after treatment as a function of time.

Figures 4A-4B show inhibition of MMP-2 activity in U87 glioblastoma cells by

Angiopep-2 (An-2). Figure 4A is a photograph of a zymographic assay showing inhibition of MMP-2 for degrading gelatin. Figure 4B is a graph showing gelatinolytic activity of MMP-2 for various concentrations of Angiopep-2.

Figure 5A-5C show inhibition of MMP-2 activity and plasmin activity in U87 glioblastoma cells by Angiopep-2 (An-2). Figure 5A is a photograph of a zymographic assay showing inhibition of MMP-2 and plasmin for degrading gelatin and plasminogen. Figure 5B is a graph showing gelatinolytic activity of MMP-2 for various concentrations of Angiopep-2. Figure 5C is a graph showing activity of plasmin for various

concentrations of Angiopep-2.

Detailed Description

We have unexpectedly discovered that the polypeptides of the invention, as exemplified by Angiopep-2, have therapeutic activity, based on the discovery that these polypeptides reduce expression or activity of matrix metalloproteinase (MMP) or plasmin and reduce phosphorylation of ΙκΒ (e.g., αΙκΒ), which is an inhibitor of NF-κΒ. Because increased expression or activity of MMP, plasmin, and/or NF-κΒ is associated with cancer and/or cancer metastasis, these polypeptides can be used to treat cancer. In addition, because these peptides cross the BBB, they are useful in treating cancers protected by the BBB. These polypeptides may both act as a therapeutic or biologically active compound and target tissues (e.g., the brain across the BBB or into particular cell types or tissues). These polypeptides can be used alone as the active agent or in combination with a therapeutic conjugate to cross the BBB or enter particular cell types (e.g., as described herein). Therapeutic polypeptides and therapeutic conjugates, and their use in treatment of disease, are described in detail below.

Matrix metalloproteinases (MMPs) have a central role in many cellular processes, including extracellular matrix degradation, cellular migration, cellular proliferation, cell- cell adhesion, apoptosis, tumor development, and metastasis. In cancer, MMPs have been implicated in migration or invasion of cancer cells and angiogenesis. In particular, MMP-2 and MMP-9 play a role in degradation of basement membrane proteins, migration of tumor cells to blood vessels, and activation of growth factors that stimulate tumor cell growth. Generally, MMPs are present as a zymogen (i.e., proMMPs) that is activated by a protease. Furthermore, the plasmin/plasminogen cascade may interact with pathways that activate MMPs, where plasmin is known to activate MMPs (e.g., MMP-3 or MMP-9, under particular conditions). Plasmin is a proteolytic enzyme involved in fibrinolysis, wound healing, organ repair, and degradation of ECM. In its inactive form, plasmin is present as plasminogen and activated by numerous proteases (e.g., tissue plasminogen activator, urokinase plasminogen activator, and Factor XII). Increased expression of plasminogen and/or conversion of plasminogen to plasmin could contribute to the invasiveness of tumors and to promote metastasis. Thus, agents that reduce the expression or activity of MMPs (e.g., MMP-2 or MMP-9), proMMPs (e.g., proMMP-2 or proMMP-9), plasmin, or plasminogen could be used to treat cancer and cancer metastasis. NF-KB is a key transcription factor implicated in many diseases and physiological responses, including responding to various stimuli and regulating the immune response. Numerous oncogenes, cytokines, and carcinogens activate NF-κΒ, which further leads to increased transcription of genes that promote tumor growth and progression. Furthermore, agents that interfere with NF-κΒ activity can have chemotherapeutic activity. ΙκΒ proteins are a class of proteins that inhibit NF-κΒ. In its inactive state, NF- KB is present as a dimer bound to an ΙκΒ protein in the cytoplasm. Upon phosphorylation by IKB kinase, the ΙκΒ protein is ubiquinated, undergoes proteasomal degradation, and releases the active NF-κΒ complex. Examples of IKB proteins include ΙκΒα, ΙκΒβ, ΙκΒγ, ΙκΒε, and Bcl-3. Based on this relationship, activity of NF-κΒ can be reduced by inhibiting the activation or phosphorylation of IKB. Thus, agents that reduce the activation of NF-κΒ and the phosphorylation of IKB could be used to treat cancer.

The Examples herein describe a therapeutic polypeptide that inhibits or reduces the activity of MMPs or plasmin and reduces the phosphorylation of IKB. Thus, the present invention encompasses polypeptides having therapeutic activity resulting from reducing activity of any useful target protein (e.g., MMPs, plasmin, NF-κΒ, and/or IKB). Therapeutic activity can also be determined by its effect in treating or prophylactically treating any disease, disorder, or condition, as described herein.

Therapeutic polypeptides

The invention features polypeptides having therapeutic activity, as described herein. Thus, these polypeptides can be used alone to treat a disease or used in combination with a therapeutic conjugate.

The therapeutic polypeptides and therapeutic conjugates can feature any sequence of SEQ ID NOS: 1-93, 97-105, and 107-122, or a fragment or analog thereof. In certain embodiments, the therapeutic polypeptide or conjugate may have at least 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or even 100% identity to a sequence described herein. The therapeutic polypeptide or conjugate may have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) substitutions relative to one of the sequences described herein. The therapeutic polypeptide or conjugate may have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) additions and deletions of amino acids relative to one of the sequences described herein. Other modifications are described in greater detail below.

The invention also features functional fragments of the sequences described herein. In certain embodiments, the fragments have therapeutic activity, as described herein. Truncations of the sequence may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more amino acids from either the N-terminus of the sequence, the C-terminus of the sequence, or a combination thereof. Other fragments include sequences where internal portions are deleted. Deletions of the sequence may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more amino acids from the internal portion of the sequence.

Fragments and analogs of the invention include those polypeptides with a consensus sequence of Lys-Arg-X3-X4-X5-Lys, where

X3 is Asn or Gin;

X4 is Asn or Gin; and

X5 is Phe, Tyr, or Trp.

This consensus sequence includes the amino acid sequence Lys-Arg-Asn-Asn-Phe-Lys (SEQ ID NO: 123) and conservative substitutions. Conservative substitutions and derivatives of amino acids and peptides are well known in the art and can be determined by any useful methods (e.g., by using a substitution matrix or any other method described herein). An analog or derivative of a polypeptide includes a sequence containing one or more conservative substitutions selected from the following groups or a subset of these groups: Ser, Thr, and Cys; Leu, He, and Val; Glu and Asp; Lys and Arg; Phe, Tyr, and Trp (e.g., Phe and Tyr); and Gin, Asn, Glu, Asp, and His (e.g., Gin and Asn).

Conservative substitutions may also be determined by other methods, such as by the BLAST (Basic Local Alignment Search Tool) algorithm, the BLOSUM substitution matrix (e.g., BLOSUM 62 matrix), and PAM substitution matrix (e.g., PAM 250 matrix).

The polypeptides of the invention include additions and deletions of amino acids to the consensus sequence of Lys-Arg-X3-X4-X5-Lys (SEQ ID NO: 147), where X3-X5 are as defined above. The deletions or additions can include any part of the consensus sequence of Lys-Arg-X3-X4-X5-Lys (SEQ ID NO: 147) or Lys-Arg-Asn-Asn-Phe-Lys (SEQ ID NO: 123). In some embodiments, deletions or additions of 1, 2, 3, 4, or 5 amino acids may be made from the consensus sequence. In particular embodiments, the deletions or additions may be from 1 to 3 amino acids.

Any useful substitutions, additions, and deletions can be made that does not destroy significantly a desired biological activity (e.g., therapeutic activity, ability to cross the BBB, or ability to enter a particular cell type). The modification may reduce (e.g., by at least 5%, 10%, 20%, 25%, 35%, 50%, 60%, 70%, 75%, 80%, 90%, or 95%), may have no effect, or may increase (e.g., by at least 5%, 10%, 25%, 50%, 100%, 200%, 500%, or 1000%) the biological activity of the consensus sequence or original sequence. Furthermore, additions and deletions may have or may optimize a characteristic of the consensus sequence or polypeptide, such as charge (e.g., positive or negative charge), hydrophilicity, hydrophobicity, in vivo stability, bioavailability, toxicity, immunological activity, immunological identity, and conjugation properties. For example, positive charge can be promoted by deleting one or more amino acids (e.g., from 1 to 3 amino acids) that are not basic/positively charged (as described below based on common side chain properties) or less positively charged (e.g., as determined by pKa). In another example, positive charge can be promoted by inserting one or more amino acids (e.g., from 1 to 3 amino acids) that are basic/positively charged or more positively charged (e.g., as determined by pKa).

Substantial modifications in function or immunological identity are accomplished by selecting substitutions, additions, and deletions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the bulk of the side chain.

Naturally occurring residues are divided into groups based on common side chain properties:

(1) hydrophobic: norleucine, methionine (Met), Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine (He), Histidine (His), Tryptophan (Trp), Tyrosine (Tyr), and Phenylalanine (Phe);

(2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), and Threonine (Thr);

(3) acidic/negatively charged: Aspartic acid (Asp) and Glutamic acid (Glu);

(4) basic: Asparagine (Asn), Glutamine (Gin), Histidine (His), Lysine (Lys), and Arginine (Arg);

(5) residues that influence chain orientation: Glycine (Gly) and Proline (Pro);

(6) aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe), and

Histidine (His);

(7) polar: Ser, Thr, Asn, Gin;

(8) basic positively charged: Arg, Lys, His; and

(9) charged: Asp, Glu, Arg, Lys, His.

Other amino acid substitutions are listed in Table 2. Table 2: Amino acid substitutions

Identification of therapeutic polypeptides

Additional therapeutic polypeptides may be identified by using one of the assays or methods described herein. For example, a candidate polypeptide may be produced by conventional peptide synthesis and administered to a laboratory animal. A biologically active polypeptide may be identified, for example, based on its ability to increase survival of an animal injected with tumor cells and treated with the polypeptide as compared to a control which has not been treated with a polypeptide (e.g., treated with a vehicle). For example, a biologically active polypeptide may be identified based on its location in the parenchyma in an in situ cerebral perfusion assay. In another example, a biologically- active polypeptide can be identified by its protective effect, where an animal is treated with the polypeptide, stimulated with an agent to induce cellular migration or proliferation (e.g., phorbol 12-myristate 13 -acetate), and then compared to a control that has not been treated with the polypeptide. In another example, a biologically active peptide can be identified by using an in vitro cellular assay and based on its ability to regulate intracellular processes (e.g., inhibit one or more of a matrix metalloproteinase, IKB, or NF-KB activity). Similarly, additional therapeutic conjugates can be identified using the various assays or methods described herein, where the candidate polypeptide is first conjugated with a therapeutic agent (e.g., paclitaxel).

Assays to determine accumulation in other tissues may be performed as well.

Labeled polypeptides can be administered to an animal, and accumulation in different organs can be measured. For example, a candidate polypeptide conjugated to a detectable label (e.g., a near-IR fluorescence spectroscopy label such as Cy5.5) allows live in vivo visualization. Such a polypeptide can be administered to an animal, and the presence of the polypeptide in an organ can be detected, thus allowing determination of the rate and amount of accumulation of the polypeptide in the desired organ. In other embodiments, the polypeptide can be labeled with a radioactive isotope (e.g., ¹²⁵I). The polypeptide is then administered to an animal. After a period of time, the animal is sacrificed, and the organs are extracted. The amount of radioisotope in each organ can then be measured using any means known in the art. By comparing the amount of a labeled candidate polypeptide in a particular organ relative to the amount of a labeled control polypeptide, the ability of the candidate polypeptide to access and accumulate in a particular tissue can be ascertained. Appropriate negative controls include any peptide or polypeptide known not to be efficiently transported into a particular cell type (e.g., a peptide related to Angiopep that does not cross the BBB or any other peptide).

Diseases and conditions

The therapeutic polypeptides described herein can be used to treat a variety of diseases and conditions, in particular to treat cancer. Because the therapeutic polypeptide itself has biological activity (e.g., inhibition of matrix metalloproteinase, plasmin, NF-KB, and/or IKB phosphorylation), this polypeptide can be used to treat diseases and conditions that can benefit from that activity. In addition, the polypeptide of the invention can be conjugated to a therapeutic agent and used for diseases and conditions that can be treated by that agent.

Biological activity of the therapeutic polypeptide

The compounds of the invention (e.g., a therapeutic polypeptide or a therapeutic conjugate) have various therapeutic activities. As described below in the Examples, the therapeutic polypeptide itself can inhibit matrix metalloproteinase (MMP), plasmin, and/or ΙκΒ activity. Thus, the therapeutic polypeptide, either alone or in combination with a therapeutic conjugate, may be used to treat diseases or conditions associated with dysregulation of MMPs (e.g., MMP-2 and MMP-9), plasmin, NF-κΒ, or ΙκΒ.

Exemplary diseases involving MMP regulation include bone and growth plate disorders (e.g., arthritis, articular cartilage degeneration, chondrodysplasia, osteoarthritis, rheumatoid arthritis, and synovial joint arthritis), cancer (e.g., adenocarcinoma, bladder cancer, breast cancer, bladder cancer, cervical cancer, colon cancer, colorectal cancer, cutaneous basal cell carcinoma, cutaneous squamous cell carcinoma, endometrial cancer, endothelial-related cancer (e.g., hemangioma), esophageal cancer, gastric cancer, gastric cardia adenocarcinoma, lung cancer, nasopharyngeal cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, urothelial cancer, and any cancer described herein), cardiovascular disorders (e.g., aortic aneurysm, such as abdominal or thoracic aortic aneurysms; atherosclerosis, including carotid or coronary atherosclerosis; heart failure, including progressive and chronic heart failure; left ventrical dysfunction; myocardial infarct; and restenosis), central nervous system disorders (e.g., Alzheimer's disease; brain cancer, including gliomas or any brain cancers described herein, such as medulloblastoma); brain hemorrhage; dementia; inflammatory myopathy; ischemic brain injury; meningitis; multiple sclerosis; and any other CNS disorder described herein), inflammatory or immune disorders (e.g., asthma, chronic obstructive pulmonary disorder, periodontitis, psoriasis, and rheumatoid arthritis), metabolic disorders (e.g., bone density disorders; diabetes, such as type 1 or type 2; kidney failure; and nephropathy), and vision disorders (e.g., glaucoma, including primary open angle glaucoma; and macular degeneration).

Exemplary disease involving upregulation of plasmin or plasminogen include cancer (e.g., breast cancer; colon cancer; colorectal cancer; endothelial-related cancer, such as hemangioma; lung cancer, such as non-small cell lung cancer and squamous cell lung cancer; medulloblastoma; and ovarian cancer).

Exemplary disease involving NF-κΒ and ΙκΒ include cancer (e.g., breast cancer, colorectal cancer, endothelial-related cancer, melanoma, and multiple myeloma), cardiovascular disorders (e.g., vascular disease), infection (e.g., bronchiolitis, pneumonia, and septic shock), inflammatory or immune disorders (e.g., arthritis, atopic dermatitis, diabetes, diabetic retinopathy, ectodermal dysplasia, inflammatory bowel disease, psoriasis, sarcoidosis, and ulcerative colitis), and pulmonary disease.

Cancer therapy

Compounds of the invention (e.g., a therapeutic polypeptide or a therapeutic conjugate) may be used to treat any brain or central nervous system disease (e.g., a brain cancer such as glioblastoma, astrocytoma, glioma, medulloblastoma, and oligodendroma, neuroglioma, ependymoma, and meningioma). The compounds of the invention (e.g., a therapeutic polypeptide or a therapeutic conjugate) can be used for transport to the liver, eye, lung, kidney, spleen, muscle, or ovary and may also be used, in conjunction with an appropriate therapeutic agent, to treat a disease associated with these tissues (e.g., a cancer such as hepatocellular carcinoma, liver cancer, small cell carcinoma (e.g., oat cell cancer), mixed small cell/large cell carcinoma, combined small cell carcinoma, and metastatic tumors).

Metastatic cancer can originate from cancer of any tissue, including any described herein. Exemplary metastatic cancers include those originating from brain cancer, breast cancer, colon cancer, prostate cancer, ovarian cancer, sarcoma, bladder cancer, neuroblastoma, Wilm's tumor, lymphoma, non-Hodgkin's lymphoma, and certain T-cell lymphomas.

Additional exemplary cancers that may be treated using a composition of the invention include hepatocellular carcinoma, breast cancer, cancers of the head and neck including various lymphomas such as mantle cell lymphoma, non-Hodgkin's lymphoma, endothelial-related cancer such as hemangioma, adenoma, squamous cell carcinoma, laryngeal carcinoma, cancers of the retina, cancers of the esophagus, multiple myeloma, ovarian cancer, uterine cancer, melanoma, colorectal cancer, bladder cancer, prostate cancer, lung cancer (including non-small cell lung carcinoma), pancreatic cancer, cervical cancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adenocarcinoma, parotid adenocarcinoma, endometrial sarcoma, multidrug resistant cancers, proliferative diseases and conditions (e.g., such as neovascularization associated with tumor angiogenesis, macular degeneration (e.g., wet/dry AMD), corneal neovascularization, diabetic retinopathy, neovascular glaucoma, myopic degeneration), and other proliferative diseases and conditions (e.g., such as restenosis and polycystic kidney disease). Brain cancers that may be treated with vector that is transported efficiently across the BBB include glioma, mixed glioma, glioblastoma multiforme, astrocytoma, pilocytic astrocytoma, dysembryoplastic neuroepithelial tumor, oligodendroglioma, ependymoma, oligoastrocytoma, hemangioma, medulloblastoma, retinoblastoma, neuroblastoma, germinoma, and teratoma.

Administration and dosage

The present invention also features pharmaceutical compositions that contain a therapeutically effective amount of a therapeutic polypeptide and/or a conjugate of the invention. The composition can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the composition for proper formulation. Suitable formulations for use in the present invention are found in Remington 's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed., 1985. For a brief review of methods for drug delivery, see, e.g., Langer, Science 249: 1527-1533 (1990).

The pharmaceutical compositions are intended for parenteral, intranasal, topical, oral, or local administration, such as by a transdermal means, for prophylactic and/or therapeutic treatment. The pharmaceutical compositions can be administered parenterally (e.g., by intravenous, intramuscular, or subcutaneous injection), or by oral ingestion, or by topical application or intraarticular injection at areas affected by the vascular or cancer condition. Additional routes of administration include intravascular, intra-arterial, intratumoral, intraperitoneal, intraventricular, intraepidural, as well as nasal, ophthalmic, intrascleral, intraorbital, rectal, topical, or aerosol inhalation administration. Sustained release administration is also specifically included in the invention, by such means as depot injections or erodible implants or components. Thus, the invention provides compositions for parenteral administration that include the above mention agents dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, e.g., water, buffered water, saline, PBS, and the like. The compositions may contain

pharmaceutically acceptable auxiliary substances as required to approximate

physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents and the like. The invention also provides compositions for oral delivery, which may contain inert ingredients such as binders or fillers for the formulation of a tablet, a capsule, and the like. Furthermore, this invention provides compositions for local administration, which may contain inert ingredients such as solvents or emulsifiers for the formulation of a cream, an ointment, and the like.

These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules. The composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.

The compositions containing an effective amount can be administered for prophylactic or therapeutic treatments. These treatments can be administered to a subject having cancer, a subject having an increased risk of having cancer, or a subject in remission from cancer. In prophylactic applications, compositions can be administered to a subject with a clinically determined predisposition or increased susceptibility to cancer or metastatic cancer (e.g., having a genetic mutation or an increase in a tumor marker indicative of increased susceptibility). These prophylactic applications also include administration to a subject in remission of cancer or metastatic cancer (e.g., in remission for any period of time, such as 1 year, 5 years, 10 years, or 20 years). Compositions of the invention can be administered to the subject (e.g., a human) in an amount sufficient to delay, reduce, or preferably prevent the onset of clinical disease or the recurrence of clinical disease.

In therapeutic applications, compositions are administered to a subject (e.g., a human) already suffering from disease (e.g., cancer or metastatic cancer) in an amount sufficient to ameliorate or at least partially arrest the symptoms of the condition and its complications. For example, in the treatment of cancer or metastatic cancer (e.g., those described herein), an agent or compound that decreases, prevents, delays, suppresses, or arrests any symptom of the disease or condition would be therapeutically effective. An effective amount of an agent or compound is not required to cure a disease or condition but will provide a treatment for a disease or condition, such that the onset of the disease or condition is delayed, hindered, or prevented, or the disease or condition symptoms are ameliorated, or the term of the disease or condition is changed or, for example, is less severe or recovery is accelerated in an individual.

Effective amounts for this use may depend on the severity of the disease or condition and the weight and general state of the subject, but generally range from about 0.05 μg to about 1000 μg (e.g., 0.5-100 μg) of an equivalent amount of the compound per dose per subject. Suitable regimes for initial administration and booster administrations are typified by an initial administration followed by repeated doses at one or more hourly, daily, weekly, or monthly intervals by a subsequent administration. The total effective amount of a compound (e.g., a therapeutic polypeptide, therapeutic conjugate, or therapeutic agent) present in the compositions of the invention can be administered to a mammal as a single dose, either as a bolus or by infusion over a relatively short period of time, or can be administered using a fractionated treatment protocol, in which multiple doses are administered over a more prolonged period of time (e.g., a dose every 4-6, 8-12, 14-16, or 18-24 hours, or every 2-4 days, 1-2 weeks, once a month). Alternatively, continuous intravenous infusions sufficient to maintain therapeutically effective concentrations in the blood are contemplated.

The effective amount of one or more compounds present within the compositions of the invention and used in the methods of this invention applied to mammals (e.g., humans) can be determined by the ordinarily-skilled artisan with consideration of individual differences in age, weight, and the condition of the mammal. Because certain compounds of the invention exhibit an enhanced ability to cross the BBB, the dosage of the compounds of the invention can be lower than (e.g., less than or equal to about 90%, 75%, 50%, 40%, 30%, 20%, 15%, 12%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of) the equivalent dose of required for a therapeutic effect of the unconjugated agent. The agents of the invention are administered to a subject (e.g. a mammal, such as a human) in an effective amount, which is an amount that produces a desirable result in a treated subject (e.g., preservation of neurons, new neuronal growth). Effective amounts can also be determined empirically by those of skill in the art.

The subject may also receive a compound in the range of about 0.05 to 1,000 μg equivalent dose as compared to unconjugated agent per dose one or more times per week (e.g., 2, 3, 4, 5, 6, or 7 or more times per week), 0.1 to 2,500 (e.g., 2,000, 1,500, 1,000, 500, 100, 10, 1, 0.5, or 0.1) μg dose per week. A subject may also receive a compound of the composition in the range of 0.1 to 3,000 μg per dose once every two or three weeks. Single or multiple administrations of the compositions of the invention including an effective amount can be carried out with dose levels and pattern being selected by the treating physician. The dose and administration schedule can be determined and adjusted based on the severity of the disease or condition in the subj ect, which may be monitored throughout the course of treatment according to the methods commonly practiced by clinicians or those described herein.

The compounds of the present invention may be used in combination with either conventional methods of treatment or therapy or may be used separately from

conventional methods of treatment or therapy.

When the compounds of this invention are administered in combination therapies with other agents, they may be administered sequentially or concurrently to an individual. Alternatively, pharmaceutical compositions according to the present invention may be comprised of a combination of a compound of the present invention in association with a pharmaceutically acceptable excipient, as described herein, and another therapeutic or prophylactic agent known in the art.

Therapeutic conjugates

In certain embodiments, the therapeutic polypeptide is administered or the polypeptide of the invention can be linked to a therapeutic agent or a transport vector to form a therapeutic conjugate. In a conjugate, the polypeptide is joined by a chemical bond either directly (e.g., a covalent bond such as a disulfide or a peptide bond) or indirectly (e.g., through a linker such as those described herein). In a conjugate having a transport vector, a therapeutic agent may be releasable after transport across the BBB, for example, by enzymatic cleavage or breakage of a chemical bond between the transport vector and the agent. The released agent may then function in its intended capacity in the absence of the vector. Exemplary linkers, therapeutic agents, and transport vectors are described below.

Joining of the polypeptide to a therapeutic peptidic agent

When the conjugate includes a therapeutic peptidic agent linked to a polypeptide through a peptide bond or an amino acid or peptide linker, the resultant conjugate is a fusion protein. In some embodiments, the agent may be linked to the polypeptide by a covalent bond. The covalent bond may be a peptide bond (e.g., produced synthetically or recombinantly as a fusion protein). Exemplary therapeutic peptidic agents are described below.

Joining of the polypeptide to a transport vector

To form a conjugate including a transport vector, at least two general approaches can be used. In a first approach, a transport vector containing the agent (e.g., any described herein) is formed. Then, a polypeptide described herein is conjugated to the transport vector. In a second approach, the polypeptide is first conjugated to a transport vector molecule (i.e., a molecule capable of forming a transport vector (e.g., any described herein)), and the transport vector is formed subsequently using the conjugated transport vector molecule. In either approach, the polypeptide may be conjugated through a tether or linker molecule.

A conjugate including a transport vector can be formed in a step-wise process. For example, the transport vector molecule is first attached to the linker, and transport vectors are formed containing the transport vector molecule. Then, the transport vector is incubated with the polypeptide to form a covalent bond with the linker. In a particular example, the transport vector molecule is a lipid molecule, the lipid molecule is attached to the linker, and the resultant compound is used to form liposomes. Then, the liposomes are incubated with a solution containing the polypeptide to attach the polypeptide to the distal end of the linker.

In another example, the transport vector is covalently linked to a linker with an activated group, the polypeptide is covalently linked to a second linker, and then the modified transport vector and modified polypeptide are reacted together to form a covalent bond between the first linker and a second linker. For example, the amino group of a transport vector forms a covalent bond by displacing the N-hydroxysuccinimidyl group of the linker succinimidyl 4-formylbenzoate. This modified transport vector has a terminal carbonyl group on the linker. Then, the amino group of the polypeptide forms a covalent bond by displacing the N-hydroxysuccinimidyl group of the linker succinimidyl 4-hydrazinonicotinate acetone hydrazone. This modified polypeptide has a terminal hydrazine group on the linker. Finally, the modified transport vector and the modified polypeptide are combined to form a covalent bond between the hydrazine group of the modified polypeptide and the terminal carbonyl group of the transport vector. In another example, polyoxyethylene-(p-nitrophenyl carbonate)- phosphoethanolamine is used in the formation of lipid micelles containing siRNA molecules. Briefly, in this example, polyoxyethylene-bis (p-nitrophenyl carbonate) ((pNP)₂-PEG) is conjugated to a lipid capable of forming liposomes or micelles such as l^-dipalmitoyl-OT-glycero-S-phosphoethanolamine (DPPE), resulting in production of pNP-PEG-PE. This molecule can then, in turn, be conjugated to a polypeptide (e.g., any described herein) to form a peptide-PEG-PE conjugate. This conjugate can then be used in the formation of liposomes that contain PEG moieties which serve as anchors for binding polypeptide molecules on the external face of the liposome. See, e.g., Zhang et al, J. Control. Release 112:229-239 (2006).

Production of lipid vectors can also be achieved by conjugating a polypeptide to a liposome following its formation. In one example of this procedure, a mixture of lipids suitable for encapsulating a molecule and having sufficient in vivo stability are provided, where some of the lipids are attached to a tether (such as PEG) containing a linker (e.g., any linker described herein). The mixture is dried, reconstituted in aqueous solution with the desired polynucleotide, and subject to conditions capable of forming liposomes (e.g., sonication or extrusion). A polypeptide described herein is then conjugated to the linker on the tether. In one particular example of this method, the mixture of 93% 1-palmitoyl- 2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 3% didodecyldimethylammonium bromide (DDAB), 3% distearoylphosphatidylethanolamine (DSPE)-PEG2000, and 1% DSPE-PEG2000-maleimide is provided. This mixture is then prepared in chloroform, evaporated under nitrogen, and then dissolved in Tris buffer to which the desired polynucleotide is added. The mixture is then passed through a series of polycarbonate filters of reduced pore size 400 nm to 50 nm to generate 80-100 nm liposomes. The liposomes are mixed with a nuclease or protease to remove unencapsulated therapeutic agents. If the therapeutic agent is a DNA molecule, then DNA endonuclease I and exonuclease III can be used. The transport vector described herein can then be conjugated to the DSPE-PEG200 that contains the linker (e.g., maleimide or any linker herein). These lipid vectors, which contain a therapeutic agent and are conjugated to a polypeptide described herein, can then be administered to a subject to deliver the therapeutic agent across the BBB or to specific tissues. Further examples of this approach are described in Boado, Pharm. Res. 24: 1772-1787 (2007); Pardridge, Pharm. Res. 24: 1733-1744 (2007); and Zhang et al., Clin. Cane. Res. 10:3667-3677 (2004). Alternatively, the conjugate is formed without the use of a linker. Rather, a zero- length coupling agent is used to activate the functional groups within the transport vector or the polypeptide without introducing additional atoms. Examples of zero-length coupling agents include dicyclohexylcarbodiimide and ethylchloroformate.

Linkers

The polypeptide may be bound to a therapeutic agent or a transport vector either directly (e.g., through a covalent bond such as a peptide bond) or may be bound through a linker. Linkers include chemical linking agents (e.g., cleavable linkers) and peptides. Any of the linkers described below may be used in the compounds of the invention.

Chemical linking agents

In some embodiments, the linker is a chemical linking agent. The polypeptide may be conjugated through sulfhydryl groups, amino groups (amines), or any appropriate reactive group. Homomultifunctional and heteromultifunctional cross-linkers

(conjugation agents, including bifunctional and trifunctional agents) are available from many commercial sources. Sites available for cross-linking may be found on the polypeptides and therapeutic agents or transport vectors described herein. The cross- linker may comprise a flexible arm, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms. The flexible arm can be polyethylene glycol spacer, such as (PEG)_n, where n is 1-20.

Exemplary cross-linkers include BS³ ([bis(sulfosuccinimidyl)suberate]; BS³ is a homobifunctional N-hydroxysuccinimide ester that targets accessible primary amines), NHS/EDC (N-hydroxysuccinimide and l-ethyl-3-(3-dimethylaminopropyl)carbodiirnide; NHS/EDC allows for the conjugation of primary amine groups with carboxyl groups), sulfo-EMCS ([Ν-ε-maleimidocaproic acidjhydrazide; sulfo-EMCS are heterobifunctional reactive groups (maleimide and NHS-ester) that are reactive toward sulfhydryl and amino groups), hydrazide (most proteins contain exposed carbohydrates and hydrazide is a useful reagent for linking carboxyl groups to primary amines), SATA (N-succinimidyl-S- acetylthioacetate; SATA is reactive towards amines and adds protected sulfhydryls groups), and BMOE (bis-maleimidoethane).

To form covalent bonds, one can use as a chemically reactive group a wide variety of active carboxyl groups (e.g., esters) where the hydroxyl moiety is physiologically acceptable at the levels required to modify the peptide. Particular agents include N- hydroxysuccinimide (NHS), N-hydroxy-sulfosuccinimide (sulfo-NHS), maleimide- benzoyl-succinimide (MBS), gamma-maleimido-butyryloxy succinimide ester (GMBS), maleimido propionic acid (MP A), maleimido hexanoic acid (MHA), and maleimido undecanoic acid (MUA).

Primary amines are the principal targets for NHS esters. Accessible a-amine groups present on the N-termini of proteins and the ε-amine of lysine react with NHS esters. Thus, compounds of the invention can include a linker having a NHS ester conjugated to an N-terminal amino of a peptide or to a ε-amine of lysine. An amide bond is formed when the NHS ester conjugation reaction reacts with primary amines releasing N-hydroxysuccinimide. These succinimide containing reactive groups are herein referred to as succinimidyl groups. In certain embodiments of the invention, the functional group on the protein will be a thiol group and the chemically reactive group will be a maleimido-containing group such as gamma-maleimide-butrylarnide (GMBA or MP A). Such maleimide containing groups are referred to herein as maleimido groups.

The maleimido group is most selective for sulfhydryl groups on peptides when the pH of the reaction mixture is 6.5-7.4. At pH 7.0, the rate of reaction of maleimido groups with sulfhydryls (e.g., thiol groups on proteins such as serum albumin) is 1000-fold faster than with amines. Thus, a stable thioether linkage between the maleimido group and the sulfhydryl can be formed. Accordingly, a compound of the invention can include a linker having a maleimido group conjugated to a sulfhydryl group of a polypeptide or of an agent.

Amine-to-amine linkers include NHS esters and imidoesters. Exemplary NHS esters are DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate),

BS³ (bis[sulfosuccinimidyl] suberate), TSAT (irw-succinimidyl aminotriacetate), variants of bis-succinimide ester-activated compounds that include a polyethylene glycol spacer, such as BS(PEG)_n, where n is 1-20 (e.g., BS(PEG)₅ and BS(PEG)₉), DSP

(dithiobis [succinimidyl propionate]), DTSSP (3,3'- dithiobis[sulfosuccinimidylpropionate]), DST (disuccinimidyl tartarate), BSOCOES (bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone), EGS (ethylene glycol

bis[succinimidylsuccinate]), and sulfo-EGS (ethylene glycol

bis[sulfosuccinimidylsuccinate]). Imidoesters include DMA (dimethyl adipimidate^»2 HC1), DMP (dimethyl pimelimidate»2 HC1), DMS (dimethyl suberimidate»2 HC1), and DTBP (dimethyl 3,3'-dithiobispropionimidate^»2 HC1). Other amine-to-amine linkers include DFDNB (l,5-difluoro-2,4-dinitrobenzene) and THPP ^-[tris(hydroxymethyl) phosphino] propionic acid (betaine)).

The linker may be a sulfhydryl-to-sulfhydryl linker. Such linkers include maleimides and pyridyldithiols. Exemplary maleimides include BMOE (bis- maleimidoethane), BMB (1,4-bismaleimidobutane), BMH (bismaleimidohexane), TMEA (irw[2-maleimidoethyl]amine), BM(PEG)₂ (1,8-bis-maleimidodiethyleneglycol) or BM(PEG)_n, where n is 1 to 20 (e.g., 2 or 3), BMDB (1,4 bismaleimidyl-2,3- dihydroxybutane), and DTME (dithio-bismaleimidoethane). Exemplary pyridyldithiols include DPDPB (l,4-di-[3'-(2'-pyridyldithio)-propionamido]butane). Other sulfhydryl linkers include HBVS (1,6-hexane-bis-vinylsulfone).

The linker may be an amine-to-sulfhydryl linker, which includes NHS ester/maleimide compounds. Such amine-to-sulfhydryl linkers can include ester linkers (e.g., any linker described herein containing an ester group). Examples of these compounds are AMAS (N-(a-maleimidoacetoxy)succinimide ester), BMPS (ν-[β- maleimidopropyloxyjsuccinimide ester), GMBS (N-[y-maleiinidobutyryloxy]succinimide ester), sulfo-GMBS (N-[y-maleiinidobutyryloxy]sulfosuccinimide ester), MBS (m- maleimidobenzoyl-N-hydroxysuccinimide ester), sulfo-MBS (m-maleimidobenzoyl-N- hydroxysulfosuccinimide ester), SMCC (succinimidyl 4-[N- maleimidomethyl]cyclohexane-l-carboxylate), sulfo-SMCC (sulfosuccinimidyl 4-[N- maleimidomethyl]cyclohexane-l-carboxylate), EMCS ([Ν-ε- maleimidocaproyloxyjsuccinimide ester), sulfo-EMCS ([Ν-ε- maleimidocaproyloxyjsulfosuccinimide ester), SMPB (succinimidyl 4-\p- maleimidophenyl]butyrate), sulfo-SMPB (sulfosuccinimidyl 4-\p- maleimidophenyl]butyrate), SMPH (succinimidyl-6-[^- maleimidopropionamidojhexanoate), LC-SMCC (succinimidyl-4-[N- maleimidomethyl]cyclohexane-l-carboxy-[6-amidocaproate]), sulfo-KMUS (N-[K- maleimidoundecanoyloxyjsulfosuccinimide ester), SM(PEG)_n (succinimidyl-([N- maleimidopropionamido-polyethyleneglycol) ester), where n is 1 to 30 (e.g., 2, 4, 6, 8, 12, or 24), SPDP (N-succinimidyl 3-(2-pyridyldithio)-propionate), LC-SPDP

(succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), sulfo-LC-SPDP

(sulfosuccinimidyl 6-(3'-[2-pyridyldithio]-propionamido)hexanoate), SMPT (4- succinimidyloxycarbonyl-a-methyl-a-[2-pyridyldithio]toluene), sulfo-LC-SMPT (4- sulfosuccininiidyl-6-[a-methyl-a-(2-pyridyldithio)toluaniido]hexanoate), SIA (N- succinimidyl iodoacetate), SBAP (succinimidyl 3-[bromoacetamido]propionate), SIAB (N-succinimidyl[4-iodoacetyl]aminobenzoate), and sulfo-SIAB (N-sulfosuccinimidyl[4- iodoacetyljaminobenzoate).

In particular embodiments, the linker has the formula:

where n is an integer between 2 and 15 (e.g., n is 3, 6, or 11); and either Y is a thiol on A and Z is a primary amine on B or Y is a thiol on B and Z is a primary amine on A.

In other embodiments, the linker is an amino-to-nonselective linker. Examples of such linkers include NHS ester/aryl azide and NHS ester/diazirine linkers. NHS ester/aryl azide linkers include NHS-ASA (N-hydroxysuccinimidyl-4-azidosalicylic acid), ANB-NOS (N-5-azido-2-nitrobenzoyloxysuccinimide), sulfo-HSAB (N- hydroxysulfosuccinimidyl-4-azidobenzoate), sulfo-NHS-LC-ASA (sulfosuccinimidyl [4- azidosalicylamidojhexanoate), SANPAH (N-succinimidyl-6-(4'-azido-2'- nitrophenylamino)hexanoate), sulfo-SANPAH (N-sulfosuccinimidyl-6-(4'-azido-2'- nitrophenylamino)hexanoate), sulfo-SFAD (sulfosuccinimidyl- (perfluoroazidobenzamido)-ethyl- 1 ,3 ' -dithioproprionate), sulfo-S AND

(sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-l,3'-proprionate), and sulfo- SAED (sulfosuccinimidyl 2-[7-amino-4-methylcoumarin-3-acetamido]ethyl- 1,3'dithiopropionate). NHS ester/diazirine linkers include SDA (succinimidyl 4,4'- azipentanoate), LC-SDA (succinimidyl 6-(4,4'-azipentanamido)hexanoate), SDAD (succinimidyl 2-([4,4'-azipentanamido]ethyl)-l, 3 '-dithioproprionate), sulfo-SDA (sulfosuccinimidyl 4,4'-azipentanoate), sulfo-LC-SDA (sulfosuccinimidyl 6-(4,4'- azipentanamido)hexanoate), and sulfo-SDAD (sulfosuccinimidyl 2-([4,4'- azipentanamido] ethyl)- 1 ,3 '-dithioproprionate).

Exemplary amine-to-carboxyl linkers include carbodiimide compounds (e.g., DCC (N,N-dicyclohexylcarbodimide) and EDC (l-ethyl-3-[3- dimethylaminopropyl] carbodiimide)). Exemplary sulfhydryl-to-nonselective linkers include pyridyldithiol/aryl azide compounds (e.g., APDP ((N-[4-(p- azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide)). Exemplary sulfhydryl-to- carbohydrate linkers include maleimide/hydrazide compounds (e.g., BMPH ( ν^"-[β- maleimidopropionic acidjhydrazide), EMCH ([N-s-maleimidocaproic acidjhydrazide), MPBH 4-(4-N-maleimidophenyl)butyric acid hydrazide), and KMUH (N-[K- maleimidoundecanoic acidjhydrazide)) and pyridyldithiol/hydrazide compounds (e.g., PDPH (3-(2-pyridyldithio)propionyl hydrazide)). Exemplary carbohydrate-to- nonselective linkers include hydrazide/aryl azide compounds (e.g., ABH (p-azidobenzoyl hydrazide)). Exemplary hydroxyl-to-sulfhydryl linkers include isocyanate/maleimide compounds (e.g., (N-[p-maleimidophenyl]isocyanate)). Exemplary amine-to-DNA linkers include NHS ester/psoralen compounds (e.g., SPB (succinimidyl-[4-(psoralen-8- y loxy )] -butyrate)).

Linkers are also described in U.S. Patent No. 4,680,338 having the formula Y=C=N-Q-A-C(0)-Z, where Q is a homoaromatic or heteroaromatic ring system; A is a single bond or an unsubstituted or substituted divalent C_{1 -3}o bridging group; Y is O or S; and Z is CI, Br, I, N₃ , N-succinimidyloxy, imidazolyl, 1-benzotriazolyloxy, OAr where Ar is an electron-deficient activating aryl group, or OC(0)R where R is -A-Q-N=C=Y or C4-20 tertiary -alkyl.

Linkers are atent No. 5,306,809, which describes linkers

having the formula

H, Ci-6 alkyl, C_2-6 alkenyl, C6-12 aryl or aralkyl

R

or these coupled with a divalent organic -0-, -S-, or where R' is Ci-6 alkyl,

o o linking moiety; R₂is H, C_1-12 alkyl, C6-12 aryl, or C6-12 aralkyl; R₃ is

or another chemical structure which is able to delocalize the lone pair electrons of the adjacent nitrogen; and R4 is a pendant reactive group capable of linking R₃to a polypeptide or to an agent.

The linker can be polyvalent or monovalent. A monovalent linker has only one activated group available for forming a covalent bond. However, the monovalent linker can include one or more functional groups that can be chemically modified by using a coupling agent, as described herein, to form a second activated group. For example, a terminal hydroxyl group of the linker can be activated by any number of coupling agents. Examples of coupling agents include N-hydroxysuccinimide, ethylchloroformate, dicyclohexylcarbodiimide, and trifluoromethanesulfonyl chloride. See, e.g. U.S. Patent Nos. 5,395,619 and 6,316,024.

A polyvalent linker (e.g., a multifunctional linker) has two or more activated groups. The activated groups in the linker can be the same, as in a homopolyvalent linker, or different, as in a heteropolyvalent linker. Heteropolyvalent linkers allow for conjugating a polypeptide and a transport vector with different functional groups.

Examples of heteropolyvalent linkers include polyoxyethylene-bis(p-nitrophenyl carbonate), mal-PEG-DSPE, diisocyanate, and succinimidyl 4-hydrazinonicotinate acetone hydrazone.

Examples of homopolyvalent linkers with two activated groups include disuccinimidyl glutarate, disuccinimidyl suberate, bis(sulfosuccinimidyl) suberate, bis(NHS)PEG₅, bis(NHS)PEG₉, dithiobis(succinimidyl propionate), 3,3 ^'- dithiobis(sulfosuccinimidylpropionate), disuccinimidyl tartrate, bis[2-(succinimido oxycarbonyloxy)ethyl]sulfone, ethylene glycol bis[succinimidylsuccinate]), ethylene glycol bis[sulfosuccinimidylsuccinate]), dimethyl adipimidate, dimethyl pimelimidate, dimethyl suberimidate, dimethyl 3,3 ^'-dithiobispropionimidate, l,5-difluoro-2,4- dinitrobenzene, bis-maleimidoethane, 1,4-bismaleimidobutane, bismaleimidohexane, 1,8- bis-maleimidodiethyleneglycol, 1,11-bis-maleimido-triethyleneglycol, l,4-di-[3 ^'-(2^'- pyridyldithio)-propionamido]butane, 1,6-hexane-bis-vinylsulfone, and bis-[b-(4- azidosalicylamido)ethyl]disulfide.

Examples of homopolyvalent linkers with three activated groups include tris- succinimidyl aminotriacetate, β-[tris(hydroxymethyl) phosphino] propionic acid, and tris[2-maleimidoethyl]amine.

Examples of heteropolyvalent linkers include those with a maleimide activated group and a succinimide activated group, such as N-[a-maleimidoacetoxy]succinimide ester, N-[B-maleimidopropyloxy]-succinimide ester, Ν-[γ- maleimidobutyryloxy] succinimide ester, m-maleimidobenzoyl-N-hydroxysuccinimide ester, succinimidyl 4-[N-maleimidomethyl]cyclohexane-l-carboxylate, Ν-[ε- maleimidocaproyloxy] succinimide ester, and succinimidyl 4-\p- maleimidophenyl]butyrate, including N-sulfosuccinimidyl derivatives; those with a PEG spacer molecule, such as succinimidyl-([N-maleimidopropionamido]- (ethyleneglycol)_x)ester, wherein x is from 2 to 24; those with a pyridyldithio activated group and a succinimide activated group, such as N-succinimidyl-3-(2- pyridyldithio)propionate, succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate, 4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene, and 4-sulfosuccinimidyl- 6-methyl-a-(2-pyridyldithio)toluamido]hexanoate); those with a haloacetyl activated group and a succinimide activated group, such as N-succinimidyl iodoacetate and N- succinimidyl[4-iodoacetyl]aminobenzoate; those with an aryl azide activated group and a succinimide activated group, such as N-hydroxysuccinimidyl-4-azidosalicylic acid, sulfosuccinimidyl[4-azidosalicylami do] -hexanoate, and N-succinimidyl-6-(4'-azido-2'- nitrophenyl amino) hexanoate; those with an diazirine activated group and a succinimide activated group, such as succinimidyl 4,4'-azipentanoate and succinimidyl 6-(4,4'- azipentanamido)hexanoate; N-[4-(p-azidosalicylamido) butyl]-3'-(2'- pyridyldithio)propionamide; N-[β-maleimidopropionic acid] hydrazide; Ν-(ε- maleimidocaproic acid) hydrazide; 4-(4-N-maleimidophenyl)butyric acid hydrazide hydrochloride; (N-[K-maleimidoundecanoic acid] -hydrazide); 3-(2- pyridyldithio)propionyl hydrazide; p-azidobenzoyl hydrazide; and N-[p- maleimidophenyl]isocyanate.

In other embodiments, the linker is a trifunctional, tetrafunctional, or greater linking agent. Exemplary trifunctional linkers include TMEA, THPP, TSAT, LC-TSAT (fTO-succinimidyl (6-aminocaproyl)aminotriacetate), im-succinimidyl-1 ,3,5- benzenetricarboxylate, MDSI (maleimido-3,5-disuccinimidyl isophthalate), SDMB (succinimidyl-3,5-dimaleimidophenyl benzoate, Mal-4 (tetrakis-(3- maleimidopropyl)pentaerythritol, and NHS-4 (tetrakis-(N- succinimidylcarboxypropyl)pentaerythritol)).

TMEA has the structure:

TMEA, through its maleimide groups, can react with sulfhydryl groups (e.g., through cysteine amino acid side chains). THPP has the structure:

The hydroxyl groups and carboxy group of THPP can react with primary or secondary amines.

Amino acid and peptide linkers

In other embodiments, the linker includes at least one amino acid (e.g., a peptide of at least 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 40, or 50 amino acids). In certain embodiments, the linker is a single amino acid (e.g., any naturally occurring amino acid such as Cys). In other embodiments, a gly cine-rich peptide such as a peptide having the sequence [Gly- Gly-Gly-Gly-Ser]_n (SEQ ID NO: 124) where n is 1, 2, 3, 4, 5 or 6 is used, as described in U.S. Patent No. 7,271,149. In other embodiments, a serine-rich peptide linker is used, as described in U.S. Patent No. 5,525,491. Serine rich peptide linkers include those of the formula [X-X-X-X-Gly]_y, where up to two of the X are Thr, and the remaining X are Ser, and y is 1 to 5 (e.g., Ser-Ser-Ser-Ser-Gly (SEQ ID NO: 125), where y is greater than 1). In some cases, the linker is a single amino acid (e.g., any amino acid, such as Gly or Cys).

Amino acid linkers may be selected for flexibility (e.g., flexible or rigid) or may be selected on the basis of charge (e.g., positive, negative, or neutral). Flexible linkers typically include those with Gly resides (e.g., [Gly-Gly-Gly-Gly-Ser]_n where n is 1, 2, 3, 4, 5 or 6). Other linkers include rigid linkers (e.g., PAPAP (SEQ ID NO: 126) and (PT)_nP (SEQ ID NO: 127), where n is 2, 3, 4, 5, 6, or 7) and a-helical linkers (e.g., A(EAAAK)_nA (SEQ ID NO: 128), where n is 1, 2, 3, 4, or 5).

Examples of suitable linkers are succinic acid, Lys, Glu, and Asp, or a dipeptide such as Gly-Lys. When the linker is succinic acid, one carboxyl group thereof may form an amide bond with an amino group of the amino acid residue, and the other carboxyl group thereof may, for example, form an amide bond with an amino group of the peptide or substituent. When the linker is Lys, Glu, or Asp, the carboxyl group thereof may form an amide bond with an amino group of the amino acid residue, and the amino group thereof may, for example, form an amide bond with a carboxyl group of the substituent. When Lys is used as the linker, a further linker may be inserted between the ε-amino group of Lys and the substituent. In one particular embodiment, the further linker is succinic acid, which can form an amide bond with the ε-amino group of Lys and with an amino group present in the substituent. In one embodiment, the further linker is Glu or Asp (e.g., which forms an amide bond with the ε-amino group of Lys and another amide bond with a carboxyl group present in the substituent), that is, the substituent is an Ν^ε- acylated lysine residue.

In other embodiments, the peptide linker is a branched polypeptide. Exemplary branched peptide linkers are described in U.S. Patent No. 6,759,509. Such linkers include those of the formula: A-W_c (CH₂)— (Q)p-(

where A is a thiol acceptor; W is a bridging moiety; c is an integer of 0 to 1; a is an integer of 2 to 12; Q is O, NH, or N-lower alkyl; p is an integer of 0 or 1; d is an integer of 0 or 1; E is a polyvalent atom; each b is an integer of 1 to 10; each X is of the formula:

_CO-Y-Z_m-G_n

where Y is two amino acid residues in the L form; Z is one or two amino acid residues; m is an integer of 0 or 1 ; G is a self-immolative spacer; and n is a integer of 0 or 1; provided that when n is 0 then— Y— Z_m is Ala-Leu- Ala-Leu (SEQ ID NO: 129) or Gly-Phe-Leu- Gly (SEQ ID NO: 130); or each X is of the formula: ft /(CHsJb-X¹

A-W_c (CH₂)— (Q)p-(C)_d-E

(CH₂)_b-X¹

where each X¹ is of the formula— CO— Y— Z_m— G_n; and where Y, Z, Q, E, G, m, d, p, a, b, and n are as defined above; or each X¹ is of the formula: A-W_c (CH₂)— (Q)p-(

where each X² is of the formula— CO— Y— Z_m— G_n; and where Y, Z, G, Q, E, m, d, p, a, b, and n are as defined above; or each X²is of the formula: A-W_c (CH₂)— (Q)_p-(

where each X³ is of the formula— CO— Y— Z_m— G_n; and wherein Y, Z, G, Q, E, m, d, p, a, b, and n are as defined above; or each X³ is of the formula:

where each X⁴ is of the formula— CO— Y— Z_m— G_n; and where Y, Z, G, Q, E, m, d, p, a, b, and n are as defined above.

The branched linker may employ an intermediate self-immolative spacer moiety (G), which covalently links together the agent or peptide vector and the branched peptide linker. A self-immolative spacer can be a bifunctional chemical moiety capable of covalently linking together two chemical moieties and releasing one of said spaced chemical moieties from the tripartate molecule by means of enzymatic cleavage (e.g., any appropriate linker described herein. In certain embodiments, G is a self-immolative spacer moiety which spaces and covalently links together the agent or peptide vector and the peptide linker, where the spacer is linked to the peptide vector or agent via the T moiety (as used in the following formulas "T" represents a nucleophilic atom which is already contained in the agent or peptide vector), and which may be represented by

O, N or S;— HN— R¹—COT, where T is O, N or S,

T

, ^N^COOR² _?

and R¹ is Ci-5 alkyl; H , where T is O R² is H or Ci_-5 alkyl;

where T is O, N or S; or where T is O, N, or S.

Preferred Gs include PABC (p-aminobenzyl-carbamoyl), GABA (γ-aminobutyric acid), α,α-dimethyl GABA, and β,β-dimethyl GABA.

In the branched linker, the thiol acceptor "A" is linked to a peptide vector or agent by a sulfur atom derived from the peptide vector or agent. The thiol acceptor can be, for

O

example, an a-substituted acetyl group. Such a group has the formula: , where Y is a leaving group such as CI, Br, I, mesylate, tosylate, and the like. If the thiol acceptor is an alpha-substituted acetyl group, the thiol adduct after linkage to the ligand forms the bond— S— CH₂— . Preferably, the thiol acceptor is a Michael Addition acceptor. A representative Michael Addition acceptor of this invention has the formula

After linkage the thiol group of the ligand, the Michael Addition acceptor becomes a

Michael Addition adduct, e.g.,

where L is an agent or a polypeptide of the invention.

The bridging group "W" is a bifunctional chemical moiety capable of covalently linking together two spaced chemical moieties into a stable tripartite molecule.

Examples of bridging groups are described in S. S. Wong, Chemistry of Protein

Conjugation and Crosslinking, CRC Press, Florida (1991); and Means et al, Bioconj. Chem., 1 :2-12 (1990), the disclosures of which are incorporated herein by reference. W can covalently link the thiol acceptor to a keto moiety. An exemplary a bridging group has the formula— (CH₂)_f— (Z)_g— (CH₂)_h— , where f is 0 to 10; h is 0 to 10; g is 0 or 1, provided that when g is 0, then f+h is 1 to 10; Z is S, O, NH, S0₂, phenyl, naphthyl, a polyethylene glycol, a cycloaliphatic hydrocarbon ring containing 3 to 10 carbon atoms, or a heteroaromatic hydrocarbon ring containing 3 to 6 carbon atoms and 1 or 2 heteroatoms selected from O, N, or S. Preferred cycloaliphatic moieties include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. Preferred heteroaromatic moieties include pyridyl, polyethylene glycol (1-20 repeating units), furanyl, pyranyl, pyrimidinyl, pyrazinyl, pyridazinyl, oxazinyl, pyrrolyl, thiazolyl, morpholinyl, and the like. In the bridging group, it is preferred that when g is 0, f+h is an integer of 2 to 6 (e.g., 2 to 4 such as 2). When g is 1, it is preferred that f is 0, 1 or 2; and that h is 0, 1 or 2. Preferred bridging groups coupled to thiol acceptors are shown in the Pierce Catalog, pp. E-12, E-13, E-14, E-15, E-16, and E-17 (1992).

Modifications to linkers

Any of the linkers described herein (e.g., chemical linking agents or amino acid) may be modified. For example, the linkers can include a spacer molecule. The spacer molecule within linker can be of any suitable molecule. Examples of spacer molecules include aliphatic carbon groups (e.g., C₂-C₂o alkyl groups), cleavable heteroatomic carbon groups (e.g., C₂-C₂o alkyl groups with dithio groups), and hydrophilic polymer groups. Examples of hydrophilic polymer groups include poly (ethylene glycol) (PEG), polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethylene glycol, polyaspartamide, and a hydrophilic peptide sequence.

In one example, the hydrophilic polymer is PEG, such as a PEG chain having a molecular weight between 500-10,000 Da (e.g., between 1,000-5,000 Da, such as 2,000 Da). Methoxy or ethoxy-capped analogues of PEG can also be used. These are commercially available in sizes ranging between 120-20,000 Da. Preparation of lipid- tether conjugates for use in liposomes is described, for example, in U.S. Patent No. 5,395,619, hereby incorporated by reference. Other spacer molecules include polynucleotides (e.g., DNA or RNA), polysaccharides such as dextran or xanthan, cellulose derivatives (e.g., carboxymethyl cellulose), polystyrene, polyvinyl alcohol, poly methylacrylic acid, and poly(NIPAM). Synthetic reaction schemes for activating PEG with coupling agents are set forth in U.S. Patent Nos. 5,631,018, 5,527,528, and

5,395,619. Synthetic reaction schemes for linkers with PEG spacer molecules are set forth in U.S. Patent Nos. 6,828,401, and 7,217,845.

PEG, for example, can be conjugated to a polypeptide of the invention by any means known in the art. In certain embodiments, the PEG molecule is derivatized with a linker, which is then reacted with the protein to form a conjugate. Suitable linkers include aldehydes, tresyl or tosyl linkers, dichlorotriazine or chlorotriazine, epoxide, carboxylates such as succinimidyl succinate, carbonates such as a p-nitrophenyl carbonate, benzotriazolyl carbonate, 2,3,5-trichlorophenyl carbonate, and PEG- succinimidyl carbonate, or reactive thiols such as pyridyldisufide, maleimide, vinylsulfone, and iodo acetamide. Conjugation can take place at amino groups (e.g., the N-terminal amino group or amino groups within the lysine side chain), or at thiol hydroxyl, or amide groups, depending on the linker used. See, e.g., Veronese et al, Drug Discov. Today 10: 1451-1458 (2005).

Therapeutic agents

The conjugate can include any useful therapeutic agent. Any of the therapeutic agents described below may be used in the compounds of the invention. Agents of particular interest include anticancer agents (e.g., paclitaxel, etoposide, doxorubicin, and analogs thereof), small molecule drugs, labels, therapeutic nucleic acid agents, and therapeutic peptidic agents (e.g., as described herein). Anticancer agents

In accordance with the present invention, the agent may be an anticancer agent. An anticancer agent encompassed by the present invention may include, for example, a drug having a group allowing its conjugation to the polypeptide of the invention.

Particular anticancer agents include those selected from the group consisting of paclitaxel (Taxol^®), vinblastine, vincristine, etoposide, doxorubicin, cyclophosphamide, docetaxel (Taxotere^®), melphalan, and chlorambucil; derivatives (or analogs) thereof;

pharmaceutically acceptable salts thereof; or a combination thereof. In particular embodiments, the anticancer agent is paclitaxel, etoposide, or doxorubicin; a

pharmaceutically acceptable salt thereof; or a derivative thereof.

Exemplary embodiments of paclitaxel derivatives (or analogs) include derivatives disclosed and referred to in U.S. Patent No. 6,911,549, issued on June 28, 2005, the entire contents of which is incorporated herein by reference. Particular paclitaxel derivatives include ((azidophenyl)ureido)taxoid, (2α,5α,7β,9α,10β,13α)-5,10,13,20-ί6ϋ^ΰβτχ^ΐΒχ- 1 l-ene-2,7,9-triol, (2 ,5 ,9 ,10β)-2,9,10-triacetoxy-5-((β-D-glucopyranosyl)oxy)-3,l 1- cyclotax-l l-en-13-one, 1 β-hydroxybaccatin I, 1,7-dihydroxytaxinine, l-acety-5,7,10- deacetyl-baccatin I, 1-dehydroxybaccatin VI, 1-hydroxy- 2-deacetoxy-5-decinnamoyl- taxinine j, l-hydroxy-7,9-dideacetylbaccatin I, 1 -hydroxybaccatin I, 10-acetyl-4- deacetyltaxotere, 10-deacetoxy paclitaxel, 10-deacetyl baccatin lll dimethyl sulfoxide disolvate, 10-deacetyl- 10-(3-aminobenzoyl)paclitaxel, 10-deacetyl-10-(7- (diethylamino)coumarin-3-carbonyl)paclitaxel, 10-deacetyl-9-dihydrotaxol, 10- deacetylbaccatine III, 10-deacetylpaclitaxel, 10-deacetyltaxinine, 10-deacetyltaxol, 10- deoxy-10-C-mo holinoethyl docetaxel, 10-O-acetyl-2-O-(cyclohexylcarbonyl)-2- debenzoyltaxotere, 10-O-sec-aminoethyl docetaxel, 11-desmethyllaulimalide, 13-deoxo- 13-acetyloxy-7,9-diacetyl-l,2-dideoxytaxine, 13-deoxybaccatin III, 14-hydroxy-10- deacetyl-2-O-debenzoylbacatin III, 14-hydroxy-10-deacetylbaccatin III, 14β-benzoyloxy- 13-deacetylbaccatin IV, 14β-benzoyloxy-2-deacetylbaccatin VI, 14β-benzoyloxybaccatin IV, 19-hydroxybaccatin III, 2',2"-methylenedocetaxel, 2',2"-methylenepaclitaxel, 2'- (valyl-leucyl-lysyl-PABC)paclitaxel, 2'-acetyltaxol, 2'-0-acetyl-7-0-(N-(4'- fluoresceincarbonyl)alanyl)taxol, 2,10,13-triacetoxy-taxa-4(20),l l-diene-5,7,9-triol, 2,20- O-diacetyltaxumairol N, 2-(4-azidobenzoyl)taxol, 2-deacetoxytaxinine J, 2-debenzoyl-2- m-methoxybenozyl-7-triethylsilyl-13-oxo-14-hydroxybaccatin III 1,14-carbonate, 2-0- (cyclohexylcarbonyl)-2-debenzoylbaccatin III 13-0-(N-(cyclohexylcarbonyl)-3- cyclohexylisoserinate), 2a, 7β,9 ,10β,13 - entaacetoxyltaxa-4 (20), l l-dien-5-ol, 2α,5α,7β,9α, 13 -pentahy droxy- 10β-acetoxytaxa-4(20), 11 -diene, 2α,7β,9α, 10β, 13- pentaacetoxy- 11 P-hydroxy-5a-(3 ' -N,N-dimethylamino-3 ' -phenyl)-propionyloxytaxa- 4(20),12-diene, 2 ,7β-diacetoxy-5 ,10β,13β-trihydroxy-2(3-20)abeotaxa-4(20),l l-dien-

9- one, 2 ,9 -dihydroxy-10β,13 -diacetoxy-5 -(3'-methylamino-3'-phenyl)- propionyloxytaxa-4(20), 11 -diene, 2 -hydroxy-7β,9 , 10β, 13a-tetraacetoxy-5a-(2' - hydroxy-3 '-N,N-dimethylamino-3 ' -phenyl)-propionyloxytaxa-4(20), 11 -diene, 3 ' -(4- azidobenzamido)taxol, 3 ' -N-(4-benzoy ldihy drocinnamoyl)-3 ' -N-debenzoy lpaclitaxel, 3 ' - N-m-aminobenzamido-3'-debenzamidopaclitaxel, 3'-p-hydroxypaclitaxel, 3,11- cyclotaxinine NN-2, 4-deacetyltaxol, 5,13 -diacetoxy-taxa-4(20),l l-diene-9,10-diol, 5-0- benzoylated taxinine K, 5-O-phenylpropionyloxytaxinine A, 5 ,13 -diacetoxy-10β- cinnamoyloxy-4(20),l l-taxadien-9a-ol, 6,3'-p-dihydroxypaclitaxel, 6-a-hydroxy-7- deoxy-10-deacetylbaccatin-III, 6-fluoro-lO-acetyldocetaxel, 6-hydroxytaxol, 7,13- diacetoxy-5-cinnamyloxy-2(3-20)-abeo-taxa-4(20),l l-diene-2,10-diol, 7,9- dideacetylbaccatin VI, 7-(5'-Biotinylamidopropanoyl)paclitaxel, 7-acetyltaxol, 7-deoxy-

10- deacetylbaccatin-III, 7-deoxy-9-dihydropaclitaxel, 7-epipaclitaxel, 7- methylthiomethylpaclitaxel, 7-0-(4-benzoyldihydrocinnamoyl)paclitaxel, 7-0-(N-(4'- fluoresceincarbonyl)alanyl)taxol, 7-xylosyl-10-deacetyltaxol, 8,9-single-epoxy brevifolin, 9-dihydrobaccatin III, 9-dihydrotaxol, 9 -hydroxy-2 ,10β,13 -triacetoxy-5 -(3'-N,N- dimethylamino-3'-phenyl)-propionyloxytaxa-4(20), 11 -diene, baccatin III, baccatin III 13- 0-(N-benzoyl-3-cyclohexylisoserinate), BAY59, benzoyltaxol, BMS 181339, BMS 185660, BMS 188797, brevifoliol, butitaxel, cephalomannine, dantaxusin A, dantaxusin B, dantaxusin C, dantaxusin D, dibromo-lO-deacetylcephalomannine, DJ927, docetaxel, Flutax 2, glutarylpaclitaxel 6-aminohexanol glucuronide, IDN 5109, IDN 5111, IDN 5127, IDN 5390, isolaulimalide, laulimalide, MST 997, N-(paclitaxel-2'-0-(2- amino)phenylpropionate)-0-(β-glucuronyl)carbamate, N-(paclitaxel-2'-0-3,3-dimethyl butanoate)-0-^-glucuronyl)carbamate, N-debenzoy l-N-(3- (dimethylamino)benzoyl)paclitaxel, nonataxel, octreotide-conjugated paclitaxel,

Paclitaxel, paclitaxel-transferrin, PNU 166945, poly(ethylene glycol)-conjugated paclitaxel-2'-glycinate, polyglutamic acid-paclitaxel, protax, protaxel, RPR 109881A, SB T-101187, SB T-1102, SB T-1213, SB T-1214, SB T-1250, SB T-12843, tasumatrol E, tasumatrol F, tasumatrol G, taxa-4(20),l l(12)-dien-5-yl acetate, taxa-4(20),l l(12)-diene- 5-ol, taxane, taxchinin N, taxcultine, taxezopidine M, taxezopidine N, taxine, taxinine, taxinine A, taxinine M, taxinine NN-1, taxinine NN-7, taxol C-7-xylose, taxol-sialyl conjugate, taxumairol A, taxumairol B, taxumairol G, taxumairol H, taxumairol I, taxumairol K, taxumairol M, taxumairol N, taxumairol O, taxumairol U, taxumairol V, taxumairol W, taxumairol-X, taxumairol-Y, taxumairol-Z, taxusin, taxuspinanane A, taxuspinanane B, taxuspine C, taxuspine D, taxuspine F, taxuyunnanine C, taxuyunnanine S, taxuyunnanine T, taxuyunnanine U, taxuyunnanine V, tRA-96023, and wallifoliol.

Other paclitaxel analogs include 1-deoxy paclitaxel, 10-deacetoxy-7-deoxypaclitaxel, 10- O-deacetylpaclitaxel 10-monosuccinyl ester, 10-succinyl paclitaxel, 12b-acetyloxy- 2a,3,4,4a,5,6,9,10,l l,12,12a,12b-dodecahydro-4,l l-dihydroxy-12-(2,5- dimethoxybenzy loxy )-4a, 8, 13 , 13 -tetramethyl-5 -oxo-7, 11 -methano- 1 H- cyclodeca(3,4)benz(l,2-b)oxet-9-yl 3-(tert-butyloxycarbonyl)amino-2-hydroxy-5-methyl- 4-hexaenoate, 130-nm albumin-bound paclitaxel, 2' -paclitaxel methyl 2-glucopyranosyl succinate, 3'-(4-azidophenyl)-3'-dephenylpaclitaxel, 4-fluoropaclitaxel, 6,6,8-trimethyl- 4,4a,5,6,7,7a,8,9-octahydrocyclopenta(4,5)cyclohepta(l,2-c)-furan-4,8-diol 4-(N-acetyl- 3-phenylisoserinate), 6,6,8-trimethyl-4,4a,5,6,7,7a,8,9- octahydrocyclopenta(4,5)cyclohepta(l ,2-c)-furan-4,8-diol 4-(N-tert-butoxycarbonyl-3- phenylisoserinate), 7-(3-methyl-3-nitrosothiobutyryl)paclitaxel, 7-deoxypaclitaxel, 7- succinylpaclitaxel, A-Z-CINN 310, AI-850, albumin-bound paclitaxel,AZ 10992,isotaxel, MAC321, MBT-0206, NK105, Pacliex, paclitaxel poliglumex, paclitaxel-EC-1 conjugate, polilactofate, and TXD 258. Other paclitaxel analogs are described in U.S. Patents Nos. 4,814,470, 4,857,653, 4,942,184, 4,924,011, 4,924,012, 4,960,790, 5,015,744, 5,157,049, 5,059,699, 5,136,060, 4,876,399, and 5,227,400.

Exemplary etoposide derivatives (or analogs) include podophyllotoxin

derivatives. Other derivatives of etoposide include etoposide phosphate

(ETOPOPHOS^®), etoposide 4'-dimethylglycine, etoposideoMG, teniposide, and NK611, or any pharmaceutically acceptable salts thereof (e.g., -OP(0)(ONa)₂). Still other podophyllotoxin derivatives suitable for use in the invention are described in U.S. Patent Nos. 4,567,253, 4,609,644, 4,900,814, 4,958,010, 5,489,698, 5,536,847, 5,571,914, 6,051,721, 6,107,284, 6,475,486, 6,610,299, 6,878,746, 6,894,075, 7,087,641, 7,176,236, 7,241,595, 7,342,114, and 7,378,419; and in U.S. Patent Publication Nos. 20030064482, 20030162722, 20040044058, 20060148728, and 20070249651, each of which is hereby incorporated by reference.

Exemplary doxorubicin (hydroxydaunorubicin or Adriamycin^®) derivatives (or analogs) include epirubicin (Ellence^® or Pharmorubicin^®). Other doxorubicin derivatives can be found in U.S. Patent Nos. 4,098,884, 4,301,277, 4,314,054, 4,464,529, 4,585,859, 4,672,057, 4,684,629, 4,826,964, 5,200,513, 5,304,687, 5,594,158, 5,625,043, and 5,874,412, each of which is hereby incorporated by reference. Small molecule drugs

Any small molecule drug can be linked with the polypeptide of the invention. Small molecule drugs include an anticancer agent, an antibiotic, a cytotoxic agent, an alkylating agent, an antineoplastic agent, an antimetabolic agent, an antiproliferative agent, a neurotransmitter (e.g., agmatine), a tubulin inhibitor, a topoisomerase I or II inhibitor, a hormonal agonist or antagonist, an apoptotic agent, an immunomodulator, and a radioactive agent (e.g., an isotope), or any agent described herein. Exemplary small molecule drugs include paclitaxel (Taxol^®), a Taxol^® derivative, vinblastine, vincristine, etoposide, doxorubicin, cyclophosphamide, Taxotere^®, melphalan, chlorambucil, methotrexate, camptothecin, homocamptothecin, thiocolchicine, colchicine,

combretastatin, combretastin A-4, podophyllotoxin, rhizoxin, rhizoxin-d, dolistatin,

CC1065, ansamitocin p3, maytansinoid, and any pharmaceutically acceptable salts, etc. and combinations thereof, as well as any drug which may be a P-gp substrate. The invention may also include analogs of any of these agents (e.g., therapeutically effective analogs).

Labels

A label can be linked to the polypeptide to allow for diagnostic and/or therapeutic treatment. Examples of labels include detectable labels, such as an isotope, a radioimaging agent, a marker, a tracer, a fluorescent label (e.g., rhodamine), and a reporter molecule (e.g., biotin).

Examples of radioimaging agents emitting radiation (detectable radio-labels) that may be suitable are exemplified by indium- 111, technetium-99, or low dose iodine- 131. Detectable labels, or markers, for use in the present invention may be a radiolabel, a fluorescent label, a nuclear magnetic resonance active label, a luminescent label, a chromophore label, a positron emitting isotope for PET scanner, a chemiluminescence label, or an enzymatic label. Fluorescent labels include but are not limited to, green fluorescent protein (GFP), fluorescein, and rhodamine. Chemiluminescence labels include, but are not limited to, luciferase and β-galactosidase. Enzymatic labels include, but are not limited to, peroxidase and phosphatase. A histamine tag may also be a detectable label. For example, therapeutic conjugates may comprise a polypeptide, a therapeutic agent, and may further comprise a label. The label may be for example a medical isotope, such as for example and without limitation, technetium-99, iodine- 123 and -131, thallium-201, gallium-67, fluorine-18, indium-I l l, etc.

Therapeutic nucleic acid agents

The polypeptide may be conjugated to any therapeutic nucleic acid agent, including expression vectors (e.g., a plasmid) and RNAi agents. The expression vector may encode a polypeptide (e.g., a therapeutic polypeptide such as an interferon, a therapeutic cytokine (e.g., IL-12), or FGF-2) or may encode a therapeutic nucleic acid (e.g., an RNAi agent such as those described herein). Nucleic acids include any type known in the art, such as double and single-stranded DNA and RNA molecules of any length, conformation, charge, or shape (i.e., linear, concatemer, circular (e.g., a plasmid), nicked circular, coiled, supercoiled, or charged). Additionally, the nucleic acid can contain 5' and 3' terminal modifications and include blunt and overhanging nucleotides at these termini, or combinations thereof. In certain embodiments of the invention the nucleic acid is or encodes an RNA interference sequence (e.g., an siRNA, shRNA, miRNA, or dsRNA nucleotide sequence) that can silence a targeted gene product. The nucleic acid can be, for example, a DNA molecule, an RNA molecule, or a modified form thereof.

Exemplary RNAi targets include growth factors (e.g., epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF- β)), growth factor receptors, including receptor tyrosine kinases (e.g., EGF receptor (EGFR), including Her2/neu (ErbB), VEGF receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), cytokines, chemokines, kinases, including cytoplasmic tyrosine and serine/threonine kinases (e.g., focal adhesion kinase, cyclin-dependent kinase, SRC kinases, syk-ZAP70 kinases, BTK kinases, RAF kinase, MAP kinases (including ERK), and Wnt kinases), phosphatases, regulatory GTPases (e.g., Ras protein), transcription factors (e.g., MYC), hormones and hormone receptors (e.g., estrogen and estrogen receptor), anti-apoptotic molecules (e.g., survivin, Bcl-2, Bcl-xL), oncogenes (e.g., tumor suppressor regulators such as mdm2), enzymes (e.g., superoxide dismutase 1 (SOD-1), a, β (BACE), and γ secretases, alpha-L-iduronidase, iduronate sulfatase, heparan N- sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoAlpha-glucosaminide

acetyltransferase, N-acetylglucosamine 6-sulfatase, N-acetylgalactosamine 4-sulfatase, beta-galactosidase, sphingomyelinase, glucocerebrosidase, alpha-galactosidase-A, ceramidase, galactosylceramidase, arylsulfatase A, aspartoacylase, phytanoyl-CoA hydroxylase, peroxin-7, beta-hexosaminidase A, aspartylglucosaminidase, fucosidase, and alpha-mannosidase, sialidase), and other proteins (e.g., Huntingtin (Htt protein), amyloid precursor protein (APP), sorting nexins (including SNX6), a-synuclein, LINGO- 1, Nogo-A, and Nogo receptor 1 (NgR-1)), and glial fibrillary acidic protein. Table 3 illustrates the relationship between exemplary RNAi targets and diseases.

Exemplary RNAi sequences to silence EGFR are SEQ ID NO: 131

(GGAGCUGCCCAUGAGAAAU) and SEQ ID NO: 132

(AUUUCUCAUGGGCAGCUCC). Likewise, VEGF can be silenced with an RNAi molecule having the sequence, for example, set forth in SEQ ID NO: 133

(GGAGTACCCTGATGAGATC). Additional RNAi sequences for use in the agents of the invention may be either commercially available (e.g., Dharmacon, Ambion) or the practitioner may use one of several publicly available software tools for the construction of viable RNAi sequences (e.g., The siRNA Selection Server, maintained by

MIT/Whitehead; available at: http://jura.wi.mit.edu/bioc/siRNAext/). Examples of diseases or conditions, and RNAi target that may be useful in treatment of such diseases, are shown in Table 3.

Table 3: Exemplary Diseases and Target Molecules

Disease/ Condition RNAi Target Molecules

Cancer

Glioblastoma Epidermal growth factor receptor (EGFR), Vascular

endothelial growth factor (VEGF)

Glioma EGFR, VEGF

Astrocytoma EGFR, VEGF

Neuroblastoma EGFR, VEGF

Lung cancer EGFR, VEGF

Breast cancer EGFR, VEGF Hepatocellular carcinoma EGFR, VEGF

Therapeutic peptidic agents

Therapeutic peptidic agents include a broad class of agents based on proteins or peptides (e.g., any useful peptidic- or protein-based drug). Exemplary therapeutic peptidic agents include, without limitation, a peptidic- or protein-based drug (e.g., a positive pharmacological modulator (agonist) or a pharmacological inhibitor (antagonist)) etc.

The conjugate may be a therapeutic polypeptide (e.g., a fusion protein) consisting essentially of the polypeptide of the invention and a protein. Exemplary therapeutic peptidic agents include cellular toxins (e.g., monomethyl auristatin E (MMAE), bacteria endotoxins and exotoxins, diphtheria toxins, botulinum toxin, tetanus toxins, perussis toxins, staphylococcus enterotoxins, toxic shock syndrome toxin TSST-1, adenylate cyclase toxin, shiga toxin, and cholera enterotoxin), anti-angiogenic compounds (e.g., endostatins, chemokines, inhibitors of matrix metalloproteinase (MMPIs), anastellin, vitronectin, antithrombin, tyrosine kinase inhibitors, and VEGF inhibitors), hormones (e.g., growth hormone), and cytokines (e.g., granulocyte-macrophage colony-stimulating factor, interleukins, lymphokines, and chemokines).

Other therapeutic peptidic agents that may be included in a conjugate of the invention are adrenocortiocotropic hormones (ACTH, corticotropin), growth hormone peptides (e.g., human placental lactogen (hPL), growth hormones, and prolactin (Prl)), melanocyte stimulating hormones (MSH), oxytocin, vasopressin (ADH), corticotropin releasing factor (CRF), gonadotropin releasing hormone associated peptides (GAP), growth hormone releasing factor (GRF), lutenizing hormone release hormones (LH-RH), orexins (HCRT, e.g., orexin-A/hypocretin-1 and orexin-B/hypocretin-2), prolactin releasing peptides, somatostatin, thyrotropin releasing hormone (THR), calcitonin (CT), caltitonin precursor peptide, calcitonins gene related peptide (CGRP), parathyroid hormones (PTH), parathyroid hormone related proteins (PTHrP), amylin, glucagon, insulin and insulin-like peptides, neuropeptide Y, pancreatic polypeptide (PP), peptide YY, somatostatin, cholecystokinin (CCK), gastrin releasing peptide (GRP), gastrin, gastrin inhibitory peptide, motilin, secretin, vasoactive intestinal peptide (VIP), natriuretic peptides (e.g., atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), brain natriuretic peptide, and C-type natriuretic peptide (CNP)), tachykinins (e.g., neurokinin A, neurokinin B, and substance P), substance P, angiotensins (e.g., angiotensin I and angiotensin II), renin, endothelins (e.g., endothelin-1, endothelin-2, endothelin-3, sarafotoxin (a snake venom), and scorpion toxin), sarafotoxin peptides, opioid peptides (e.g., casomorphin peptides, demorphins, endorphins, enkephalins, deltorphins, and dyno hins), thymic peptides (e.g., thymopoietin, thymulin,

thymopentin, thymosin, and thymic humoral factor (THF)), adrenomedullin peptides (AM), allatostatin peptides, amyloid beta-protein fragments (Αβ fragments), antimicrobial peptides (e.g., defensin, cecropin, buforin, and magainin), antioxidant peptides (e.g., natural killer-enhancing factor B (NKEF-B)), bombesin, bone Gla protein peptides (e.g., osteocalcin, bone Gla-protein, or BGP), CART peptides, cell adhesion peptides, cortistatin peptides, fibronectin fragments and fibrin related peptides, FMRF peptides, galanin, guanylin and uroguanylin, and inhibin peptides.

In particular embodiments, the therapeutic peptidic agent is a GLP-1 agonist, leptin or a leptin analog, neurotensin or a neurotensin analog, a neurotensin receptor agonist, glial-derived neurotrophic factor (GDNF) or a GDNF analog, or brain-derived neurotrophic factor (BDNF) or a BDNF analog. These therapeutic peptidic agents or the receptors that bind these agents have been implicated in various types of cancers. More details regarding these agents are provided below. GLP-1 agonist

The polypeptides described herein can be conjugated to a GLP-1 agonist.

Particular GLP-1 agonists include GLP-1, exendin-4, and analogs or fragments thereof. Exemplary analogs are described below.

GLP-1 and GLP-1 analogs can be used in the conjugates and therapeutic polypeptides of the invention. In certain embodiments, the GLP-1 analog is a peptide, which can be truncated, may have one or more substitutions of the wild type sequence (e.g., the human wild type sequence), or may have other chemical modifications. GLP-1 agonists can also be non-peptide compounds, for example, as described in U.S. Patent No. 6,927,214. Particular analogs include LY548806, CJC-1131, and Liraglutide.

The GLP-1 analog can be truncated form of GLP-1. The GLP-1 peptide may be truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 20, or more residues from its N- terminus, its C-terminus, or a combination thereof. In certain embodiments, the truncated GLP-1 analog is the GLP-l(7-34), GLP-l(7-35), GLP-l(7-36), or GLP-l(7-37) human peptide or the C-terminal amidated forms thereof.

The GLP-1 analog can include substitutions, such as an amino acid other than alanine at position 8 or an amino acid other than glycine at position 22 (e.g., [Glu²²]GLP- l(7-37)OH, [Asp²²] GLP-1 (7-37)OH, [Arg²²]GLP-l(7-37)OH, [Lys²²]GLP-l(7-37)OH, [Cya²²]GLP-l(7-37)OH, [Val⁸,Glu²²]GLP-l(7-37)OH, [Val⁸,Asp²²]GLP-l(7-37)OH, [Val⁸,Arg²²]GLP-l(7-37)OH, [Val⁸,Lys²²]GLP-l(7-37)OH, [Val⁸,Cya²²]GLP-l(7-37)OH, [Gly⁸,Glu²²]GLP-l(7-37)OH, [Gly⁸,Asp²²]GLP-l(7-37)OH, [Gly⁸,Arg²²]GLP-l(7- 37)OH, [Gly⁸,Lys²²]GLP-l(7-37)OH, [Gly⁸,Cya²²]GLP-l(7-37)OH, [Glu²²]GLP-l(7- 36)NH₂, [Asp²²]GLP-l(7-36)NH₂, [Arg²²]GLP-l(7-36)NH₂, [Lys²²]GLP-l(7-36)NH₂, [Cya²²]GLP-l(7-36)NH₂, [Val⁸,Glu²²]GLP-l(7-36)NH₂, [Val⁸,Asp²²]GLP-l(7-36)NH₂, [Val⁸, Arg²²] GLP- 1 (7-36)NH₂, [Val⁸,Ly s²²] GLP-1 (7-36)NH₂, [Val⁸,Cy a²²] GLP- 1 (7- 36)NH₂, [Gly⁸,Glu²²]GLP-l(7-36)NH₂, [Gly⁸,Asp²²] GLP-1 (7-36)NH₂, [Gly⁸,Arg²²]GLP- 1(7-36)NH₂, [Gly⁸,Lys²²]GLP-l(7-36)NH₂, [Gly⁸,Cya²²]GLP-l(7-36)NH₂,

[Val⁸ Lys²³]GLP-l(7-37)OH, [Val⁸,Ala²⁷]GLP-l(7-37)OH, [Val⁸,Glu³⁰]GLP-l(7-37)OH, [Gly⁸,Glu³⁰]GLP-l(7-37)OH, [Val⁸,His³⁵]GLP-l(7-37)OH, [Val⁸,His³⁷] GLP-1 (7-37)OH, [Val⁸,Glu²²,Lys²³]GLP-l(7-37)OH, [Val⁸,Glu²²,Glu²]GLP-l(7-37)OH,

[Val⁸,Glu²²,Ala²⁷]GLP-l(7-37)OH, [Val⁸,Gly³⁴,Lys³⁵]GLP-l(7-37)OH, [Val⁸,His³⁷]GLP- l(7-37)OH, [Gly⁸,His³⁷]GLP-l(7-37)OH); or a substitution at position 7 with the N- acylated or N-alkylated amino acids (e.g., [D-His⁷]GLP-l(7-37), [Tyr⁷]GLP-l(7-37), [N- acetyl-His⁷]GLP-l(7-37), [N-isopropyl-His⁷]GLP-l(7-37), [D-Ala⁸]GLP-l(7-37), [D- Glu⁹]GLP-l(7-37), [Asp⁹]GLP-l(7-37), [D-Asp⁹]GLP-l(7-37), [D-Phe¹⁰]GLP-l(7-37), [Ser²²,Arg²³,Arg²⁴,Gln²⁶]GLP-l(7-37), and [Ser⁸,Gln⁹,Tyr¹⁶,Lys¹⁸,Asp²¹]GLP-l(7-37)). Other GLP-1 analogs are described in U.S. Patent Nos. 5,545,618, 5,574,008, 5,981,488, 7,084,243, 7,101,843, and 7,238,670.

Exendin-4 and exendin-4 analogs can also be used in the conjugates and therapeutic polypeptides of the invention. The compounds of the invention can include fragments of the exendin-4 sequence. Exendin-4 has the sequence:

His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val- Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser- NH₂ (SEQ ID NO: 134).

Particular exendin-4 analogs include those having a cysteine substitution (e.g., [Cys³²] exendin-4); a lysine substitution (e.g., [Lys³⁹]exendin-4); a leucine substitution (e.g., [Leu ,Phe ]exendin-4 amide, [Leu ,Phe ]exendin-4(l-28) amide, and

[Leu¹⁴,Ala²²,Phe²⁵]exendin-4(l-28) amide); or exendin fragments (e.g., exendin-4(l-30), exendin-4(l-30) amide, exendin-4(l-28) amide, and exendin-4(l-31)). Other exendin analogs are described in U.S. Patents Nos. 7,157,555, 7,220,721, and 7,223,725; and U.S. Patent Application Publication No. 2007/0037747.

Leptin and leptin analogs

Leptin is an adipokine, and thus the therapeutic peptidic agent can include an adipokine or an analog thereof. Adipokines include adiponectin, leptin, and resistin. Adiponectins include human, mouse, and rat adiponectin. Leptins include leptin(l 16- 130), leptin(22-56), leptin(57-92), leptin(93-105), LY396623, metreleptin, murine leptin analog, pegylated leptin, and methionyl human leptin. Resistins include human, mouse, and rat resistin. The leptin may be a cleaved sequence (e.g., amino acids 22-167 of the human sequence) or the full length protein. The polypeptide used in the invention may be any of these peptides or proteins or may be substantially identical to any of these peptides or proteins.

The leptin analog may be an OB receptor agonist. In certain embodiments, the OB receptor agonist is an agonist for the OB-Rb form, which is the predominant receptor found in the hypothalamus or the OB-R, which is found at the blood-brain barrier and is involved in leptin transport.

Neurotensin or a neurotensin analog

Neurotensin (NT) is a 13 amino acid peptide found in the central nervous system and in the gastrointestinal tract. In brain, NT is associated with dopaminergic receptors and other neurotransmitter system. Peripheral NT acts as a paracrine and endocrine peptide on both the digestive and cardiovascular systems. To exert its biological effects in the brain NT has to be injected or delivered directly to the brain because NT does not cross the BBB and is rapidly degraded by peptidases following systematic administration. Preclinical pharmacological studies, most of which involve direct injection of NT into the brain, strongly suggest that an agonist of NT receptors would be clinically useful for the treatment of neuropsychiatric conditions including psychosis, schizophrenia,

Parkinson's disease, pain, and the abuse of psychostimulants. In particular, in various animal studies, intraventricular injection of NT led to hypothermia and analgesia in antinociception experiments.

The peptide therapeutic may be neurotensin or analog thereof. Human neurotensin is a thirteen amino acid peptide having the sequence QLYENKPRRPYIL (SEQ ID NO: 135). Exemplary neurotensin analogs include (VIP-neurotensin) hybrid antagonist, acetylneurotensin(8-13), JMV 1193, KK13 peptide, neuromedin N, neuromedin N precursor, neurotensin(l-lO), neurotensin(l-l l), neurotensin(l-13), neurotensin(l-6), neurotensin(l-8), neurotensin(8-13), Asp(12)-neurotensin(8-13), Asp(13)-neurotensin(8-13), Lys(8)-neurotensin(8-13), N-methyl-Arg(8)-Lys(9)-neo- Trp(l l)-neo-Leu(12)-neurotensin(8-13), neurotensin(9-13), neurotensin 69L, Arg(9)- neurotensin, azidobenzoyl-Lys(6)-T (l l)-neurotensin, Gln(4)-neurotensin, iodo- Tyr(l l)-neurotensin, iodo-Tyr(3)-neurotensin, N-a-

(fluoresceinylthiocarbamyl)glutamyl(l)-neurotensin, Phe(l l)-neurotensin, Ser(7)- neurotensin, Trp(l l)-neurotensin, Tyr(l l)-neurotensin, rat NT77, PD 149163, proneurotensin, stearyl-Nle(17)-neurotensin(6-l l)VIP(7-28), ^99mTc-NT-XI, TJN 950, and vasoactive intestinal peptide-neurotensin hybrid.

Other neurotensin analogs include NT64L [L-neo-Trpl 1]NT(8- 13), NT72D [D- Lys9,D-neo-Trpl l,tert-Leul2]NT(9-13), NT64D [D-neo-Trpl l]NT(8-13), NT73L [D- Lys9,L-neo-Trpl l]NT(9-13), NT65L [L-neo-Trpl 1, tert-Leul2]NT(8-13), NT73D [D- Lys9,D-neo-Trpl l]NT(9-13), NT65D [D-neo-ΤφΙ Ι, tert-Leul2]NT(8-13), NT74L

[DAB9,L-neo-Trpl l,tert-Leul2]NT(9-13), NT66L [D-Lys8, L-neo-Trpl 1, tert- Leul2]NT(8-13), NT74D [DAB9,Pro,D-neo-Trpl l,tert-Leul2]NT(9-13), NT66D [D- Lys8, D-neo-Trpl l, tert-Leul2]NT(8-13), NT75L [DAB 8 L-neo-Trpl 1]NT(8-13), NT67L [D-Lys8, L-neo-Trpl 1]NT(8-13), NT75D [DAB8,D-neo-Trpl l]NT(8-13), NT67D [D-Lys8, D-neo-Trpl 1]NT(8-13), NT76L [D-Orn9,L-neo-Trpl 1]NT(8-13),

NT69L [N-methyl-Arg8,L-Lys9 L-neo-Trpl l,tert-Leul2]NT(8-13), NT76D [D-Orn9,D- ηβο-Τ 11]ΝΤ(8-13), NT69D [N-methyl-Arg8 L-Lys9,D-neo^l l,tert-Leul2]NT(8- 13), NT77L [D-Orn9,L-neo-Trpl l,tert-Leul2]NT(8-13), NT71L [N-methyl-Arg8,DAB9 L-neo-Trpl l,tert-leul2]NT(8-13), NT77D [D-Orn9,D-neo^l l,tert-Leul2]NT(8-13), NT71D [N-methyl-Arg8,DAB9,D-neo-Tφl l,tert-leul2]NT(8-13), NT78L [N-methyl- Arg8,D-Orn9 L-neo^l l,tert-Leul2]NT(8-13), NT72L [D-Lys9,L-neo-Trpl l,tert- Leul2]NT(9-13), and NT78D [N-methyl-Arg8,D-Om9,D-neo^l l,tert-Leul2]NT(8- 13), where neo-Τφ is (2-amino-3-[lH-indolyl]propanoic acid). Other neurotensin analogs include Beta-lactotensin (NTR2 selective), JMV-449, and PD-149 or PD-163 (NTRl selective; reduced amide bond 8-13 fragment of neurotensin).

Other neurotensin analogs include those with modified amino acids (e.g., any of those described herein). The neurotensin analog may also be a neurotensin receptor agonist. For example, the neurotensin analog can be selective for NTRl, NTR2, or NTR3 (e.g., may bind to or activate one of NTRl, NTR2, or NTR3 at least 2, 5, 10, 50, 100, 500, 1000, 5000, 10,000, 50,000, or 100,000 greater) as compared to at least one of the other NTR receptors or both. Glial-derived neurotrophic factor (GDNF) or a GDNF analog

GDNF is secreted as a disulfide-linked homodimer, and is able to support survival of dopaminergic neurons, Purkinje cells, motoneurons, and sympathetic neurons. GDNF analogs or fragments having one or more of these activities may be used in the present invention, and activity of such analogs and fragments can be tested using any means known in the art.

Human GDNF is expressed as a 211 amino acid protein (isoform 1); a 185 amino acid protein (isoform 2), and a 133 amino acid protein. Mature GDNF is a 134 amino acid sequence that includes amino acids 118-211 of isoform 1, amino acids 92-185 of isoform 2. Isoform 3 includes a transforming growth factor like domain from amino acids 40-133. Other forms of GDNF include amino acids 78-211 of isoform 1.

In certain embodiments, the GDNF analog is a splice variant of GDNF. Such proteins are described in PCT Publication No. WO 2009/053536, and include the pre- (a)pro-GDNF, pre-^)pro-GDNF, and pre-(y)pro-GDNF splice variant, as well as the variants lacking the pre-pro region: (a)pro-GDNF, ^)pro-GDNF, and pre-(y)pro-GDNF.

GDNF analogs also include fragments of a GDNF precursor protein or the biologically active variant. Exemplar GDNF analogs include Pro-Pro-Glu-Ala-Pro-Ala- Glu-Asp-Arg-Ser-Leu-Gly-Arg-Arg (SEQ ID NO: 136); Phe-Pro-Leu-Pro-Ala-Gly-Lys- Arg (SEQ ID NO: 137); FPLPA-amide (SEQ ID NO: 138), PPEAPAEDRSL-amide (SEQ ID NO: 139), LLEAPAEDHSL-amide (SEQ ID NO: 140), SPDKQMAVLP (SEQ ID NO: 141), SPDKQAAALP (SEQ ID NO: 142), SPDKQTPIFS (SEQ ID NO: 143),

ERNRQAAAANPENSRGK-amide(SEQ ID NO: 144), ERNRQAAAASPENSRGK- amide (SEQ ID NO: 145), and ERNRQSAATNVENSSKK-amide (SEQ ID NO: 146). Other GDNF analogs are described in U.S. Patent Application Publication Nos.

2009/0069230 and 2006/0258576; and PCT Publication No. WO 2008/069876.

Brain-derived neurotrophic factor (BDNF) or a BDNF analog

BDNF is glycoprotein of the nerve growth factor family of proteins. The protein is encoded as a 247 amino acid polypeptide (isoform A), a 255 amino acid polypeptide (isoform B), a 262 amino acid polypeptide (isoform C), a 276 amino acid polypeptide (isoform D), a 329 amino acid polypeptide (isoform E). The mature 119 amino acid glycoprotein is processed from the larger precursor to yield a neutrophic factor that promotes the survival of neuronal cell populations. The mature protein includes amino acids 129-247 of the isoform A preprotein, amino acids 137-255 of the isoform B preprotein, amino acids 144-162 of isoform C preprotein, amino acids 158-276 of the isoform D preprotein, or amino acids 211 (or 212) - 329 of the isoform E preprotein. BDNF acts at the TrkB receptor and at low affinity nerve growth factor receptor (LNGFR or p75). BDNF is capable of supporting neuronal survival of existing neurons and can also promote growth and differentiation of new neurons. The BDNF fragments or analogs of the invention may have any of the aforementioned activities. Activity of such analogs and fragments can be tested using any means known in the art. Other BDNF analogs are described in U.S. Patent No. 6,800,607, U.S. Patent Application Publication No. 2004/0072291, and PCT Publication No. WO 96/15146.

Transport vectors

The conjugate can include any useful transport vector to bind or contain any therapeutic agent (e.g., as described herein). The transport vectors of the invention may include any lipid, carbohydrate, or polymer-based composition capable of transporting an agent (e.g., a therapeutic agent, such as those described herein). Transport vectors include lipid vectors (e.g., liposomes, micelles, and polyplexes) and polymer-based vectors, such as dendrimers. Other transport vectors include nanoparticles, which can include silica, lipid, carbohydrate, or other pharmaceutically-acceptable polymers.

Transport vectors can protect against degradation of an agent (e.g., any described herein), thereby increasing the pharmacological half-life and bio-availability of these compounds.

Lipid vectors can be formed using any biocompatible lipid or combination of lipids capable for forming lipid vectors (e.g., liposomes, micelles, and lipoplexes). Encapsulation of an agent into a lipid vector can protect the agent from damage or degradation or facilitate its entry into a cell. Lipid vectors, as a result of charge interactions (e.g., a cationic lipid vector and anionic cell membrane), interact and fuse with the cell membrane, thus releasing the agent into the cytoplasm. A liposome is a bilayered vesicle comprising one or more of lipid molecules, polypeptide-lipid conjugates, and lipid components. A lipoplex is a liposome formed with cationic lipid molecules to impart an overall positive charge to the liposome. A micelle is vesicle with a single layer of surfactants or lipid molecules. Liposomes

In certain embodiments, the lipid vector is a liposome. Typically, the lipids used are capable of forming a bilayer and are cationic. Classes of suitable lipid molecules include phospholipids (e.g., phosphotidylcholine), fatty acids, glycolipids, ceramides, glycerides, and cholesterols, or any combination thereof. Altematively or in addition, the lipid vector can include neutral lipids (e.g., dioleoylphosphatidyl ethanolamine (DOPE)). Other lipids that can form lipid vectors are known in the art and described herein.

As used herein, a "lipid molecule" is a molecule with a hydrophobic head moiety and a hydrophilic tail moiety and may be capable of forming liposomes. The lipid molecule can optionally be modified to include hydrophilic polymer groups. Examples of such lipid molecules include l,2-distearoyl-ST?-glycero-3-phosphoethanolanrine-N- [methoxy(polyethylene glycol)-2000] and l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[carboxy (polyethylene glycol)-2000] .

Examples of lipid molecules include natural lipids, such as cardiolipin (CL), phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidyl serine (PS); sphingolipids, such as sphingosine, ceramide, sphingomyelin, cerebrosides, sulfatides, gangliosides, and phytosphingosine; cationic lipids, such as l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP), dimethyldioctadecyl ammonium bromide (DDAB), 3-β-[Ν-(Ν',Ν'- dimethylaminoethane)carbamoly]cholesterol (DC-Choi), N-[l-(2,3,- ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N- [ 1 -(2,3,-dioleyloxy)propyl] -N,N-dimethyl-N-hy droxy ethylammonium bromide

(DORIE), and l,2-di-0-octadecenyl-3-trimethylammonium propane (DOTMA); phosphatidylcholines, such as l,2-dilauroyl-ST?-glycero-3-ethylphosphocholine, 1,2- dilauroyl-sn-glycero-3-phosphocholine (DLPC), l,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), l,2-dipalmitoyl-ST?-glycero-3-phosphocholine (DPPC), 1,2- distearoyl-OT-gly cero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-ST?-gly cero-3- phosphocholine (DOPC), and l-palinitoyl-2-oleoyl-OT-glycerol-3-phosphocholine (POPC); phosphoethanolamines, such as l,2-dibutyryl-s7?-glycero-3- phosphoethanolamine, l,2-distearoyl-ST?-glycero-3-phosphoethanolainine (DSPE), 1,2- dimyristoyl-ST?-glycero-3-phosphoethanolamine (DMPE), 1 ,2-dipalmitoyl-s7?-glycero-3- phosphoethanolamine (DPPE), l,2-dioleoyl-ST?-glycero-3-phosphoethanolamine (DOPE), and l^-dioleoyl-OT-glycero-S-phosphoethanolamine-N-iglutaryl); phosphatidic acids, such as l,2-dimyristoyl-s7?-glycero-3 -phosphate, l,2-dipalinitoyl-OT-glycero-3 -phosphate, and l,2-dioleoyl-57?-glycero-3-phosphate; phosphatidylglycerols, such as dipalmitoyl phosphatidylglycerol (DMPC), l,2-dimyristoyl-OT-glycero-3-phospho-( -rac-glycerol), and l,2-dioleoyl-s«-glycero-3-phospho-(r-rac-glycerol); phosphatidylserines, such as l,2-dimyristoyl-s7?-glycero-3-phospho-L-serine, l,2-dipalmitoyl-ST?-glycero-3-phospho-L- serine, and l,2-dioleoyl-ST?-glycero-3-phospho-L-serine; cardiolipins, such as ,3'- bis[l,2-dimyristoyl-s7?-glycero-3-phospho]-s7?-glycerol; and PEG-lipid conjugates, such as 1 ^-dipalmitoyl-i'w-glycero-S-phosphoethanolamine-N-fmethoxyipoly ethylene glycol)- 750], l^-dipalmitoyl-i'w-glycero-S-phosphoethanolamine-N-fmethoxyipoly ethylene glycol)-2000], l^-dipalmitoyl-OT-glycero-S-phosphoethanolamine-N- [methoxy(poly ethylene glycol)-5000], l,2-distearoyl-ST?-glycero-3-phosphoethanolainine- N-[methoxy(polyethylene glycol)-2000], and l,2-distearoyl-s7?-glycero-3- phosphoethanolamine-N-[carboxy (polyethylene glycol)-2000] .

Commercially available lipid compositions include Lipofectamine™ 2000 and Lipofectin® from Invitrogen Corp.; Transfectam® and Transfast™ from Promega Corp.; NeuroPORTER™ and Escort™ from Sigma- Aldrich Co.; FuGENE® 6 from Roche; and LipoTAXI® from Strategene. Known lipid compositions include the Trojan Horse Lipsome technology, as described in Boado, Pharm. Res. 24: 1772-1787 (2007).

The liposomes can also include other components that aid in the formation or stability of liposomes. Examples of components include cholesterol, antioxidants (e.g., a-tocopherol and β-hydroxytoluidine), surfactants, and salts.

A lipid molecule can be bound to a polypeptide by a covalent bond or a non- covalent bond (e.g., ionic interaction, entrapment or physical encapsulation, hydrogen bonding, absorption, adsorption, van der Waals forces, or any combinations thereof) with or without the use of a linker.

The liposome can be of any useful combination comprising lipid molecules, including polypeptide-lipid conjugates and other components that aid in the formation or stability of liposomes. A person of skill in that art will know how to optimize the combination that favor encapsulation of a particular agent, stability of the liposome, scaled-up reaction conditions, or any other pertinent factor. Exemplary combinations are described in Boado, Pharm. Res. 24: 1772-1787 (2007). In one example, the liposome comprises 93% POPC, 3% DDAB, 3% distearoylphosphatidylethanolamine (DSPE)- PEG2000, and 1% DSPE-PEG2000 covalently linked to a polypeptide.

Polyplexes

Complexes of polymers with agents are called polyplexes. Polyplexes typically consist of cationic polymers and their production is regulated by ionic interactions with an anionic agent (e.g., a polynucleotide). In some cases, polyplexes cannot release the bound agent into the cytoplasm. To this end, co-transfection with endosome-lytic agents (to lyse the endosome that is made during endocytosis), such as inactivated adenovirus, must occur. In certain cases, polymers, such as polyethylenimine, have their own method of endosome disruption, as does chitosan and trimethylchitosan. Polyplexes are described, for example, in U. S. Patent Application Publication Nos. 2002/0009491 , 2003/0134420, and 2004/0176282.

Polyplexes can be formed with any polymer and copolymer described herein, where non-charged or anionic polymers can be further derivatized to include cationic side chains. Examples of cationic side chains are amines, which are typically protonated under physiological conditions. Exemplary polymers that can be used to form polyplexes include polyamines, such as polylysine, polyarginine, polyamidoamine, and polyethylene imine.

Dendrimers

A dendrimer is a highly branched macromolecule with a spherical shape. The surface of the particle may be functionalized in many ways and many of the properties of the resulting construct are determined by its surface. In particular, it is possible to construct a cationic dendrimer (i.e., one with a positive surface charge). When in the presence of genetic material such as DNA or RNA, charge complimentarity leads to a temporary association of the polynucleotide with the cationic dendrimer. On reaching its destination the dendrimer-polynucleotide complex is then taken into the cell via endocytosis or across the BBB by transcytosis. Dendrimers are described, for example, in U.S. Patent Nos. 6,113,946 and 7,261,875. Examples of methods for making dendrimers are described in Svenson et a\., Adv. Drug. Deliv. Rev. 57:2106-2129 (2005).

For polyamidoamine (PAMAM) dendrimers, the core of the dendrimer typically comprises an amino group. Exemplary core molecules include ammonia; diamine molecules, such as ethylenediamine, 1,4-diaminobutane, 1,6-diaminohexane, 1,12- diaminododecane, and cystamine; and triamine molecules, such as triethanolamine. In the first step of the addition reaction, polymeric monomers are used to build upon the core by reacting the monomers with the amino groups of the core to form a tetra-branched molecule. Subsequent addition reactions with the diamine molecule and the polymeric monomer further build upon the dendrimer.

Examples of polymeric monomers that react with amino groups include methacrylate to form PAMAM dendrimers; and acrylonitrile to form poly (propylene imine) dendrimers. Examples of PAMAM dendrimers and synthetic reactions of dendrimers are set forth in U.S. Pat. Nos. 4,507,466, 5,527,524, and 5,714,166. Examples of PAMAM dendrimers formed with a triethanolamine core are set forth in Wu et al, Chem. Comm. 3:313-315 (2005); and Zhou et al, Chem. Comm. 22:2362-2364 (2006). Synthesis of the dendrimers can include additional steps, such as adding protecting groups to activated groups in order to prevent intramolecular reactions; and adding a deprotection step to remove protecting groups.

In addition to PAMAM dendrimers, other types of dendrimers can be used. For phosphorous dendrimers, the core of the dendrimer comprises a P=0 group. Exemplary core molecules include a cyclotriphosphazene group and a thiophosphoryl group.

Examples of polymeric monomers include phenoxymethyl(methylhydrazono) groups. Alternatively, the dendrimer is a hyperbranched polymer with a polyester core structure. Examples of such dendrimers include hyperbranched 2,2-bis(hydroxymethyl)propionic acid polyester- 16-hydroxyl.

The outer surface groups of the dendrimer can have a variety of functional groups, including amidoethanol, amidoethylethanolamine, amino, hexylamide, carboxylate, succinamidyl, trimethoxysilyl, tris(hydroxymethyl)amidomethane, and 3- carbomethoxypyrrolidinone groups. In addition, these functional groups can be further treated with a coupling agent to form activated groups (as defined herein).

In one particular example, the polyamidoamine dendrimer is conjugated to a polyvalent linker containing a hydrophilic polymer group: a-malemidyl-ω-Ν- hydroxysuccinimidyl polyethylene glycol (MW 3400). Examples of these dendrimers are described in Ke et al, J. Pharm. Sci. 97:2208-2216 (2008); Huang et al, J. Gene Med. 11 :754-763 (2009); Huang et al, Biomaterials 29:238-246 (2008); and Liu et al.

Biomaterials 30:4195-4202 (2009).

In another particular example, the polyamidoamine dendrimer is conjugated to a polyvalent linker containing an aliphatic group: 4-sulfosuccinimidyl-6-methyl-a-(2- pyridyldithio)toluamido]hexanoate. Examples of these dendrimers are described in Kang et al, Pharm. Res. 22:2099-2106 (2005).

Agents can be associated with the derivatized dendrimer by any number of methods, such as by covalent and non-covalent associations (e.g., ionic interaction, entrapment or physical encapsulation, hydrogen bonding, absorption, adsorption, van der Waals forces, or any combinations thereof).

Nanoparticles

Nanoparticles may be used as a transport vector in the invention. As used herein, a "nanoparticle" is a colloidal, polymeric, or elemental particle ranging in size from about 1 nm to about 1000 nm. Nanoparticles can be made up of silica, carbohydrate, lipid, or polymer molecules. Molecules can be either embedded in the nanoparticle matrix or may be adsorbed onto its surface. In one example, the nanoparticle may be made up of a biodegradable polymer such as poly(butylcyanoacrylate) (PBCA). Examples of elemental nanoparticles include carbon nanoparticles and iron oxide nanoparticles, which can then be coated with oleic acid (OA)-Pluronic^®. In this approach, a drug (e.g., a hydrophobic or water insoluble drug) is loaded into the nanoparticle, as described in Jain et al, Mol. Pharm. 2: 194-205 (2005). Other nanoparticles are made of silica, and include those described, for example, in Burns et al, Nano Lett. 9:442-448 (2009).

Nanoparticles can be formed from any useful polymer. Examples of polymers include biodegradable polymers, such as poly(butyl cyanoacrylate), poly(lactide), poly(glycolide), poly-s-caprolactone, poly(butylene succinate), poly(ethylene succinate), and poly(p-dioxanone); poly(ethyleneglycol); poly-2-hydroxyethylmethacrylate (poly(HEMA)); copolymers, such as poly(lactide-co-glycolide), poly(lactide)- poly(ethyleneglycol), poly(poly(ethyleneglycol)cyanoacrylate-co- hexadecylcyanoacrylate, and poly [HEMA-co-methacry lie acid]; proteins, such as fibrinogen, collagen, gelatin, and elastin; and polysaccharides, such as amylopectin, a- amylose, and chitosan. Examples of nanoparticles and methods of making thereof are described in Li et al, Int. J. Pharm. 259:93-101 (2003); Yu et al, Int. J. Pharm. 288:361- 368 (2005); Kreuter et al, Brain Res. 674: 171-174 (1995); Kreuter et al, Pharm. Res. 20:409-416 (2003); and Steiniger et al, Int. J. Cancer 109:759-767 (2004).

Other nanoparticles include solid lipid nanoparticles (SLN). SLN approaches are described, for example, in Kreuter, Ch. 24, In V. P. Torchilin (ed), Nanoparticles as Drug Carriers pp. 527-548, Imperial College Press, 2006). Examples of lipid molecules for solid lipid nanoparticles include stearic acid and modified stearic acid, such as stearic acid-PEG 2000; soybean lechitin; and emulsifying wax. Solid lipid nanoparticles can optionally include other components, including surfactants, such as Epicuron^® 200, poloxamer 188 (Pluronic® F68), Brij 72, Brij 78, polysorbate 80 (Tween 80); and salts, such as taurocholate sodium. Agents can be introduced into solid lipid nanoparticles by a number of methods discussed for liposomes, where such methods can further include high-pressure homogenization, and dispersion of microemulsions. Exemplary agents in SLNs include an anticancer agent, such as doxorubicin, tobramycin, idarubicin, or paclitaxel, or a paclitaxel derivative. Examples of SLNSs and method of making thereof are described in Koziara et al, Pharm. Res. 20: 1772-1778 (2003).

Nanoparticles can also include nanometer-sized micelles. Micelles can be formed from any polymers described herein. Exemplary polymers for forming micelles include block copolymers, such as poly(ethylene glycol) and poly(s-caprolactone). (e.g., a PEO- b-PCL block copolymer including a polymer of ε-caprolactone and a-methoxy-ω- hydroxy-poly(ethylene glycol)).

In certain embodiments, the properties of the nanoparticle are altered by coating with a surfactant. Any biocompatible surfactant may be used, for example, polysorbate surfactants, such as polysorbate 20, 40, 60, and 80 (Tween 80); Epicuron^® 200;

poloxamer surfactants, such as 188 (Pluronic^® F68) poloxamer 908 and 1508; and Brij surfactants, such as Brij 72 and Brij 78. In other embodiments, the surfactant is covalently attached to the nanoparticle, as is described in PCT Publication No. WO 2008/085556. Such an approach may reduce toxicity by preventing the surfactant from leeching out of the nanoparticle. Nanoparticles can be optionally coated with a surfactant.

Nanoparticles can optionally be modified to include hydrophilic polymer groups (e.g., poly(ethyleneglycol) or poly(propyleneglycol)), for example, by covalently attaching hydrophilic polymer groups to the surface or by using polymers that contain such hydrophilic polymer groups (e.g., poly[methoxy poly (ethyleneglycol)

cyanoacrylate-co-hexadecyl cyanoacrylate]). Nanoparticles can be optionally cross- linked, which can be particularly useful for protein-based nanoparticles.

Therapeutic agents can be introduced to nanoparticles by any useful method. Agents can be incorporated into the nanoparticle at, during, or after the formation of the nanoparticle. Examples of such approaches are described in Kreuter, Nanoparticular Carriers for Drug Delivery to the Brain, Chapter 24, in Torchilin (ed.), Nanoparticulates as Drug Carriers (2006), Imperial College Press.

Carbohydrate-based delivery methods

Carbohydrate-based polymers such as chitosan can be used as a transport vector e.g., in the formation of micelles or nanoparticles. As chitosan polymers can be amphiphilic, these polymers are especially useful in the delivery of hydrophobic agents (e.g., those described herein). Exemplary chitosan polymers include quaternary ammonium palmitoyl glycol chitosan, which can be synthesized as described in Qu et al, Biomacromolecules 7:3452-3459 (2006).

Hybrid methods

Some hybrid methods combine two or more techniques and can be useful for administering the conjugates of the invention to a cell, tissue, or organ of a subject.

Virosomes, for example, combine liposomes with an inactivated virus. This combination has more efficient gene transfer in respiratory epithelial cells than either viral or liposomal methods alone. Other methods involve mixing other viral vectors with cationic lipids or hybridizing viruses.

Multimeric polypeptides, therapeutic conjugates, and therapeutic polypeptides

The compounds of the invention also encompass multimeric (e.g., dimeric or trimeric) forms of the polypeptides, therapeutic polypeptides, and therapeutic conjugates described herein. In multimeric polypeptides or multimeric therapeutic polypeptides, two or more polypeptides are joined together by a chemical bond either directly (e.g., a covalent bond such as a disulfide or a peptide bond) or indirectly (e.g., through a linker such as those described herein). In multimeric therapeutic conjugates, two or more polypeptides are joined to the therapeutic agent(s) by a chemical bond either directly (e.g., a covalent bond such as a disulfide or a peptide bond) or indirectly (e.g., through a linker such as those described herein). Exemplary multimeric polypeptides, therapeutic polypeptides, and therapeutic conjugates are described below. Any linker described herein can be used for multimeric polypeptides, therapeutic polypeptides, and therapeutic conjugates (e.g., polyvalent linkers).

Multimeric polypeptides and multimeric therapeutic polypeptides

In certain embodiments, the multimeric polypeptide or multimeric therapeutic polypeptide is a dimer having the formula:

A¹-X-A²,

where A¹ and A² are each, independently, a polypeptide (e.g., any polypeptide described herein) and X is a linker. The linker may be any linker described herein. In particular embodiments, the linker contains a maleimido moiety and binds to a cysteine present in the peptide vector (e.g., a peptide vector to which an N-terminal or C-terminal cysteine residue has been added).

In other embodiments, the multimeric polypeptide or multimeric therapeutic polypeptide has or includes a formula selected from the group consisting of:

where A¹, A², A³, A^m, and each A^p are, independently, a polypeptide (e.g., any polypeptide described herein); X, X¹, and each X^p are, independently, a linker (e.g., any linker described herein) that joins together two polypeptides; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is n + 2; and p is an integer from 2 to n + 1. In particular embodiments, n is 1, and the compound has the formu

Higher order multimeric polypeptides or multimeric therapeutic polypeptides also be described by the formula:

where A¹, A², each A^q, each A^r, and each A^s are, independently, polypeptides (e.g., any of those described herein); A³ is a polypeptide or is absent; X, each X^q, each X^r, and each X^s are, independently, linkers that join polypeptides; m, n, and p are each, independently, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; q is an integer from 4 to m + 3; r is an integer from m + 4 to m + n + 3; and s is an integer from m + n + 4 to m + n + p + 3.

In some embodiments, multimeric polypeptides include any of modifications or further conjugations described herein for polypeptides (e.g., posttranslational processing or by chemical modification, including ubiquitination, pegylation, acetylation, acylation, cyclization, amidation, oxidation, sulfation, formation of cysteine, or covalent attachment of one or more therapeutic agents).

Multimeric therapeutic polypeptides that are fusion proteins are also encompassed in the present invention. In one embodiment, the multimeric therapeutic polypeptide is in the form of a fusion protein. The fusion protein may contain 2, 3, 4, 5, or more polypeptides, either joined directly by a peptide bond, or through peptide linkers. In one example, fusion protein dimers are described by the formula:

A¹-X-A²

where A¹ and A² are, independently, a polypeptide (e.g., any described herein) and X is either (a) a peptide bond that joins A¹ and A² or (b) one or more amino acids joined to A and A by peptide bonds. In certain embodiments, the peptide linker is a single amino acid (e.g., a naturally occurring amino acid), a flexible linker, a rigid linker, or an alpha- helical linker. Exemplary peptide linkers that can be used in the invention are described in the section entitled "Amino acid and peptide linkers" below. In certain embodiments A¹ and A² are the same polypeptide.

Fusion protein multimers can be described by the formula:

A¹-(Xⁿ-A^m), n

where n is or is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is an integer from 2 to n + 1 ; A¹ and each A^m are, independently, a polypeptide (e.g., any described herein); and each Xⁿ is, independently, either (a) a polypeptide that joins A¹ and A² or (b) one or more amino acids joined to the adjacent polypeptide (A¹ or A") by peptide bonds.

The polypeptides forming the multimer, in certain embodiments, may each be fewer than 50, 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 amino acids in length. The fusion protein may be fewer than 1,000, 500, 250, 150, 100, 90, 80, 75, 70, 65, 60, 55, 50, 45, 40, or 35 amino acids in length.

Multimeric therapeutic conjugates

The therapeutic agent or transport vector can be joined to one or more multimeric polypeptides joined (e.g., by a covalent bond) to form a multimeric conjugate or a therapeutic polypeptide. When the therapeutic agent is a therapeutic peptidic agent, then the multimeric therapeutic conjugate can be a fusion protein multimer, as described herein.

Compounds including a therapeutic agent and dimeric polypeptide can be conjugated either through the polypeptide portion of the molecule or through the linker portion of the molecule. Compounds of the invention in which the agent is joined (e.g., through a linker where the linker is a chemical linker, peptide, or a covalent bond, such as a peptide bond) to the polypeptide can be represented by the formula:

where A¹ and A² are each, independently, polypeptides (e.g., any described herein); X is a linker (e.g., chemical linker, peptide, or covalent bond) that joins A¹ and A²; B¹ is a therapeutic agent or transport vector; and Y¹ is a linker that joins B¹ and A¹.

In certain embodiments, two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) therapeutic agents or transport vectors are joined to one or both of the polypeptides. Such compounds can be represented by the formula:

where A¹, A², and X are as defined above; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is an integer from 1 to m; q is an integer from m + 1 to m + n; each B^p and each B^q are, independently, a therapeutic agent or transport vector (e.g., any described herein); and each Y^p and each Y^q are, independently, a linker that joins each B^p or each B^q to A¹ or A², respectively. In other embodiments, the therapeutic agent or transport vector is joined (e.g., through a covalent bond or a chemical linker such as those described herein) to the dimer through the linker that joins the polypeptides forming the dimer. Such compounds can have the formula:

where A¹ and A² are polypeptides (e.g., any described herein); B is a therapeutic agent or transport vector; and X is a linker that joins A¹, A², and B.

In other embodiments, the therapeutic agent or transport vector can be joined to both the linker and a polypeptide. Such compounds can be represented by the formula:

where A¹ and A² are, independently, polypeptides; B^z is an agent or is absent; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is an integer from 1 to m; q is an integer from m + 1 to m + n; Each B^p and B^q is, independently, a therapeutic agent or transport vector (e.g., any described herein); and each Y^p and Y^q is, independently, a linker that joins each B^p or each B^q to A¹ or A², respectively, where at least one (e.g., at least two) of the following is true (i) Bl is present; (ii) m is at least 1 ; and (iii) n is at least 1.

Compounds of the invention can also include a trimeric polypeptide. Where the trimeric polypeptide is joined to a single agent through one of the polypeptides, the compound can have one of the following formulas:

/

-X¹ B

where A¹, A², and A³ are each, independently, a polypeptide (e.g., any described herein); X¹ and X² are, independently, linkers (e.g., any described herein); B¹ is a therapeutic agent or transport vector; and Y¹ is a linker that joins B¹ to a polypeptide (e.g., A¹, A², and A³) or to the linker X¹.

In other embodiments, the trimeric polypeptide is conjugated to one or more than one therapeutic agent or transport vector. Such conjugation can be through either the polypeptide, or through the linker(s). Such compounds can include one of the following formulas:

where A¹, A², and A³ are, independently, polypeptides; n, m, and j are 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each B^p, each B^q, and each B^r are, independently, therapeutic agents or transport vectors (e.g., any described herein); B^z and B^y are, independently, therapeutic agents or transport vectors or are absent; X¹ is a linker joining A¹, A², and B^z, if present; and X² is a linker joining A², A³, and B^y, if present. In certain embodiments, at least one of n, m, or j is at least one, B^z is present, or B^y is present. In other embodiments, at least two (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30) of B^p, B^q, B^r, B^y, and B^z are present.

The compounds of the invention can also include polypeptide multimers of a higher order (e.g., quatromers, pentomers, etc.). Such multimers can be described by the formula:

where A¹, A², each A^q, each A^r, and each A^s are, independently, polypeptides; A³ is a polypeptide or is absent; X, each X^q, X^r, and X^s are, independently, linkers that join polypeptides; m, n, and p are each, independently, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; q is an integer from 4 to m + 3; r is an integer from m + 4 to m + n + 3; and s is an integer from m + n + 4 to m + n + p + 3. One or more agents can be joined to either the linkers (X, any X^q, X^r, or X^s) or the polypeptides (A¹, A², A³, each A^q, each A^r, and each A^s) of this formula in order to form higher order multimer conjugates.

Modified polypeptides

The polypeptides, therapeutic polypeptides, therapeutic conjugates, and therapeutic peptidic agents used in the invention may have a modified amino acid sequence. In certain embodiments, the modification does not destroy significantly a desired biological activity (e.g., ability to cross the BBB or agonist activity). The modification may reduce (e.g., by at least 5%, 10%, 20%, 25%, 35%, 50%, 60%, 70%, 75%, 80%, 90%, or 95%), may have no effect, or may increase (e.g., by at least 5%, 10%, 25%, 50%, 100%, 200%, 500%, or 1000%) the biological activity of the original polypeptide. The modified polypeptide may have or may optimize a characteristic of a sequence, such as in vivo stability, bioavailability, toxicity, immunological activity, immunological identity, and conjugation properties.

Modifications include those by natural processes, such as posttranslational processing, or by chemical modification techniques known in the art. Modifications may occur anywhere in a polypeptide including the polypeptide backbone, the amino acid side chains and the amino- or carboxy -terminus. The same type of modification may be present in the same or varying degrees at several sites in a given polypeptide, and a polypeptide may contain more than one type of modification. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslational natural processes or may be made synthetically. Other modifications include pegylation, acetylation, acylation, addition of acetomidomethyl (Acm) group, ADP-ribosylation, alkylation, amidation, biotinylation, carbamoylation, carboxyethylation, esterification, covalent attachment to fiavin, covalent attachment to a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of drug, covalent attachment of a marker (e.g., fluorescent or radioactive), covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation and ubiquitination.

A modified polypeptide can also include an amino acid insertion, deletion, or substitution, either conservative or non-conservative (e.g., D-amino acids, desamino acids) in the polypeptide sequence (e.g., where such changes do not substantially alter the biological activity of the polypeptide). In particular, the addition of one or more cysteine residues to the amino or carboxy terminus of any of the polypeptides of the invention can facilitate conjugation of these polypeptides by, e.g., disulfide bonding. For example, Angiopep-1 (SEQ ID NO:67), Angiopep-2 (SEQ ID NO:97), or Angiopep-7 (SEQ ID NO: 112) can be modified to include a single cysteine residue at the amino-terminus (SEQ ID NOS: 71, 113, and 115, respectively) or a single cysteine residue at the carboxy- terminus (SEQ ID NOS: 72, 114, and 116, respectively). Amino acid substitutions can be conservative (i.e., wherein a residue is replaced by another of the same general type or group) or non-conservative (i.e., wherein a residue is replaced by an amino acid of another type). In addition, a non-naturally occurring amino acid can be substituted for a naturally occurring amino acid (i.e., non-naturally occurring conservative amino acid substitution or a non-naturally occurring non-conservative amino acid substitution).

Polypeptides made synthetically can include substitutions of amino acids not naturally encoded by DNA (e.g., non-naturally occurring or unnatural amino acid).

Examples of non-naturally occurring amino acids include D-amino acids, an amino acid having an acetylaminomethyl group attached to a sulfur atom of a cysteine, a pegylated amino acid, the omega amino acids of the formula NH₂(CH₂)_nCOOH wherein n is 2-6, neutral nonpolar amino acids, such as sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine. Phenylglycine may substitute for Trp, Tyr, or Phe; citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline may be substituted with hydroxyproline and retain the conformation conferring properties.

Analogs may be generated by substitutional mutagenesis and retain the biological activity of the original polypeptide. Examples of substitutions identified as "conservative substitutions" are shown in Table 2. If such substitutions result in a change not desired, then other type of substitutions, denominated "exemplary substitutions" in Table 2, or as further described herein in reference to amino acid classes, are introduced and the products screened. Polypeptide derivatives and peptidomimetics

In addition to polypeptides consisting of naturally occurring amino acids, peptidomimetics or polypeptide analogs are also encompassed by the present invention and can form the polypeptide or peptide/polypeptide agents used in the compounds of the invention. Polypeptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template polypeptide. The non-peptide compounds are termed "peptide mimetics" or peptidomimetics (Fauchere et al, Infect. Immun. 54:283-287 (1986) and Evans et al, J. Med. Chem. 30: 1229-1239 (1987)). Peptide mimetics that are structurally related to therapeutically useful peptides or polypeptides may be used to produce an equivalent or enhanced therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to the paradigm polypeptide (i.e., a polypeptide that has a biological or pharmacological activity) such as naturally-occurring receptor-binding polypeptides, but have one or more peptide linkages optionally replaced by linkages such as -CH₂NH- -CH₂S- -CH₂-CH₂- -CH=CH- (cis and trans), -CH₂SO- -CH(OH)CH₂- -COCH₂- etc., by methods well known in the art (Spatola, Peptide Backbone Modifications , Vega Data, 1 :267 (1983); Spatola et al, Life Sci. 38: 1243-1249 (1986); Hudson et al, Int. J. Pept. Res. 14: 177-185 (1979); and Weinstein, 1983, Chemistry and Biochemistry, of Amino Acids, Peptides and Proteins, Weinstein eds, Marcel Dekker, New York). Such polypeptide mimetics may have significant advantages over naturally occurring polypeptides including more economical production, greater chemical stability, enhanced pharmacological properties (e.g., half- life, absorption, potency, and/or efficiency), reduced antigenicity, and others.

While the polypeptides described herein may efficiently cross the BBB or target particular cell types (e.g., those described herein), their effectiveness may be reduced by the presence of proteases. Likewise, the effectiveness of the peptide/polypeptide agents used in the invention may be similarly reduced. Serum proteases have specific substrate requirements, including L-amino acids and peptide bonds for cleavage. Furthermore, exopeptidases, which represent the most prominent component of the protease activity in serum, usually act on the first peptide bond of the polypeptide and require a free N- terminus (Powell et al, Pharm. Res. 10: 1268-1273 (1993)). In light of this, it is often advantageous to use modified versions of polypeptides. The modified polypeptides retain the structural characteristics of the original L-amino acid polypeptides, but

advantageously are not readily susceptible to cleavage by protease and/or exopeptidases.

Systematic substitution of one or more amino acids of a consensus sequence with D-amino acid of the same type (e.g., an enantiomer, such as a D-lysine in place of L- lysine) may be used to generate more stable polypeptides. Thus, a polypeptide derivative or peptidomimetic as described herein may be all L-, all D-, or mixed D, L polypeptides. The presence of an N-terminal or C-terminal D-amino acid increases the in vivo stability of a polypeptide because peptidases cannot utilize a D-amino acid as a substrate (Powell et al, Pharm. Res. 10: 1268-1273 (1993)). Reverse-D polypeptides are polypeptides containing D-amino acids, arranged in a reverse sequence relative to a polypeptide containing L-amino acids. Thus, the C-terminal residue of an L-amino acid polypeptide becomes N-terminal for the D-amino acid polypeptide, and so forth. Reverse D- polypeptides retain the same tertiary conformation and therefore the same activity, as the L-amino acid polypeptides, but are more stable to enzymatic degradation in vitro and in vivo, and thus have greater therapeutic efficacy than the original polypeptide (Brady and Dodson, Nature 368:692-693 (1994) and Jameson et al, Nature 368:744-746 (1994)). In addition to reverse-D-polypeptides, constrained polypeptides including a consensus sequence or a substantially identical consensus sequence variation may be generated by methods well known in the art (Rizo et al., Ann. Rev. Biochem. 61:387-418 (1992)). For example, constrained polypeptides may be generated by adding cysteine residues capable of forming disulfide bridges and, thereby, resulting in a cyclic polypeptide. Cyclic polypeptides have no free N- or C-termini. Accordingly, they are not susceptible to proteolysis by exopeptidases, although they are, of course, susceptible to endopeptidases, which do not cleave at polypeptide termini. The amino acid sequences of the

polypeptides with N-terminal or C-terminal D-amino acids and of the cyclic polypeptides are usually identical to the sequences of the polypeptides to which they correspond, except for the presence of N-terminal or C-terminal D-amino acid residue, or their circular structure, respectively.

A cyclic derivative containing an intramolecular disulfide bond may be prepared by conventional solid phase synthesis while incorporating suitable S-protected cysteine or homocysteine residues at the positions selected for cyclization such as the amino and carboxy termini (Sah et al, J. Pharm. Pharmacol. 48: 197 (1996)). Following completion of the chain assembly, cyclization can be performed either (1) by selective removal of the S-protecting group with a consequent on-support oxidation of the corresponding two free SH-functions, to form a S-S bonds, followed by conventional removal of the product from the support and appropriate purification procedure or (2) by removal of the polypeptide from the support along with complete side chain de-protection, followed by oxidation of the free SH-functions in highly dilute aqueous solution.

The cyclic derivative containing an intramolecular amide bond may be prepared by conventional solid phase synthesis while incorporating suitable amino and carboxyl side chain protected amino acid derivatives, at the position selected for cyclization. The cyclic derivatives containing intramolecular -S-alkyl bonds can be prepared by conventional solid phase chemistry while incorporating an amino acid residue with a suitable amino-protected side chain, and a suitable S-protected cysteine or homocysteine residue at the position selected for cyclization.

Another effective approach to confer resistance to peptidases acting on the N- terminal or C-terminal residues of a polypeptide is to add chemical groups at the polypeptide termini, such that the modified polypeptide is no longer a substrate for the peptidase. One such chemical modification is glycosylation of the polypeptides at either or both termini. Certain chemical modifications, in particular N-terminal glycosylation, have been shown to increase the stability of polypeptides in human serum (Powell et al, Pharm. Res. 10: 1268-1273 (1993)). Other chemical modifications which enhance serum stability include, but are not limited to, the addition of an N-terminal alkyl group, consisting of a lower alkyl of from one to twenty carbons, such as an acetyl group, and/or the addition of a C-terminal amide or substituted amide group. In particular, the present invention includes modified polypeptides consisting of polypeptides bearing an N- terminal acetyl group and/or a C-terminal amide group.

Also included by the present invention are other types of polypeptide derivatives containing additional chemical moieties not normally part of the polypeptide, provided that the derivative retains the desired functional activity of the polypeptide. Examples of such derivatives include (1) N-acyl derivatives of the amino terminal or of another free amino group, wherein the acyl group may be an alkanoyl group (e.g., acetyl, hexanoyl, octanoyl) an aroyl group (e.g., benzoyl) or a blocking group such as F-moc

(fluorenylmethyl-O-CO-); (2) esters of the carboxy terminal or of another free carboxy or hydroxyl group; (3) amide of the carboxy -terminal or of another free carboxyl group produced by reaction with ammonia or with a suitable amine; (4) phosphorylated derivatives; (5) derivatives conjugated to any biological ligand; and (6) other types of derivatives.

Longer polypeptide sequences which result from the addition of additional amino acid residues to the polypeptides described herein are also encompassed in the present invention. Such longer polypeptide sequences can be expected to have the same biological activity and specificity (e.g., cell tropism) as the polypeptides described above. While polypeptides having a substantial number of additional amino acids are not excluded, it is recognized that some large polypeptides may assume a configuration that masks the effective sequence, thereby preventing binding to a target (e.g., a member of the LRP receptor family, such as LRP or LRP2). These derivatives could act as competitive antagonists. Thus, while the present invention encompasses polypeptides or derivatives of the polypeptides described herein having an extension, desirably the extension does not destroy the cell targeting activity of the polypeptides or its derivatives.

Other derivatives included in the present invention are dual polypeptides consisting of two of the same, or two different polypeptides, as described herein, covalently linked to one another either directly or through a spacer, such as by a short stretch of alanine residues or by a putative site for proteolysis (e.g., by cathepsin, see e.g., U.S. Patent No. 5,126,249 and European Patent No. 495 049).

The present invention also encompasses polypeptide derivatives that are chimeric or fusion proteins containing a polypeptide described herein, or fragment thereof, linked at its amino- or carboxy -terminal end, or both, to an amino acid sequence of a different protein. Such a chimeric or fusion protein may be produced by recombinant expression of a nucleic acid encoding the protein. For example, a chimeric or fusion protein may contain at least 6 amino acids shared with one of the described polypeptides which desirably results in a chimeric or fusion protein that has an equivalent or greater functional activity.

Assays to identify peptidomimetics

As described above, non-peptidyl compounds generated to replicate the backbone geometry and pharmacophore display (peptidomimetics) of the polypeptides described herein often possess attributes of greater metabolic stability, higher potency, longer duration of action, and better bioavailability. Peptidomimetics compounds can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the One-bead one-compound' library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer, or small molecule libraries of compounds (Lam, Anticancer Drug Des 12: 145 (1997)). Examples of methods for the synthesis of molecular libraries can be found in the art, for example, in: DeWitt et al, Proc. Natl. Acad. Sci. USA 90:6909 (1993); Erb et al., Proc. Natl. Acad. Sci. USA 91 : 11422 (1994); Zuckermann et al, J. Med. Chem. 37:2678 (1994); Cho et al, Science 261 : 1303 (1993); Carell et &\., Angew. Chem. Int. Ed. Engl. 33:2059 (1994) and ibid. 2061; and Gallop et a\., Med. Chem. 37: 1233 (1994). Libraries of compounds may be presented in solution (e.g., Houghten, Biotechniques 13:412-421 (1992)) or on beads (Lam, Nature 354:82-84 (1991)), chips (Fodor, Nature 364:555-556 (1993)), bacteria or spores (U.S. Patent No. 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. Sci. USA 89: 1865-1869 (1992)), phage (Scott and Smith, Science 249:386-390 (1990)), or an enzyme (e.g., luciferase) and an enzymatic label detected by determination of conversion of an appropriate substrate to product.

Once a polypeptide as described herein is identified, it can be isolated and purified by any number of standard methods including, but not limited to, differential solubility (e.g., precipitation), centrifugation, chromatography (e.g., affinity, ion exchange, and size exclusion), or by any other standard techniques used for the purification of peptides, peptidomimetics, or proteins. The functional properties of an identified polypeptide of interest may be evaluated using any functional assay known in the art. Desirably, assays for evaluating downstream receptor function in intracellular signaling are used (e.g., cell proliferation).

For example, the peptidomimetics compounds of the present invention may be obtained using the following three-phase process: (1) scanning the polypeptides described herein to identify regions of secondary structure necessary for targeting the particular cell types described herein; (2) using conformationally constrained dipeptide surrogates to refine the backbone geometry and provide organic platforms corresponding to these surrogates; and (3) using the best organic platforms to display organic pharmocophores in libraries of candidates designed to mimic the desired activity of the native polypeptide. In more detail the three phases are as follows. In phase 1, the lead candidate polypeptides are scanned and their structure abridged to identify the requirements for their activity. A series of polypeptide analogs of the original are synthesized. In phase 2, the best polypeptide analogs are investigated using the conformationally constrained dipeptide surrogates. Indolizidin-2-one, indolizidin-9-one and quinolizidinone amino acids (I²aa, I⁹aa and Qaa respectively) are used as platforms for studying backbone geometry of the best peptide candidates. These and related platforms (reviewed in Halab et al,

Biopolymers 55: 101-122 (2000) and Hanessian et al, Tetrahedron 53: 12789-12854 (1997)) may be introduced at specific regions of the polypeptide to orient the

pharmacophores in different directions. Biological evaluation of these analogs identifies improved lead polypeptides that mimic the geometric requirements for activity. In phase 3, the platforms from the most active lead polypeptides are used to display organic surrogates of the pharmacophores responsible for activity of the native peptide. The pharmacophores and scaffolds are combined in a parallel synthesis format. Derivation of polypeptides and the above phases can be accomplished by other means using methods known in the art.

Structure function relationships determined from the polypeptides, polypeptide derivatives, peptidomimetics or other small molecules described herein may be used to refine and prepare analogous molecular structures having similar or better properties. Accordingly, the compounds of the present invention also include molecules that share the structure, polarity, charge characteristics and side chain properties of the polypeptides described herein.

In summary, based on the disclosure herein, those skilled in the art can develop peptides and peptidomimetics screening assays which are useful for identifying compounds for targeting an agent to particular cell types (e.g., those described herein). The assays of this invention may be developed for low-throughput, high-throughput, or ultra-high throughput screening formats. Assays of the present invention include assays amenable to automation. Example 1

Experimental procedure for gelatin zymography

Gelatin zymography was used to assess the extent of proMMP-2 and proMMP-9 activity. Briefly, an aliquot (20 μΐ) of the culture medium was subjected to SDS-PAGE in a gel containing 0.1 mg/ml gelatin. The gels were then incubated in 2.5% Triton X-100 and rinsed in nanopure distilled H₂0. Gels were further incubated at 37°C for 20 hrs in 20 mM NaCl, 5 mM CaCl₂, 0.02% Brij-35, 50 mM Tris-HCl buffer, pH 7.6, then stained with 0.1% Coomassie Brilliant blue R-250 and destained in 10% acetic acid, 30% methanol in H₂0. Gelatinolytic activity was detected as unstained bands on a blue background.

Example 2

Inhibition of PMA-mediated MMP-9 secretion in medulloblastoma cells

In order to determine the effect of Angiopep-2 in DAOY medulloblastoma cells, we performed various stimulation assays using vehicle (control), phorbol 12-my striate 13-acetate (PMA), or TNF. PMA (a diester of phorbol) promotes cancer cell migration and cellular invasion and increases MMP-2 and MMP-9 secretion by inducing various intracellular pathways. MMP-2 and MMP-9 expression and secretion are associated with cancer and cancer metastasis, as described herein.

Medulloblastoma-derived DAOY cells were serum-starved in the presence of

Angiopep-2 in combination with vehicle, TNF or 1 mM PMA for 18 hours. Scanning densitometry was used to quantify the extent of proMMP-9 gelatinolytic activity for each set of data. Data shown is representative of two independent experiments.

As shown in Figure 1A, PMA induced MMP-2 and MMP-9 secretion (as detected by their prometalloproteinases, proMMP-2 and proMMP-9, respectively). In contrast, vehicle and TNF only induced MMP-2 secretion. As shown in Figure IB, treatment with Angiopep-2 resulted in a dose-dependent and significant decrease in MMP-9 secretion for PMA-treated medulloblastoma cells.

Example 3

Inhibition of PMA-mediated phosphorylation of ΙκΒ in glioblastoma cells

To determine the effect of Angiopep-2 in intracellular processes, we performed in vitro phosphorylation assays for ΙκΒ. Phosphorylation of ΙκΒ, resulting in active NF-KB, is implicated in various cellular processes, including inflammation, cell proliferation, and apoptosis.

Medulloblastoma-derived DAOY cells were serum-starved for 30 minutes in the presence of vehicle or Angiopep-2. Cells were then incubated for the indicated time with vehicle, TNF or 1 mM PMA. Lysates were isolated, electrophoresed via SDS-PAGE and immunodetection of phosphorylated IkB (Ρ-ΙκΒ), ΙκΒ, and of GAPDH proteins was performed. Quantification was performed by scanning densitometry of the

autoradiograms. Data were expressed as x-fold induction over basal untreated cells for P- IKB, and as the percent (%) expression of untreated basal conditions for IKB.

As shown in Figure 2A, this assay was performed for Angiopep-2, PMA, and TNF as a function of time. As shown in Figure 2B, Angiopep-2 does not trigger significant IKB phosphorylation in U87 glioblastoma cells. In contrast, both PMA and TNF resulted in increased IKB phosphorylation (about 45-fold and 65-fold over control, respectively, at 10 minutes).

To assess inhibition of IKB phosphorylation by Angiopep-2, cells were pre- incubated with Angiopep-2 for 30 minutes and then stimulated with PMA or TNF. As shown in Figure 3A, this assay was performed for PMA, TNF, or vehicle. As shown in Figure 3B, Angiopep-2 prevented PMA-mediated phosphorylation of IKB in U87 glioblastoma cells. As compared to data provided in Figure 2B, pre-incubation with Angiopep-2 decreased TNF-mediated phosphorylation, whereas the effect of PMA was completely abrogated by Angiopep-2 (Figure 3B). Together, these data indicate that Angiopep-2 inhibited PMA-mediated IKB phosphorylation in glioblastoma cells.

Example 4

Inhibition of PMA-mediated MMP-2 activity in glioblastoma cells

We performed zymography assays to test whether Angiopep-2 affected the activity of MMP-2 in U87 glioblastoma cells. Activity of MMP-2 was determined by detecting gelatinolytic activity after stimulation with 1 μΜ PMA and treatment with Angiopep-2 (An-2). As shown in Figure 4A, a zymography assay was performed in the presence of gelatin by detecting proMMP-2. As shown in Figure 4B, Angiopep-2 inhibited activity of MMP-2 in glioblastoma cells. Example 5

Inhibition of PMA-mediated MMP-2 and plasmin activity in glioblastoma cells

We also performed zymography assays to test whether Angiopep-2 affected the activity of MMP-2 and plasmin in U87 glioblastoma cells. Activity of MMP-2 and plasmin was determined by detecting gelatinolytic or plasmin activity, respectively, after stimulation with PMA and treatment with Angiopep-2. As shown in Figure 5A, a zymography assay was performed in the presence of gelatin and plasminogen. As shown in Figures 5B and 5C, Angiopep-2 inhibited activity of MMP-2 and plasmin in glioblastoma cells.

Other embodiments

All publications, patent applications, and patents mentioned in this specification are herein incorporated by reference.

Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific desired embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the fields of medicine, pharmacology, or related fields are intended to be within the scope of the invention.

Claims

CLAIMS What is claimed is:

1. A method of treating or prophylactically treating a subject having cancer, having an increased risk of cancer, or in remission from cancer, said method comprising administering to said subject an effective amount of a therapeutic polypeptide, said polypeptide comprising an amino acid sequence at least 70% identical to a sequence selected from the group consisting of SEQ ID NOS: l-93, 97-105, and 107-122, or a fragment thereof, wherein said polypeptide is not conjugated to a second therapeutic agent.

2. The method of claim 1, wherein said therapeutic polypeptide has an amino acid sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOS: l-93, 97-105, and 107-122, or a fragment thereof.

3. The method of claim 1, wherein said therapeutic polypeptide is Angiopep- 2 or a fragment thereof.

4. The method of any of claims 1-3, wherein said cancer is brain cancer.

5. The method of claim 4, wherein said brain cancer is selected from the group consisting of glioma, mixed glioma, glioblastoma multiforme, astrocytoma, pilocytic astrocytoma, dysembryoplastic neuroepithelial tumor, oligodendroglioma, ependymoma, oligoastrocytoma, hemangioma, medulloblastoma, retinoblastoma, neuroblastoma, germinoma, and teratoma.

6. The method of any of claims 1-5, wherein said cancer is metastatic cancer.

7. The method of any of claims 1-6, wherein said subject is human.

8. The method of any of claims 1-7, wherein said therapeutic polypeptide reduces matrix metalloproteinase activity.

9. The method of claim 8, wherein said matrix metalloproteinase is MMP-2 or MMP-9.

10. The method of any of claims 1-9, wherein said therapeutic polypeptide reduces plasmin activity.

11. The method of any of claims 1-10, wherein said therapeutic polypeptide reduces phosphorylation of ΙκΒ.

12. The method of claim 11, wherein said therapeutic polypeptide reduces activation of NF-KB.

13. The method of any of claims 1-12, wherein said therapeutic polypeptide reduces migration or invasion of cancer cells.

14. The method of any of claims 1-13, wherein said therapeutic polypeptide reduces angiogenesis.

15. The method of any of claims 1-14, wherein said therapeutic polypeptide is present in a composition and said composition does not include ANG1005 or includes less than 5% of ANG1005 in the composition.

16. A composition comprising:

(c) a therapeutic polypeptide not conjugated to a therapeutic agent and

(d) a therapeutic conjugate comprising a second polypeptide conjugated to a second therapeutic agent or a transport vector,

wherein said therapeutic polypeptide and said therapeutic conjugate are together present in an effective amount, and said therapeutic polypeptide and said second polypeptide independently have an amino acid sequence at least 70% identical to a sequence selected from the group consisting of SEQ ID NOS: l-93, 97-105, and 107-122, or a fragment thereof.

17. The composition of claim 16, wherein said therapeutic polypeptide and said second polypeptide independently have an amino acid sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-93, 97-105, and 107-122, or a fragment thereof.

18. The composition of claim 16, wherein said therapeutic polypeptide is Angiopep-2 or a fragment thereof.

19. The composition of any of claims 16-18, wherein said second polypeptide is conjugated to said second therapeutic agent or said transport vector by a covalent bond or a linker.

20. The composition of claim 19, wherein said covalent bond is a peptide bond.

21. The composition of claim 19, wherein said linker is at least one amino acid or an ester linker.

22. The composition of any of claims 16-21, wherein said second polypeptide is conjugated to said second therapeutic agent and said second therapeutic agent is selected from the group consisting of an anticancer agent, a therapeutic nucleic acid agent, a small molecule drug, a label, and a therapeutic peptidic agent.

23. The composition of claim 22, wherein said second therapeutic agent is said anticancer agent and said anticancer agent is selected from the group consisting of paclitaxel, etoposide, and doxorubicin, or analogs thereof.

24. The composition of claim 22, wherein said second therapeutic agent is said therapeutic peptidic agent and said therapeutic peptidic agent is selected from the group consisting of a GLP-1 agonist, leptin or a leptin analog, neurotensin or a neurotensin analog, a neurotensin receptor agonist, glial-derived neurotrophic factor (GDNF) or a GDNF analog, or brain-derived neurotrophic factor (BDNF) or a BDNF analog.

25. The composition of claim 22 or 24, wherein said second therapeutic agent is said therapeutic peptidic agent and said therapeutic conjugate is a fusion protein.

26. The composition of any of claims 16-21, wherein said second polypeptide is conjugated to said transport vector and said transport vector is selected from the group consisting of a lipid vector, a polyplex, a dendrimer, and a nanoparticle.

27. The composition of claim 26, wherein said transport vector is bound to or contains a third therapeutic agent.

28. The composition of claim 27, wherein said third therapeutic agent is selected from the group consisting of an anticancer agent, a therapeutic nucleic acid agent, a small molecule drug, a label, and a therapeutic peptidic agent.

29. The composition of any of claims 16-23, wherein said therapeutic conjugate is ANG1005.

30. The composition of claim 16, wherein said therapeutic polypeptide is Angiopep-2 or a fragment thereof and said therapeutic conjugate is ANG1005.

31. The composition of any of claims 16-30, wherein the ratio of said therapeutic polypeptide and said therapeutic conjugate is from 1 :9 to 9: 1.

32. The composition of claim 31, wherein the ratio of said therapeutic polypeptide and said therapeutic conjugate is 1 : 1.

33. The composition of any of claims 16-32, further comprising a pharmaceutically acceptable carrier.

34. A method of treating or prophylactically treating a subject having cancer, having an increased risk of cancer, or in remission from cancer, said method comprising administering to said subject a composition of any of claims 16-33 in an effective amount.

35. The method of claim 34, wherein said cancer is brain cancer.

36. The method of claim 35, wherein said brain cancer is selected from the group consisting of glioma, mixed glioma, glioblastoma multiforme, astrocytoma, pilocytic astrocytoma, dysembryoplastic neuroepithelial tumor, oligodendroglioma, ependymoma, oligoastrocytoma, hemangioma, medulloblastoma, retinoblastoma, neuroblastoma, germinoma, and teratoma.

37. A kit comprising:

(a) a therapeutic polypeptide not conjugated to a therapeutic agent and

(b) a therapeutic conjugate comprising a second polypeptide conjugated to a second therapeutic agent or a transport vector,

wherein said therapeutic polypeptide and said therapeutic conjugate are together present in an effective amount, and said therapeutic polypeptide and said second polypeptide independently have an amino acid sequence at least 70% identical to a sequence selected from the group consisting of SEQ ID NOS: l-93, 97-105, and 107-122, or a fragment thereof.

38. The kit of claim 37, wherein said therapeutic polypeptide is Angiopep-2 or a fragment thereof and said therapeutic conjugate is ANG1005.