WO2018112397A1 - Treatment of cancer - Google Patents

Abstract

Provided are methods relating to compositions that include a cyclodextrin-containing polymer (CDP) conjugate, e.g., CRLX101, and compositions thereof for the treatment of a cancer (e.g., breast cancer).

Description

TREATMENT OF CANCER

Claim of Priority

This application claims priority to U.S. Patent Application No. 62/435,315, filed

December 16, 2016, which is incorporated herein by reference in its entirety.

Background of the Invention

VEGF-pathway targeting antiangiogenic drugs, such as bevacizumab, when combined with chemotherapy, have changed clinical practice for the treatment of a broad spectrum of human cancers. However, adaptive resistance often develops and one major mechanism is elevated tumor hypoxia and upregulated HIF-Ια caused by antiangiogenic treatment. Reduced tumor vessel numbers and function following antiangiogenic therapy may also affect intratumoral delivery of concurrently administered chemotherapy. As such, there is a need for improved treatments of cancer using combination therapy.

Summary of the Invention

Disclosed herein are methods of treating cancer in a subject, the methods comprising administration of a cyclodextrin-containing polymer conjugate in combination with a second agent. In some embodiments, the cancer is breast cancer, e.g., triple-negative breast cancer (TNBC). In some embodiments, the cancer is post-surgical advanced metastatic TNBC. In some embodiments, the cyclodextrin-containing polymer conjugate comprises camptothecin. In some embodiments, the cyclodextrin-containing polymer conjugate is CRLX101.

In some embodiments, the cyclodextrin-containing polymer conjugate (e.g., CRLX101) is administered with an angiogenesis inhibitor. The angiogenesis inhibitor may be a VEGF pathway inhibitor, such bevacizumab. In some embodiments, the angiogenesis inhibitor (e.g., bevacizumab) is administered at the same time as the cyclodextrin-containing polymer conjugate (e.g., CRLX101). In some embodiments, the angiogenesis inhibitor (e.g., bevacizumab) is administered separately from the cyclodextrin-containing polymer conjugate (e.g., CRLX101). In some embodiments, the cyclodextrin-containing polymer conjugate (e.g., CRLX101) is administered before the angiogenesis inhibitor (e.g., bevacizumab) and in other embodiments, it is administered after the angiogenesis inhibitor (e.g., bevacizumab). In some embodiments, the cyclodextrin-containing polymer conjugate (e.g., CRLX101) and the angiogenesis inhibitor (e.g., bevacizumab) are administered in the same manner.

Exemplary routes of administration include oral, parenteral, or topical administration, or via an implanted reservoir. In some embodiments, the cyclodextrin-containing polymer conjugate (e.g., CRLX101) and the angiogenesis inhibitor (e.g., bevacizumab) are administered parenterally (e.g., by injection). In some embodiments, the cyclodextrin-containing polymer conjugate (e.g., CRLX101) and the angiogenesis inhibitor (e.g., bevacizumab) are administered by

intraperitoneal injection.

In some embodiments, the cyclodextrin-containing polymer conjugate (e.g., CRLX101) and the angiogenesis inhibitor (e.g., bevacizumab) are administered at least once a month, e.g., at least every two weeks, e.g., at least once a week, or at least twice weekly. In some

embodiments, the cyclodextran-containing polymer camptothecin is administered prior to surgery, after surgery, or before and after surgery to remove the cancer, e.g. , to remove a primary tumor and/or a metastases.

In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is an adult (e.g. , over 18 years of age, over 30 years of age, over 40 years of age, over 50 years of age, over 60 years of age). In some embodiments, the subject is a female.

In another aspect, the present disclosure features a method of treating post-surgical, advanced metastatic triple-negative breast cancer (TNBC) in a human subject, the method comprising administering to the subject CRLX101 by , in combination with bevacizumab to thereby treat the subject. In some embodiments, the CRLX101 and the bevacizumab are administered at the same time. In other embodiments, the CRLX101 and the bevacizumab are administered at separate times. In some embodiments, the CRLX101 is administered before the bevacizumab and in other embodiments, it is administered after the bevacizumab.

In some embodiments, the CRLX101 and the bevacizumab are administered in the same manner, e.g., via oral, parenteral, or topical administration, or via an implanted reservoir. In some embodiments, the CRLX101 and the bevacizumab are administered parenterally (e.g., by injection). In some embodiments, the CRLX101 and the bevacizumab are administered by intraperitoneal injection. In some embodiments, the CRLX101 and the bevacizumab are administered at least once a month, e.g., at least every two weeks, e.g., at least once a week, or at least twice weekly. In some embodiments, the CRLXlOl and the bevacizumab are administered prior to surgery, after surgery, or before and after surgery to remove the cancer, e.g., to remove a primary tumor and/or a metastases.

In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is an adult (e.g., over 18 years of age, over 30 years of age, over 40 years of age, over 50 years of age, over 60 years of age). In some embodiments, the subject is a female.

The details of one or more embodiments of the invention are set forth herein. Other features, objects, and advantages of the invention will be apparent from the Detailed Description, the Figures, the Examples, and the Claims.

Brief Description of the Drawings

Figs. 1A-1D. CRLXlOl caused regression of orthotopic primary breast tumors. (Fig. 1A) SCID mice bearing LM2-4 primary tumors in the mammary fat pad were treated with CRLXlOl monotherapies (2, 4 and 8 mg/kg) and in combination with bevacizumab. Therapy was started day 18 after tumor cell injection and stopped after 6 months. (Fig. IB) All doses of CRLXlOl significantly extended survival of mice. (Fig. 1C) Orthotopically grown primary tumors from tumor fragment implantation of the PDX, HCI-002, were treated with 4 and 8 mg/kg CRLXlOl as single agent or in combination with bevacizumab. Therapy was started day 51 after implantation and stopped after 5 months. (Fig. ID) Bevacizumab monotherapy and all doses of CRLXlOl significantly extended survival of mice bearing PDX tumors. Error bars are SD, n=5 mice per group.

Figs. 2A-2D. CRLXlOl therapy maintained low levels of HIF-Ια and significantly increased tumor cell apoptosis. After two weeks of therapy, tumors were stained for CD31, n=5 tumors per group (Fig. 2A) and western blot analysis was performed to quantify HIF- la protein levels, n=3 tumors per group (Fig. 2B). (Fig. 2C) Ki67 and (Fig. 2D) cleaved caspase-3 staining were used to assess changes in tumor cell proliferation and apoptosis, respectively. Results are given as mean + SEM, n=5 tumors per group. *p<0.05 and **p<0.01 compared to vehicle.

Figs. 3A-3D. Contrast-enhanced ultrasound (CEUS) and photoacoustic (PA) imaging of primary LM2-4 tumors. Mice were imaged before therapy was started to establish pre-treatment baseline measurements. Mice were then imaged once weekly and treated for three weeks. (Fig. 3A) Changes in blood flow volume (peak enhancement) and blood flow rate (wash-in rate) were measured using CEUS imaging. PA imaging was used to monitor changes in average tumor oxygen saturation. (Fig. 3B) Representative CEUS images of one tumor from each therapy group overlaid with parametric color mapping to show areas of high perfusion (red), low perfusion (blue) or no perfusion (black). (Fig. 3C) Representative PA images of the same tumors overlaid with color mapping to indicate areas of high tissue oxygenation (red) and low tissue oxygenation (blue). (Fig. 3D) For each therapy group, the number of vessels with open lumens was counted and reported as a percentage of the average number of vessels present per field. Vessels were counted in five randomly selected fields from five tumors per group (bracketed numbers indicate average number of vessels counted per field). Error bars are SEM, n=4-6 tumors per group, *p<0.05, ^#p=0.07, **p<0.01 compared to vehicle.

Figs. 4A-4B. CRLX101 reduced tumor cell density and resulted in fewer, smaller lung micro-metastases. (Fig. 4A) Average number of nuclei counted within an imaged field (n=5 tumors per group). Error bars are SEM, *p<0.05 and **p<0.01 compared to vehicle. (Fig. 4B) Lungs from mice still bearing primary tumors were collected after two weeks of therapy and stained for vimentin, used to specifically stain for human tumor cells. Lungs were classified as having no vimentin (and thus no tumor cells) present, only singly dispersed tumor cells, small clusters (containing fewer than 20 cells) or large clusters (containing more than 20 cells). For each therapy group, lungs from five mice were evaluated. Five serial sections were assessed for each lung, sectioned with 100 μιη separation. Scale bar is 150 μιη.

Figs. 5A-5B. CRLX101 prevented the emergence of new metastases and caused regression of existing metastases, thus greatly extending mice survival. (Fig. 5A)

Bioluminescence images of 7-9 mice per treatment group. All therapies initiated 25 days after primary tumor resection and stopped after 7 months. (Fig. 5B) Survival curves for treated mice with advanced metastatic disease after primary tumor resections.

Fig. 6. Bioluminescence imaging of mice bearing primary LM2-4 tumors. After three months of therapy, tumors were too small to be accurately measured by calipers, thus

bioluminescence imaging was used to continue monitoring of tumor growth in all remaining animals. Mice treated with 4 mg/kg CRLX101 in combination with bevacizumab succumbed to large primary tumors despite initial tumor growth suppression. In contrast, mice treated with 8 mg/kg CRLX101 in combination with bevacizumab were free of disease after 6 months of continuous therapy followed by 4 months off-therapy. Mice treated with 8 mg/kg CRLX101 monotherapy were pre-maturely terminated after treatment cessation due to the development of thymomas unrelated to disease or therapy.

Figs. 7A-7G. (Fig. 7A) Representative staining images of PDX HCI-002 primary tumors with H&E and CD31 to evaluate tumor necrosis (N) and MVD, respectively, scale bar is 500 μιη for H&E images, 100 μιη for CD31 images. (Fig. 7B) Representative western blot of HIF-Ια for one tumor from each group. (Figs. 7C-7E) Quantification of MVD, HIF-Ια protein levels and extent of tumor necrosis, n=5 tumors per group, *p<0.05 and **p<0.01 compared to vehicle. (Figs. 7F-7G) Tumoral accumulation of total CPT (including released and conjugated CPT) and only released (free) CPT, n=3-4 tumors per group.

Figs. 8A-8D. (Fig. 8A) Representative staining images of MDA-MB-231/LM2-4 primary tumors with H&E to evaluate tumor necrosis (N), tumor boundary indicated by blue line, scale bar is 500 μιη. Scale bar for all other images (CD31, CAIX, cleaved caspase 3 and Ki67) is 100 μιη. (Fig. 8B) Representative western blot of HIF-Ια for one tumor from each group. (Figs. 8C- 8D) Quantification of CAIX staining and extent of tumor necrosis, *p<0.05, ^Ap=0.06 compared to vehicle.

Figs. 9A-9C. (Figs. 9A-9B) CEUS imaging assessment of HCI-002 primary tumors. Representative CEUS images of one tumor from each group overlaid with parametric color mapping to show areas of high perfusion (red), low perfusion (blue) or no perfusion (black). Error bars are SEM, *p<0.05 and #p=0.06 compared to vehicle. (Fig. 9C) CD31 staining (red) was co-stained with pimonidazole (green) and Hoechst (blue) staining to evaluate tumor hypoxia and perfusion, respectively. Average proportion of tumor that was perfused versus hypoxic is shown for each therapy group. Images taken at lOx objective, scale bar is 200 μιη.

Figs. 10A-10B. Assessment of vessels with open lumen and tumor cell density in PDX tumors. (Fig. 10A) For each therapy group, the number of vessels with open lumen was counted and reported as a percentage of the total number of vessels present. Vessels were counted in five randomly selected fields from 5-6 tumors per group (bracketed numbers indicate average number of vessels counted per field). (Fig. 10B) Average number of nuclei counted within an imaged field of tumors treated with vehicle, bevacizumab, CRLX101 or combination, n=5-6 tumors per group. Error bars are SEM, **p<0.01 compared to vehicle. Figs. 11A-11B. Time-intensity curves (TICs) of CEUS in primary MDA-MB-231/LM2-4 tumors. (Fig. 11A) One TIC is generated for each tumor imaged. Two parameters are taken from the TIC: peak enhancement (PE) and wash-in rate (WiR). PE is the difference between peak and baseline intensity and used as an indicator to tissue blood volume. WiR is the steepest slope of the curve and is used as an indicator of blood flow rate. (Fig. 1 IB) Representative TICs for one tumor from each therapy group taken at 2 weeks after therapy. Contrast signal increases following the injection of microbubbles, then plateaus as the tumor becomes saturated.

Corresponding CEUS images taken 55 seconds after microbubble injection, when each tumor is completely saturated with microbubbles.

Detailed Description of the Invention

Described herein are methods for treating cancer (e.g., TNBC) in subject comprising administering a cyclodextrin-containing polymer conjugate in combination with a second agent. For example, we show that an exemplary antiangiogenesis drug, bevacizumab, is highly efficacious preclinically for metastatic triple negative breast cancer when administered in combination with CRLX101, an investigational nanoparticle-drug conjugate.

Cyclodextrin-Containing Polymer (CDP) Conjugates, Particles, and Compositions

The present invention relates to compositions of therapeutic cyclodextrin-containing polymers (CDP) designed for drug delivery of a topoisomerase inhibitor such as camptothecin or a camptothecin derivative. In certain embodiments, these cyclodextrin-containing polymers improve drug stability and/or solubility, and/or reduce toxicity, and/or improve efficacy of the topoisomerase inhibitor when used in vivo.

Furthermore, by selecting from a variety of linker groups that link or couple CDP to a topoisomerase inhibitor such as camptothecin or a camptothecin derivative, and/or targeting ligands, the rate of drug release from the polymers can be attenuated for controlled delivery. The invention also relates to methods of treating subjects with compositions described herein. The invention further relates to methods for conducting a pharmaceutical business comprising manufacturing, licensing, or distributing kits containing or relating to the Cyclodextrin- containing polymer conjugates, particles and compositions described herein. More generally, the present invention provides water-soluble, biocompatible polymer conjugates comprising a water-soluble, biocompatible polymer covalently attached to the topoisomerase inhibitor through attachments that are cleaved under biological conditions to release the topoisomerase inhibitor.

Polymeric conjugates featured in the methods described herein may be useful to improve solubility and/or stability of a bioactive/therapeutic agent, such as camptothecin, reduce drug- drug interactions, reduce interactions with blood elements including plasma proteins, reduce or eliminate immunogenicity, protect the agent from metabolism, modulate drug-release kinetics, improve circulation time, improve drug half-life (e.g., in the serum, or in selected tissues, such as tumors), attenuate toxicity, improve efficacy, normalize drug metabolism across subjects of different species, ethnicities, and/or races, and/or provide for targeted delivery into specific cells or tissues.

Described herein are cyclodextrin containing polymer ("CDP") conjugates, wherein one or more topoisomerase inhibitors are covalently attached to the CDP (e.g., either directly or through a linker). The cyclodextrin-containing polymer conjugate inhibitor conjugates include linear or branched cyclodextrin-containing polymers and polymers grafted with cyclodextrin. Exemplary cyclodextrin-containing polymers that may be modified as described herein are taught in U.S. Patent Nos. 7,270,808, 6,509,323, 7,091,192, 6,884,789, U.S. Publication Nos. 20040087024, 20040109888 and 20070025952.

Accordingly, in one embodiment the cyclodextrin-containing polymer conjugate is represented by Formula I:

wherein

P represents a linear or branched polymer chain;

CD represents a cyclic moiety such as a cyclodextrin moiety;

Li, L₂ and L₃, independently for each occurrence, may be absent or represent a linker group;

D, independently for each occurrence, represents a topoisomerase inhibitor or a prodrug thereof (e.g., a camptothecin or camptothecin derivative);

T, independently for each occurrence, represents a targeting ligand or precursor thereof; a, m, and v, independently for each occurrence, represent integers in the range of 1 to 10 (preferably 1 to 8, 1 to 5, or even 1 to 3);

n and w, independently for each occurrence, represent an integer in the range of 0 to about 30,000 (preferably <25,000, <20,000, <15,000, <10,000, <5,000, <1,000, <500, <100, <50, <25, <10, or even <5); and

b represents an integer in the range of 1 to about 30,000 (preferably <25,000, <20,000, <15,000, <10,000, <5,000, <1,000, <500, <100, <50, <25, <10, or even <5),

wherein either P comprises cyclodextrin moieties or n is at least 1.

In some embodiments, one or more of the topoisomerase inhibitor moieties in the

Cyclodextrin-containing polymer conjugate can be replaced with another therapeutic agent, e.g., another anticancer agent or anti-inflammatory agent. Examples of other anticancer agents are described herein. Examples of anti-inflammatory agents include a steroid, e.g., prednisone, and a NSAID.

In certain embodiments, P contains a plurality of cyclodextrin moieties within the polymer chain as opposed to the cyclodextrin moieties being grafted on to pendant groups off of the polymeric chain. Thus, in certain embodiments, the polymer chain of formula I further comprises n' units of U, wherein n' represents an integer in the range of 1 to about 30,000, e.g., from 4-100, 4-50, 4-25, 4-15, 6-100, 6-50, 6-25, and 6-15 (preferably <25,000, <20,000, <15,000, <10,000, <5,000, <1,000, <500, <100, <50, <25, <20, <15, <10, or even <5); and U is represented by one of the general formulae below:

wherein CD represents a cyclic moiety, such as a cyclodextrin moiety, or derivative thereof;

L₄, L5, L₆, and L₇, independently for each occurrence, may be absent or represent a linker group;

D and D', independently for each occurrence, represent the same or different

topoisomerase inhibitor or prodrug forms thereof (e.g., a camptothecin or camptothecin derivative);

T and , independently for each occurrence, represent the same or different targeting ligand or precursor thereof;

f and y, independently for each occurrence, represent an integer in the range of 1 and 10; and

g and z, independently for each occurrence, represent an integer in the range of 0 and 10.

Preferably the polymer has a plurality of D or D' moieties. In some embodiments, at least 50% of the U units have at least one D or D' . In some embodiments, one or more of the topoisomerase inhibitor moieties in the CDP-topoisomerase conjugate can be replaced with another therapeutic agent, e.g., another anticancer agent or anti-inflammatory agent.

In preferred embodiments, L₄ and L₇ represent linker groups.

The CDP may include a polycation, polyanion, or non-ionic polymer. A polycationic or polyanionic polymer has at least one site that bears a positive or negative charge, respectively. In certain such embodiments, at least one of the linker moiety and the cyclic moiety comprises such a charged site, so that every occurrence of that moiety includes a charged site. In some embodiments, the CDP is biocompatible.

In certain embodiments, the CDP may include polysaccharides, and other non-protein biocompatible polymers, and combinations thereof, that contain at least one terminal hydroxyl group, such as polyvinylpyrrollidone, poly(oxyethylene)glycol (PEG), polysuccinic anhydride, polysebacic acid, PEG-phosphate, polyglutamate, polyethylenimine, maleic anhydride divinylether (DIVMA), cellulose, pullulans, inulin, polyvinyl alcohol (PVA), N-(2- hydroxypropyl)methacrylamide (HPMA), dextran and hydroxyethyl starch (HES), and have optional pendant groups for grafting therapeutic agents, targeting ligands and/or cyclodextrin moieties. In certain embodiments, the polymer may be biodegradable such as poly(lactic acid), poly(glycolic acid), poly(alkyl 2-cyanoacrylates), polyanhydrides, and polyorthoesters, or bioerodible such as polylactide-glycolide copolymers, and derivatives thereof, non-peptide polyaminoacids, polyiminocarbonates, poly alpha-amino acids, polyalkyl-cyano-acrylate, polyphosphazenes or acyloxymethyl poly aspartate and polyglutamate copolymers and mixtures thereof.

In another embodiment the cyclodextrin-containing polymer conjugate is represented by Formula II:

(Π) wherein

P represents a monomer unit of a polymer that comprises cyclodextrin moieties;

T, independently for each occurrence, represents a targeting ligand or a precursor thereof;

L₆, L₇, L₈, L9, and Lio, independently for each occurrence, may be absent or represent a linker group;

CD, independently for each occurrence, represents a cyclodextrin moiety or a derivative thereof;

D, independently for each occurrence, represents a topoisomerase inhibitor or a prodrug form thereof (e.g., a camptothecin or camptothecin derivative);

m, independently for each occurrence, represents an integer in the range of 1 to 10 (preferably 1 to 8, 1 to 5, or even 1 to 3);

o represents an integer in the range of 1 to about 30,000 (preferably <25,000, <20,000, <15,000, <10,000, <5,000, <1,000, <500, <100, <50, <25, <10, or even <5); and p, n, and q, independently for each occurrence, represent an integer in the range of 0 to 10 (preferably 0 to 8, 0 to 5, 0 to 3, or even 0 to about 2),

wherein CD and D are preferably each present at least 1 location (preferably at least 5, 10, 25, or even 50 or 100 locations) in the compound.

In some embodiments, one or more of the topoisomerase inhibitor moieties in the cyclodextrin-containing polymer conjugate can be replaced with another therapeutic agent, e.g., another anticancer agent or anti-inflammatory agent. Examples of an anticancer agent are described herein. Examples of anti-inflammatory agents include a steroid, e.g., prednisone, or a NSAID.

In another embodiment the cyclodextrin-containing polymer conjugate is represented either of the formulae below:

wherein

CD represents a cyclic moiety, such as a cyclodextrin moiety, or derivative thereof;

L₄, L5, L₆, and L₇, independently for each occurrence, may be absent or represent a linker group;

D and D', independently for each occurrence, represent the same or different

topoisomerase inhibitor or prodrug thereof (e.g., a camptothecin or camptothecin derivative);

T and T, independently for each occurrence, represent the same or different targeting ligand or precursor thereof;

f and y, independently for each occurrence, represent an integer in the range of 1 and 10 (preferably 1 to 8, 1 to 5, or even 1 to 3);

g and z, independently for each occurrence, represent an integer in the range of 0 and 10 (preferably 0 to 8, 0 to 5, 0 to 3, or even 0 to about 2); and

h represents an integer in the range of 1 and 30,000 , e.g., from 4-100, 4-50, 4-25, 4-15, 6-100, 6-50, 6-25, and 6-15 (preferably <25,000, <20,000, <15,000, <10,000, <5,000, <1,000, <500, <100, <50, <25, <20, <15, <10, or even <5),

wherein at least one occurrence (and preferably at least 5, 10, or even at least 20, 50, or 100 occurrences) of g represents an integer greater than 0.

Preferably the polymer has a plurality of D or D' moieties. In some embodiments, at least 50% of the polymer repeating units have at least one D or D'. In some embodiments, one or more of the topoisomerase inhibitor moieties in the cyclodextrin-containing polymer conjugate can be replaced with another therapeutic agent, e.g., another anticancer agent or antiinflammatory agent.

In preferred embodiments, L4 and L7 represent linker groups.

In certain such embodiments, the CDP comprises cyclic moieties alternating with linker moieties that connect the cyclic structures, e.g., into linear or branched polymers, preferably linear polymers. The cyclic moieties may be any suitable cyclic structures, such as

cyclodextrins, crown ethers (e.g., 18-crown-6, 15-crown-5, 12-crown-4, etc.), cyclic

oligopeptides (e.g., comprising from 5 to 10 amino acid residues), cryptands or cryptates (e.g., cryptand [2.2.2], cryptand-2,1,1, and complexes thereof), calixarenes, or cavitands, or any combination thereof. Preferably, the cyclic structure is (or is modified to be) water-soluble. In certain embodiments, e.g., for the preparation of a linear polymer, the cyclic structure is selected such that under polymerization conditions, exactly two moieties of each cyclic structure are reactive with the linker moieties, such that the resulting polymer comprises (or consists essentially of) an alternating series of cyclic moieties and linker moieties, such as at least four of each type of moiety. Suitable difunctionalized cyclic moieties include many that are

commercially available and/or amenable to preparation using published protocols. In certain embodiments, conjugates are soluble in water to a concentration of at least 0.1 g/mL, preferably at least 0.25 g/mL.

Thus, in certain embodiments, the invention relates to compositions of therapeutic cyclodextrin-containing polymeric compounds designed for drug delivery of a topoisomerase inhibitor. In certain embodiments, these CDPs improve drug stability and/or solubility, and/or reduce toxicity, and/or improve efficacy of the topoisomerase inhibitor when used in vivo.

Furthermore, by selecting from a variety of linker groups, and/or targeting ligands, the rate of topoisomerase inhibitor release from the CDP can be attenuated for controlled delivery.

In certain embodiments, the CDP comprises a linear cyclodextrin-containing polymer, e.g., the polymer backbone includes cyclodextrin moieties. For example, the polymer may be a water-soluble, linear cyclodextrin polymer produced by providing at least one cyclodextrin derivative modified to bear one reactive site at each of exactly two positions, and reacting the cyclodextrin derivative with a linker having exactly two reactive moieties capable of forming a covalent bond with the reactive sites under polymerization conditions that promote reaction of the reactive sites with the reactive moieties to form covalent bonds between the linker and the cyclodextrin derivative, whereby a linear polymer comprising alternating units of cyclodextrin derivatives and linkers is produced. Alternatively the polymer may be a water-soluble, linear cyclodextrin polymer having a linear polymer backbone, which polymer comprises a plurality of substituted or unsubstituted cyclodextrin moieties and linker moieties in the linear polymer backbone, wherein each of the cyclodextrin moieties, other than a cyclodextrin moiety at the terminus of a polymer chain, is attached to two of said linker moieties, each linker moiety covalently linking two cyclodextrin moieties. In yet another embodiment, the polymer is a water- soluble, linear cyclodextrin polymer comprising a plurality of cyclodextrin moieties covalently linked together by a plurality of linker moieties, wherein each cyclodextrin moiety, other than a cyclodextrin moiety at the terminus of a polymer chain, is attached to two linker moieties to form a linear cyclodextrin polymer. In some embodiments, the cyclodextrin-containing polymer conjugate comprises a water soluble linear polymer conjugate comprising: cyclodextrin moieties; comonomers which do not contain cyclodextrin moieties (comonomers); and a plurality of topoisomerase inhibitor; wherein the cyclodextrin-containing polymer conjugate comprises at least four, five six, seven, eight, etc., cyclodextrin moieties and at least four, five six, seven, eight, etc., comonomers. In some embodiments, the topoisomerase inhibitor is a topoisomerase inhibitor described herein, for example, the topoisomerase inhibitor is a camptothecin or camptothecin derivative described herein. The topoisomerase inhibitor can be attached to the CDP via a functional group such as a hydroxyl group, or where appropriate, an amino group.

In some embodiments, one or more of the topoisomerase inhibitor moieties in the cyclodextrin-containing polymer conjugate can be replaced with another therapeutic agent, e.g., another anticancer agent or anti-inflammatory agent.

In some embodiments, the least four cyclodextrin moieties and at least four comonomers alternate in the cyclodextrin-containing polymer conjugate. In some embodiments, the topoisomerase inhibitors are cleaved from said cyclodextrin-containing polymer conjugate under biological conditions to release the topoisomerase inhibitor. In some embodiments, the cyclodextrin moieties comprise linkers to which topoisomerase inhibitors are linked. In some embodiments, the topoisomerase inhibitors are attached via linkers.

In some embodiments, the comonomer comprises residues of at least two functional groups through which reaction and linkage of the cyclodextrin monomers was achieved. In some embodiments, the functional groups, which may be the same or different, terminal or internal, of each comonomer comprise an amino, acid, imidazole, hydroxyl, thio, acyl halide, -HC=CH-,

C≡C group, or derivative thereof. In some embodiments, the two functional groups are the same and are located at termini of the comonomer precursor. In some embodiments, a comonomer contains one or more pendant groups with at least one functional group through which reaction and thus linkage of a topoisomerase inhibitor was achieved. In some

embodiments, the functional groups, which may be the same or different, terminal or internal, of each comonomer pendant group comprise an amino, acid, imidazole, hydroxyl, thiol, acyl halide, ethylene, ethyne group, or derivative thereof. In some embodiments, the pendant group is a substituted or unsubstituted branched, cyclic or straight chain C1-C10 alkyl, or arylalkyl optionally containing one or more heteroatoms within the chain or ring. In some embodiments, the cyclodextrin moiety comprises an alpha, beta, or gamma cyclodextrin moiety. In some embodiments, the topoisomerase inhibitor is at least 5%, 10%, 15%, 20%, 25%, 30%, or 35% by weight of cyclodextrin-containing polymer conjugate.

In some embodiments, the comonomer comprises polyethylene glycol of molecular weight 3,400 Da, the cyclodextrin moiety comprises beta-cyclodextrin, the theoretical maximum loading of the topoisomerase inhibitor on the cyclodextrin-containing polymer conjugate is 13% by weight, and the topoisomerase inhibitor is 6-10% by weight of cyclodextrin-containing polymer conjugate. In some embodiments, the topoisomerase inhibitor is poorly soluble in water. In some embodiments, the solubility of the topoisomerase inhibitor is <5 mg/ml at physiological pH. In some embodiments, the topoisomerase inhibitor is a hydrophobic compound with a log P>0.4, >0.6, >0.8, >1, >2, >3, >4, or >5.

In some embodiments, the topoisomerase inhibitor is attached to the CDP via a second compound.

In some embodiments, administration of the cyclodextrin-containing polymer conjugate to a subject results in release of the topoisomerase inhibitor over a period of at least 6 hours. In some embodiments, administration of the cyclodextrin-containing polymer conjugate to a subject results in release of the topoisomerase inhibitor over a period of 2 hours, 3 hours, 5 hours, 6 hours, 8 hours, 10 hours, 15 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 7 days, 10 days, 14 days, 17 days, 20 days, 24 days, 27 days up to a month. In some embodiments, upon

administration of the cyclodextrin-containing polymer conjugate to a subject, the rate of topoisomerase inhibitor release is dependent primarily upon the rate of hydrolysis as opposed to enzymatic cleavage.

In some embodiments, the cyclodextrin-containing polymer conjugate has a molecular weight of 10,000-500,000. In some embodiments, the cyclodextrin moieties make up at least about 2%, 5%, 10%, 20%, 30%, 50% or 80% of the cyclodextrin-containing polymer conjugate by weight.

In one embodiment, the polysaccharide is a linear, branched or cyclic polysaccharide. In one embodiment, the polysaccharide is a linear polysaccharide that includes glucose molecules. In one embodiment, the polysaccharide is dextran, a cyclodextrin or a cyclodextrin derivative, e.g., an α-, β- and/or γ-cyclodextrin, e.g., CDP. In one embodiment, the polysaccharide is administered prior to, currently with or after administration of the composition. In one embodiment, the polysaccharide is administered at a dose of 100 mg to 10 g.

In an embodiment, the conjugate includes a topoisomerase I inhibitor and/or a topoisomerase II inhibitor. In an embodiment, the conjugate includes a topoisomerase I inhibitor or combination of topoisomerase I inhibitors, e.g., camptothecin, irinotecan, SN-38, topotecan, lamellarin D and derivatives thereof. In an embodiment, the conjugate includes a topoisomerase II inhibitor or a combination of topoisomerase II inhibitors, e.g., eptoposide, tenoposide, doxorubicin and derivatives thereof. In one embodiment, the conjugate includes a combination of one or more topoisomerase I inhibitors and one or more topoisomerase II inhibitors.

In preferred embodiments, the topoisomerase inhibitor in the cyclodextrin-containing polymer conjugate, particle or composition is camptothecin or a camptothecin derivative. The term "camptothecin derivative", as used herein, includes camptothecin analogues and

metabolites of camptothecin. For example, camptothecin derivatives can have the following structure:

wherein

R¹ is H, OH, optionally substituted alkyl (e.g., optionally substituted with NR^a ₂ or OR_a, or SiR^a ₃), or SiR^a ₃; or R¹ and R² may be taken together to form an optionally substituted 5- to 8- membered ring (e.g., optionally substituted with NR^a ₂ or OR^a);

R is H, OH, NH₂, halo, nitro, optionally substituted alkyl (e.g., optionally substituted with NR^a ₂ or OR^a, NR^a ₂, OC(=0)NR^a ₂, or OC(=0)OR^a);

R³ is H, OH, NH₂, halo, nitro, NR^a ₂, OC(=0)NR^a ₂, or OC(=0)OR^a

R⁴ is H, OH, NH₂, halo, CN, or NR^a ₂; or R³ and R⁴ taken together with the atoms to which they are attached form a 5- or 6-membered ring (e.g. forming a ring including -OCH₂0- or -OCH₂CH₂0-); each R^a is independently H or alkyl; or two R^as, taken together with the atom to which they are attached, form a 4- to 8-membered ring (e.g., optionally containing an O or NR^b)

R is H or optionally substituted alkyl (e.g., optionally substituted with OR^c or NR^C ₂); R^c is H or alkyl; or, two R^cs, taken together with the atom to which they are attached, form a 4- to 8-membered ring; and

n = 0 or 1.

In some embodiments, the camptothecin or camptothecin derivative is the compound as provided below.

In one embodiment, R 1 , R2 , R 3 and R 4 of the camptothecin derivative are each H, and n is

0.

In one embodiment, R 1 , R2 , R 3 and R 4 of the camptothecin derivative are each H, and n is

1.

In one embodiment, R 1 of the camptothecin derivative is H, R 2 is -CH₂N(CH₃)₂, R 3 is - OH, R⁴ is H; and n is 0.

In one embodiment, R 1 of the camptothecin derivative is -CH₂CH₃, R 2 is H, R 3 is:

In one embodiment, R 1 of the camptothecin derivative is -CH₂CH₃, R 2 is H, R 3 is -OH,

R⁴ is H, and n is 0.

In one embodiment, R 1 of the camptothecin derivative is ie/ -butyldimethylsilyl, R 2 is H,

R³ is -OH and R⁴ is H, and n is 0.

In one embodiment, R 1 of the camptothecin derivative is ie/ -butyldimethylsilyl, R 2 is hydrogen, R³ is -OH and R⁴ is hydrogen, and In one embodiment, R 1 of the camptothecin derivative is ie/ -butyldimethylsilyl, R 2 , R 3 and R⁴ are each H, and n is 0.

In one embodiment, R 1 of the camptothecin derivative is ie/ -butyldimethylsilyl, R 2 , R 3 and R⁴ are each H, and n is i.

In one embodiment, R 1 of the camptothecin derivative is -CH₂CH₂Si(CH₃)3 and R 2 , R 3 and R⁴ are each H.

In one embodiment, R 1 and R 2 of the camptothecin derivative are taken together with the carbons to which they are attached to form an optionally substituted ring. In one embodiment,

R 1 and R 2 of the camptothecin derivative are taken together with the carbons to which they are attached to form a substituted 6-membered ring. In one embodiment, the camptothecin derivative has the following formula:

In one embodiment, R³ and R⁴ are taken together with the carbons to which they are attached to form an optionally substituted ring. In one embodiment, R³ and R⁴ are taken together with the carbons to which they are attached to form a 6-membered heterocyclic ring. In one embodiment, the camptothecin derivative has the following formula:

In one embodiment, the camptothecin derivative has the following formula: In one embodiment, R is:

; R² is H, R³ is methyl, R⁴ is chloro; and

In one embodiment, R¹ is -CH=NOC(CH₃)₃, R², R³ and R⁴ are each H, and n is 0.

In one embodiment, R¹ is -CH₂CH₂NHCH(CH₃)₂, R², R³ and R⁴ are each H; and n is 0.

In one embodiment, R 1 and R2 are H, R 3^J and R 4" are fluoro, and n is i.

In one embodiment, each of R 1 , R3 , and R 4 is H, R 2 is NH₂, and n is 0.

In one embodiment, each of R 1 , R3 , and R 4 is H, R 2 is N0₂, and n is 0.

In an embodiment, the cyclodextrin-containing polymer conjugate is a CDP- camptothecin or camptothecin derivate conjugate, e.g., a CDP-camptothecin or camptothecin derivative conjugate described herein, e.g., CRLX101.

Combination therapy

The cyclodextrin-containing polymer conjugate, particle or composition may be used in combination with other known therapies. Administered "in combination", as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery". In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

The cyclodextrin-containing polymer conjugate, particle or composition and the at least one additional therapeutic agent can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the Cyclodextrin-containing polymer conjugate, particle or composition can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.

In some embodiments, the cyclodextrin-containing polymer conjugate, particle or composition is administered in combination with other therapeutic treatment modalities, including surgery, radiation, cryosurgery, and/or thermotherapy. Such combination therapies may advantageously utilize lower dosages of the administered agent and/or other

chemotherapeutic agent, thus avoiding possible toxicities or complications associated with the various monotherapies. The phrase "radiation" includes, but is not limited to, external-beam therapy which involves three dimensional, conformal radiation therapy where the field of radiation is designed to conform to the volume of tissue treated; interstitial-radiation therapy where seeds of radioactive compounds are implanted using ultrasound guidance; and a combination of external-beam therapy and interstitial-radiation therapy.

In some embodiments, the cyclodextrin-containing polymer conjugate, particle or composition is administered with at least one additional therapeutic agent, e.g., a second agent, such as an angiogenesis inhibitor. In some embodiments, the angiogenesis inhibitor is a vascular endothelial growth factor (VEGF) pathway inhibitor, e.g., a VEGF inhibitor or VEGF receptor inhibitor. Exemplary VEGF pathway inhibitors include anti-VEGF antibodies, e.g.,

bevacizumab, and small molecules, e.g., sunitinib (Sutent®), sorafinib (Nexavar®), ZD6474 (also known as vandetanib) (Zactima^1M), SU6668, CP-547632, AV-951 (tivozanib) and AZD2171 (also known as cediranib) (Recentin™).

In one embodiment, the angiogenesis inhibitor is bevacizumab or AV-951. In one embodiment, the angiogenesis inhibitor is selected from CP-547632 and AZD2171. In one embodiment, the conjugate, particle or composition is administered in combination with an angiogenesis inhibitor, e.g., bevacizumab, and an antimetabolite, e.g., an antifolate (e.g., pemetrexed, floruridine, raltitrexed) or pyrimidine analogue (e.g., capecitabine, 5FU, cytrarabine, gemcitabine). In one embodiment, the conjugate, particle or composition is administered with an angiogenesis inhibitor, e.g., bevacizumab, an antimetabolite, e.g., a pyrimidine analogue (e.g., 5FU), and folinic acid (leucovorin). In another embodiment, the conjugate, particle or composition is administered with an angiogenesis inhibitor, e.g., bevacizumab, an antimetabolite, e.g., a pyrimidine analogue (e.g., 5FU), folinic acid

(leucovorin), and a platinum-based agent (e.g., cisplatin, carboplatin, oxaliplatin). In one embodiment, the cancer (e.g., TNBC) is refractory, relapsed or resistant to an antimetabolite and/or a platinum-based agent.

In another embodiment, a Cyclodextrin-containing polymer conjugate, particle or composition, e.g., a CDP-camptothecin or camptothecin derivative conjugate, particle or composition, e.g., a CDP-camptothecin or camptothecin derivative conjugate, particle or composition described herein, e.g., CRLX101, is administered in combination with an angiogenesis inhibitor, e.g., bevacizumab, and an antimetabolite wherein the antimetabolite is a pyrimidine analogue, e.g., capecitabine. In one embodiment, the conjugate, particle or composition is further administered in combination with a platinum-based agent (e.g., cisplatin, carboplatin, oxaliplatin). For example, in one embodiment, the conjugate, particle or composition is administered with the following combination: an angiogenesis inhibitor, e.g., bevacizumab, a pyrimidine analogue (e.g., capecitabine), and a platinum-based agent (e.g., oxaliplatin); or an angiogenesis inhibitor (e.g., bevacizumab) and a pyrimidine analogue (e.g., capecitabine). In one embodiment, the cancer (e.g., TNBC) is refractory, relapsed or resistant to an antimetabolite and/or a platinum-based agent. Routes of Administration The pharmaceutical compositions described herein may be administered orally, parenterally (e.g., via intravenous, subcutaneous, intracutaneous, intramuscular, intraarticular, intraarterial, intraperitoneal, intrasynovial, intrasternal, intrathecal, intralesional or intracranial injection), topically, mucosally (e.g., rectally or vaginally), nasally, buccally, ophthalmically, via inhalation spray (e.g., delivered via nebulzation, propellant or a dry powder device) or via an implanted reservoir. Typically, the compositions are in the form of injectable or infusible solutions. The preferred mode of administration is, e.g., intravenous, subcutaneous,

intraperitoneal, intramuscular.

Pharmaceutical compositions suitable for parenteral administration comprise one or more cyclodextrin-containing polymer conjugate(s), particle(s) or composition(s) in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the agent from subcutaneous or intramuscular injection. This may be

accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the cyclodextrin-containing polymer conjugate, particle or composition then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the cyclodextrin-containing polymer conjugate, particle or composition in an oil vehicle.

Pharmaceutical compositions suitable for oral administration may be in the form of capsules, cachets, pills, tablets, gums, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in- water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouthwashes and the like, each containing a predetermined amount of an agent as an active ingredient. A compound may also be administered as a bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered peptide or peptidomimetic moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or

preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the cyclodextrin-containing polymer conjugate, particle or composition, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the cyclodextrin-containing polymer conjugate, particle or composition may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum

metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Pharmaceutical compositions suitable for topical administration are useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the a particle described herein include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene

polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active particle suspended or dissolved in a carrier with suitable emulsifying agents. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions described herein may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches are also included herein.

The pharmaceutical compositions described herein may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

The pharmaceutical compositions described herein may also be administered in the form of suppositories for rectal or vaginal administration. Suppositories may be prepared by mixing one or more cyclodextrin-containing polymer conjugate, particle or composition described herein with one or more suitable non-irritating excipients which is solid at room temperature, but liquid at body temperature. The composition will therefore melt in the rectum or vaginal cavity and release the cyclodextrin-containing polymer conjugate, particle or composition. Such materials include, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate.

Compositions of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of the invention.

Dosages and Dosing Regimens

The cyclodextrin-containing polymer conjugate, particle or composition can be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.

In one embodiment, the cyclodextrin-containing polymer conjugate, particle or composition is administered to a subject at a dosage of, e.g., about 1 to 40 mg/m , about 3 to 35 mg/m², about 9 to 40 mg/m², e.g., about 1, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 mg/m² of the topoisomerase inhibitor. Administration can be at regular intervals, such as weekly, or every 2, 3, 4, 5 or 6 weeks. The administration can be over a period of from about 10 minutes to about 6 hours, e.g., from about 30 minutes to about 2 hours, from about 45 minutes to 90 minutes, e.g., about 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or more. The cyclodextrin-containing polymer conjugate, particle or composition can be administered, e.g., by intravenous or intraperitoneal administration.

In one embodiment, the cyclodextrin-containing polymer conjugate, particle or composition is administered as a bolus infusion or intravenous push, e.g., over a period of 15 minutes, 10 minutes, 5 minutes or less. In one embodiment, the cyclodextrin-containing polymer conjugate, particle or composition is administered in an amount such the desired dose of the agent is administered. Preferably the dose of the cyclodextrin-containing polymer conjugate, particle or composition is a dose described herein.

In one embodiment, the subject receives 1, 2, 3, up to 10 treatments, or more, or until the disorder or a symptom of the disorder is cured, healed, alleviated, relieved, altered, remedied, ameliorated, palliated, improved or affected. For example, the subject receives an infusion once every 1, 2, 3 or 4 weeks until the disorder or a symptom of the disorder is cured, healed, alleviated, relieved, altered, remedied, ameliorated, palliated, improved or affected. Preferably, the dosing schedule is a dosing schedule described herein.

The cyclodextrin-containing polymer conjugate, particle or composition can be administered as a first line therapy, e.g., alone or in combination with an additional agent or agents. In other embodiments, a cyclodextrin-containing polymer conjugate, particle or composition is administered after a subject has developed resistance to, has failed to respond to or has relapsed after a first line therapy. The cyclodextrin-containing polymer conjugate, particle or composition can be administered in combination with a second agent. Preferably, the cyclodextrin-containing polymer conjugate, particle or composition is administered in combination with a second agent described herein.

Kits

A cyclodextrin-containing polymer conjugate, particle or composition described herein may be provided in a kit. The kit may include a cyclodextrin-containing polymer conjugate, particle or composition described herein and, optionally, a container, a pharmaceutically acceptable carrier and/or informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the cyclodextrin-containing polymer conjugate, particle or composition for the methods described herein.

The informational material of the kits is not limited in its form. In one embodiment, the informational material can include information about production of the cyclodextrin-containing polymer conjugate, particle or composition, physical properties of the cyclodextrin-containing polymer conjugate, particle or composition, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to methods for administering the cyclodextrin-containing polymer conjugate, particle or composition, e.g., by a route of administration described herein and/or at a dose and/or dosing schedule described herein.

In one embodiment, the informational material can include instructions to administer a cyclodextrin-containing polymer conjugate, particle or composition described herein in a suitable manner to perform the methods described herein, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein). In another embodiment, the informational material can include instructions to administer cyclodextrin-containing polymer conjugate, particle or composition described herein to a suitable subject, e.g., a human, e.g., a human having or at risk for a disorder described herein. In another embodiment, the informational material can include instructions to reconstitute a cyclodextrin- containing polymer conjugate, particle or composition described herein into a pharmaceutically acceptable composition.

In one embodiment, the kit includes instructions to use the cyclodextrin-containing polymer conjugate, particle or composition, such as for treatment of a subject. The instructions can include methods for reconstituting or diluting the cyclodextrin-containing polymer conjugate, particle or composition for use with a particular subject or in combination with a particular chemotherapeutic agent. The instructions can also include methods for reconstituting or diluting the cyclodextrin-containing polymer conjugate, particle or composition for use with a particular means of administration, such as by intravenous infusion or intraperitoneal administration. In another embodiment, the kit includes instructions for treating a subject with a particular indication, such as a particular cancer, or a cancer at a particular stage. For example, the instructions can be for a cancer or cancer at stage described herein, e.g., TNBC, e.g., postsurgical TNBC, e.g., post-surgical advanced metastatic TNBC. The instructions may also address first line treatment of a subject who has a particular cancer, or cancer at a stage described herein. The instructions can also address treatment of a subject who has been non-responsive to a first line therapy or has become sensitive (e.g., has one or more unacceptable side effect) to a first line therapy, such as a taxane, an anthracycline, an antimetabolite, a vinca alkaloid, a vascular endothelial growth factor (VEGF) pathway inhibitor, an epidermal growth factor (EGF) pathway inhibitor, an alkylating agent, a platinum-based agent, a vinca alkaloid. In another embodiment, the instructions will describe treatment of selected subjects with the Cyclodextrin- containing polymer conjugate, particle or composition. For example, the instructions can describe treatment of one or more of: a subject having a cancer that has increased levels of KRAS and/or ST expression, e.g., as compared to a reference standard.

The informational material of the kits is not limited in its form. In many cases, the informational material, e.g., instructions, is provided in printed matter, e.g., a printed text, drawing, and/or photograph, e.g., a label or printed sheet. However, the informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording. In another embodiment, the informational material of the kit is contact information, e.g., a physical address, email address, website, or telephone number, where a user of the kit can obtain substantive information about a cyclodextrin-containing polymer conjugate, particle or composition described herein and/or its use in the methods described herein. The informational material can also be provided in any combination of formats.

In addition to a cyclodextrin-containing polymer conjugate, particle or composition described herein, the composition of the kit can include other ingredients, such as a surfactant, a lyoprotectant or stabilizer, an antioxidant, an antibacterial agent, a bulking agent, a chelating agent, an inert gas, a tonicity agent and/or a viscosity agent, a solvent or buffer, a stabilizer, a preservative, a flavoring agent (e.g., a bitter antagonist or a sweetener), a fragrance, a dye or coloring agent, for example, to tint or color one or more components in the kit, or other cosmetic ingredient, a pharmaceutically acceptable carrier and/or a second agent for treating a condition or disorder described herein. Alternatively, the other ingredients can be included in the kit, but in different compositions or containers than a cyclodextrin-containing polymer conjugate, particle or composition described herein. In such embodiments, the kit can include instructions for admixing a cyclodextrin-containing polymer conjugate, particle or composition described herein and the other ingredients, or for using a cyclodextrin-containing polymer conjugate, particle or composition described herein together with the other ingredients. For example, the kit can include an agent which reduces or inhibits one or more symptom of hypersensitivity, a polysaccharide, and/or an agent which increases urinary excretion and/or neutralizes one or more urinary metabolite.

In another embodiment, the kit includes a second agent, such an angiogenesis inhibitor, e.g., as described herein (e.g., bevacizumab). In one embodiment, the second agent is in lyophilized or in liquid form. In one embodiment, the cyclodextrin-containing polymer conjugate, particle or composition and the second agent (e.g., bevacizumab) are in separate containers, and in another embodiment, the cyclodextrin-containing polymer conjugate, particle or composition and the second agent (e.g., bevacizumab) are packaged in the same container.

In some embodiments, a component of the kit is stored in a sealed vial, e.g., with a rubber or silicone closure (e.g., a polybutadiene or polyisoprene closure). In some embodiments, a component of the kit is stored under inert conditions (e.g., under Nitrogen or another inert gas such as Argon). In some embodiments, a component of the kit is stored under anhydrous conditions (e.g., with a desiccant). In some embodiments, a component of the kit is stored in a light blocking container such as an amber vial.

A cyclodextrin-containing polymer conjugate, particle or composition described herein can be provided in any form, e.g., liquid, frozen, dried or lyophilized form. It is preferred that a composition including the conjugate, particle or composition, e.g., a composition comprising a particle or particles that include a conjugate described herein be substantially pure and/or sterile. When a cyclodextrin-containing polymer conjugate, particle or composition described herein is provided in a liquid solution, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred. In one embodiment, the cyclodextrin-containing polymer conjugate, particle or composition is provided in lyophilized form and, optionally, a diluent solution is provided for reconstituting the lyophilized agent. The diluent can include for example, a salt or saline solution, e.g., a sodium chloride solution having a pH between 6 and 9, lactated Ringer's injection solution, D5W, or PLASMA-LYTE A Injection pH 7.4^® (Baxter, Deerfield, IL).

The kit can include one or more containers for the composition containing a cyclodextrin- containing polymer conjugate, particle or composition described herein. In some embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, IV admixture bag, IV infusion set, piggyback set or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of a

cyclodextrin-containing polymer conjugate, particle or composition described herein. For example, the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of a particle described herein. The containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight.

The kit optionally includes a device suitable for administration of the composition, e.g., a syringe, inhalant, pipette, forceps, measured spoon, dropper (e.g., eye dropper), swab (e.g., a cotton swab or wooden swab), or any such delivery device. In one embodiment, the device is a medical implant device, e.g., packaged for surgical insertion. Examples

Patients with triple-negative breast cancer (TNBC) have the highest risk of recurrence and metastasis (Foulkes WD et al, The New England Journal of Medicine 2010;363(20): 1938- 48). Such patients generally do not respond as well to chemotherapy compared to patients with other breast cancer subtypes. Various targeted therapies have been investigated, but as yet none are currently approved. One notable example of this is bevacizumab, a VEGF antibody, which was granted accelerated FDA approval with weekly paclitaxel in 2008 for first line treatment of metastatic breast cancer; this approval was later revoked by the Food and Drug Administration (FDA) after follow-up phase III clinical trials (e.g. AVADO and RIBBON- 1) with different chemotherapy backbones showed less impressive benefits in progression-free survival (PFS) (Montero AJ, et al. Current oncology reports 2012; 14(1): 1-11). More recently, however, there has been renewed interest in reconsidering bevacizumab for the treatment of breast cancer based on several phase III clinical trial results, including one in the neoadjuvant and adjuvant setting (NSABP-B40) and two in the maintenance metastatic treatment setting (IMELDA and TANIA) (Bear HD, et al. The Lancet 2015; Gligorov J, et al. The Lancet 2014;15(12): 1351-60; von

Minckwitz G, et al. The Lancet 2014; 15(11): 1269-78). Importantly, a number of trial results and meta-analyses suggest the extent of beneficial effect (and associated toxicities) of adding bevacizumab to chemotherapy may depend on the concurrent chemotherapy regimen used (Clark O, Botrel , et al. Core evidence 2014;9: 1-1). Thus, an appropriate chemotherapy partner, one with better efficacy, manageable toxicity and complementary modes of action may be critical to gaining optimal benefit out of adding bevacizumab (and vice versa).

One promising investigational chemotherapy drug that may be an effective combination partner for bevacizumab or other antiangiogenic drugs, is a nanoparticle-drug conjugate (NDC) known as CRLX101, which contains the payload camptothecin (CPT), a highly potent cytotoxic agent that inhibits topoisomerase-I (Eliasof S,et al. Proceedings of the National Academy of Sciences of the United States of America 2013; 110(37): 15127-32; Svenson S, et al. J Control Release 201 l;153(l):49-55). CPT showed impressive preclinical anti-tumor activity, but had low solubility and caused severe toxicities, which resulted in disappointing results in early phase clinical trials (10). A number of analogues were thus developed to improve the solubility of CPT, including the subsequently approved drugs, topotecan (Hycamtin^®, GlaxoSmithKline) and irinotecan (Camptosar^®, Pfizer) (11). However, both topotecan and irinotecan still have significant dose-limiting toxicities and sub-optimal pharmacokinetics that have limited more widespread use of these drugs (9). Currently there is no topoisomerase-I inhibitor chemotherapy for the treatment of breast cancer. Nonetheless, phase II studies evaluating irinotecan for refractory breast cancer showed a potentially favorable therapeutic index (12,13). Notably, a recent phase III trial (BEACON) evaluated a new formulation of irinotecan, NKTR-102, also known as etirinotecan pegol, in patients with recurrent or metastatic breast cancer (14). It showed single- agent PFS benefits similar to treatment of physician's choice. Moreover, subgroup analyses showed etirinotecan pegol significantly prolonged overall survival in patients with a history of brain or liver metastases, and with two or more sites of disease (14). CRLX101 was designed to improve the therapeutic index of CPT through improved accumulation within tumors by the enhanced permeability and retention (EPR) effect and thus reduced systemic toxicity (8,15). The drug has been administered to over 300 patients to date and appears to be generally well-tolerated, achieving an overall response rate (ORR) as a monotherapy of 16% in 19 patients in a phase II clinical trial of platinum-resistant ovarian cancer (16). In a phase 1/2 study of 22 patients with metastatic renal cell carcinoma, CRLX101 in combination with bevacizumab had an ORR of 23%, and 85% of patients experienced either a partial response or stable disease, with a median PFS of 9.9 months (17).

We recently reported that the combination of CRLX101 and bevacizumab resulted in synergistic anti-tumor efficacy in a preclinical model of advanced, metastatic ovarian cancer (16). CRLX101 was also shown to effectively and durably suppress elevated hypoxia-induced upregulation of hypoxia- inducible factor la (HIF-la) following therapy with bevacizumab, thus down-regulating expression of downstream HIF-la regulated markers, such as carbonic anhydrase IX (CAIX) (16), and blocking the induction of cancer stem cells (CSCs) by bevacizumab (18).

Here we evaluated the combination of CRLX101 and bevacizumab in long-term therapy experiments of primary TNBC xenografts either derived from tumor tissue fragment

implantation of a patient-derived xenograft (PDX) called HCT002 (19,20) or mammary fat pad injection of MDA-MB-231/LM2-4, a variant of the established cell-line MDA-MB-231 serially selected in vivo for aggressive spontaneous metastatic properties (21). We also evaluated long- term efficacy of CRLX101 and bevacizumab in a preclinical model of post-surgical, advanced metastatic TNBC (22), a model which recapitulates the more challenging circumstance of the clinical treatment of systemic metastatic disease (22,23). Here we report that CRLX101 alone and in combination with bevacizumab is an effective treatment for advanced metastatic TNBC in these mouse models, and provide new mechanistic results to help explain this encouraging antitumor activity.

Materials and Methods

Primary tumor implantation into the mammary fat pad

MDA-MB-231/LM2-41ucl6+ (LM2-4) is a highly aggressive variant of MDA-MB-231 luciferase-tagged for bioluminescence imaging and selected in vivo for its more aggressive spontaneous metastatic properties (21). Patient-derived xenograft (PDX) HCI-002 tumor fragments were originally obtained from a patient with TNBC (19,20). HCI-002 tumor fragments were serially passaged in SCID mice. Mammary fat pad injections of MDA-MD-

231/LM2-4 (5x10 5 cells) and tumor fragment implantations (pieces of 2-5 mm 3 ) of HCI-002 were carried out as previously described (20,21).

Post-surgical, advanced metastatic breast cancer therapy model

LM2-4 cells were orthotopically implanted into the right inguinal mammary fat pad of SCID mice and resected when 400-500 mm , as described in (21). Three to four weeks after primary tumor resection, distant visceral metastases can be detected by bioluminescence imaging. Mice with metastatic disease were randomized on day 25 post-resection based on metastatic load, location, and presence of regrowth at the site of resection before therapies were initiated (21,23). Drug tolerability and clinical symptoms including body weight loss and overall appearance were monitored at least twice weekly. Results

CRLX101 treatment can lead to complete primary tumor regressions

CRLX101 caused dramatic shrinkages in two different primary tumor models of human TNBC, including xenografts from cell injection of MDA-MB-231/LM2-4 and tumor fragment implantation of a PDX called HCI-002. The maximum-tolerated dose (MTD, 8 mg/kg i.p. once weekly) of CRLX101 was previously established for SCID mice and is well-tolerated even when combined with bevacizumab for long-term studies (16). MTD CRLX101 resulted in rapid and durable tumor regressions both as a monotherapy or when combined with bevacizumab in both models (Figs. 1A-1D).

In mice bearing primary LM2-4 tumors, bevacizumab monotherapy was ineffective. Whereas in contrast, all therapy groups containing CRLX101 resulted in extended survival (Figs. 1A-1B). While mice were on-therapy continuously for 6 months, 8 mg/kg CRLX101, both as monotherapy and in combination with bevacizumab, quickly and dramatically shrank established primary tumors, resulting in complete regressions (p<0.001), with all remaining mice (3 out of 5 mice treated with CRLX101 monotherapy and 4 out of 5 treated with the combination) essentially cured by 6 months of therapy (based on no observable bioluminescence signals).

Remarkably, there was no regrowth or residual disease even after treatment cessation and being off-therapy for an additional 4 months (Fig. 6). In order to better demonstrate whether the drug combination had improved anti-tumor efficacy, CRLXlOl was also evaluated at lower doses. Lower doses of CRLXlOl monotherapy resulted in slower rates of tumor shrinkage compared to 8 mg/kg, resulting in smaller tumors that still caused hind leg mobility impairment. Nonetheless, the addition of bevacizumab to 4 mg/kg CRLXlOl significantly improved anti-tumor efficacy (p<0.05 compared to 4 mg/kg CRLXlOl alone), showing improved tumor growth suppression and shrinkage despite bevacizumab alone having no anti-tumor efficacy in this treatment circumstance. However, despite early tumor suppression and continued therapy,

bioluminescence imaging of mice treated with CRLXlOl 4 mg/kg combined with bevacizumab showed these tumors eventually continued to grow.

Compared to primary tumors derived from LM2-4 cell injection, primary PDX HCI-002 tumors were highly vascular. In contrast to bevacizumab' s absence of efficacy when treating LM2-4 primary tumors, bevacizumab led to delayed HCI-002 tumor growth while mice were on- therapy continuously for 5 months (Figs. 1C-1D). CRLXlOl, both as monotherapy or in combination with bevacizumab, durably suppressed tumor growth (p=0.002 for CRLXlOl 4 mg/kg compared to vehicle and p<0.001 for all other groups). Notably, the combination of 4 mg/kg CRLXlOl and bevacizumab significantly improved the anti-tumor efficacy compared to either drug alone (p<0.001). This was comparable to administering CRLXlOl at its MTD, but was even better tolerated by SCID mice as determined by changes in total body weight. In this PDX model, despite tumor growth suppression or regression while mice were on-therapy, when all therapies were stopped, tumor regrowth did occur but was delayed longer if mice were originally treated with a higher dose of CRLXlOl monotherapy or concurrent bevacizumab.

In separate experiments, tumors from mice treated for two weeks were evaluated for changes in micro vessel density (MVD) and any corresponding changes in HIF- la protein levels. Bevacizumab reduced the number of tumor vessels by -50% in LM2-4 tumors and -65% in PDX tumors (Figs. 2A, 7A-7G and 8A-8D). This reduction in MVD by bevacizumab single agent treatment corresponded to an increase in HIF- la protein levels (Fig. 2B). While

CRLXlOl monotherapies did not significantly decrease MVD, tumors from mice which were treated with concurrent bevacizumab showed decreased MVD to levels similar to bevacizumab monotherapy. However, CRLXlOl at all doses maintained low levels of HIF- la protein levels and suppressed any bevacizumab-induced increases in HIF- la. This was also confirmed using immunohistochemistry staining of tumor sections with CAIX, a downstream marker of tumor hypoxia and HIF-Ια activity (16) (Figs. 8A-8D). Nonetheless, decreased MVD observed when tumors were concurrently treated with CRLX101 and bevacizumab did not affect tumoral accumulation of CRLX101 (Figs. 7A-7G). CRLX101 reduced the extent of tumor necrosis compared to tumors from mice treated with vehicle or bevacizumab monotherapy (Figs. 8A-8D). Furthermore, while CRLX101 did not change the extent of tumor cell proliferation (Fig. 2C), it did cause a significant increase in apoptosis at all doses administered (Fig. 2D). Concurrent bevacizumab treatment did not further increase the extent of apoptosis when combined with CRLX101.

We next evaluated whether the reduction in MVD by bevacizumab treatment correlated with a change in functional tumor perfusion. We therefore used in vivo contrast enhanced ultrasound (CEUS) imaging, which uses gas-filled microbubbles similar in size to red blood cells to non-invasively monitor changes in blood flow and volume within tumors, to monitor changes in tumor perfusion following therapy (24). Tumors from mice treated with vehicle or bevacizumab alone showed decreased tumor blood volume and flow rate over the course of two weeks of therapy (Fig. 3A). In contrast, tumors from mice treated with CRLX101 monotherapy showed greatly improved perfusion while perfusion in tumors treated with the combination remained unchanged. Parametric maps showing regions of high, low or no perfusion confirm that tumors from mice treated with CRLX101 had smaller necrotic cores compared to vehicle and bevacizumab treatment. In the combination treatment group, while a less perfused core was present, the surrounding 'viable rim' remained well-perfused (Fig. 3B). Similarly, CEUS imaging of PDX tumors showed CRLX101 maintained higher tumor perfusion and reduced tumor hypoxia, as confirmed by pimonidazole staining (Figs. 9A-9C).

In addition to measuring perfusion using CEUS, photoacoustic (PA) imaging was used to measure oxygen saturation (Figs. 3A-3D) (24). Short laser pulses are directed at the tissue, generating thermoelastic expansion to create acoustic waves detected by an ultrasound transducer. Different absorption spectra of deoxygenated and oxygenated hemoglobin are then used to non-invasively estimate the spatial distribution of oxygen saturation in vivo (25).

Consistent with CEUS data showing improved perfusion in CRLX101 treated tumors, these tumors had relatively higher tissue oxygenation levels compared to vehicle- and bevacizumab- treated tumors (Fig. 3C). Improved perfusion with CRLXlOl treatment suggests a more functional tumor vasculature, as results from Figure 2 A show CRLXlOl 4 mg/kg monotherapy does not result in an increase in MVD. Thus the therapy may cause the existing vasculature to become less dysfunctional, improving overall tumor perfusion without an increase in the total number of vessels. Tumor cells rapidly growing within a confined space causes compression (or 'solid stress') of tumoral blood vessels (26,27). One possible explanation for the overall improved perfusion following CRLXlOl treatment is the drug causes extensive tumor cell apoptosis, which may then relieve the compression on tumor blood vessels. To assess whether this may be the case, we evaluated whether CRLXlOl treatment resulted in more vessels with an open lumen, which would allow better blood flow within the tumor (Fig. 3D). Indeed, we observed that CRLXlOl monotherapy resulted in a higher proportion of tumor blood vessels with open lumen compared to both vehicle- and bevacizumab-treated mice. It is worth noting that in tumors treated with the drug combination, a similar increase in percentage of vessels with open lumens was observed, despite having fewer number of vessels overall. In other words, bevacizumab reduced the overall number of tumor vessels, but when combined with CRLXlOl, more of the remaining vessels had open lumens. These results were also confirmed in PDX tumors (Figs. 10A-10B).

In order to obtain additional confirmation that CRLXlOl therapy relieves compression of tumor blood vessels by killing tumor cells, we further evaluated whether the different therapies result in changes in tumor cell packing density, i.e. the number of cells found within a given area (Fig. 4A). While the average number of cells present within an imaged field was similar between vehicle- and bevacizumab-treated tumors, all doses of CRLXlOl caused a significant reduction in tumor cell density. Fewer tumor cells densely packed within a tumor thus may result in fewer compressed tumor blood vessels and hence improved perfusion and oxygenation.

Paradoxically, a more functional vasculature could result in a higher degree of tumor cell dissemination and metastasis to distant organs, such as the lungs. We therefore analyzed lungs from mice still bearing primary tumors for the presence of micro-metastases or tumor cells that previously shed from the primary tumor and seeded in the lungs (Fig. 4B). Mice treated with bevacizumab monotherapy showed more micro-metastases that were also larger in cluster size than those present in vehicle-treated mice. This is consistent with some previous preclinical reports that antiangiogenic agents used as monotherapies may cause an increase in metastasis and promote disease progression in mice despite an initial anti-tumor effect (28,29). In stark contrast, mice treated with CRLX101 (particularly at higher doses) either showed no micro- metastases or only singly dispersed tumor cells within the lungs, even when combined with bevacizumab. CRLX101 was therefore able to counteract any potential pro-metastatic effect of bevacizumab. This effect is similar to published data where paclitaxel counteracted the pro- invasive and pro-metastatic effects of DC 101, the VEGFR-2 antibody (20), and metronomic topotecan reduced metastatic spread elicited by sunitinib (30).

CRLX101 in a post-surgical metastatic breast cancer model shrinks existing metastases and prevents the emergence of new metastases

Given the potency of CRLX101 in both primary tumor models and evidence showing

CRLX101 was able to prevent formation of micro-metastases, CRLX101 alone and in combination with bevacizumab were evaluated in a post- surgical model of advanced metastatic breast cancer. Following surgical resection of established primary LM2-4 tumors, distant metastases were allowed to develop before treatment was initiated. In this model of advanced metastatic TNBC, metastases were monitored by bioluminescence imaging and can manifest as lymph node metastases (causing mobility issues), lung metastases (leading to labored breathing), primary tumor regrowth or local metastases at the surgical site, as well as liver metastases and ascites (causing distended abdomens). Given the inherent variability of when and where distant metastases appear, every treatment group had randomized cohorts of mice with apparent distant metastases, local metastases or regrowths, as well as mice with no signs of metastases at the start of therapy (Figs. 5A-5B). Within 1.5 months of therapy, all mice treated with vehicle and bevacizumab monotherapy succumbed to disease, even if no apparent metastases were observed at the start of treatment. In contrast, bioluminescence imaging showed that in mice that had apparent metastases at the start of therapy, treatment with CRLX101 or CRLX101 combined with bevacizumab caused such existing metastases to regress. In mice with no apparent metastases at the start of treatment, CRLX101 alone or in combination with bevacizumab was able to prevent the emergence of new metastases both while mice were on-therapy (for 7 months) and then off- therapy (for 2 months). It should be noted, however, that if mice had a very heavy metastatic tumor load at the start of therapy (particularly if ascites or heavy tumor burden in the lungs were present), CRLX101 therapy was not as effective; we hypothesize that this is due to the need for some initial time for CRLX101 to accumulate within tumor cells and release its drug payload.

CRLX101 therefore significantly extended the survival of mice with metastatic disease (Fig. 5B). While mice treated with vehicle or bevacizumab succumbed to metastases after 4 to 6 weeks, survival of mice treated with 4 mg/kg CRLX101 monotherapy was extended to 15 weeks. CRLX101 at 4 mg/kg combined with bevacizumab was effective at durably suppressing metastasis but was generally even better tolerated than MTD CRLX101, particularly important given the long-term nature of the experiment. Notably, MTD CRLX101 was highly efficacious, so much so that any added benefit of combining it with bevacizumab was not detected. Upon necropsy, all remaining mice were free of macroscopic metastases with 3 of 9 mice (treated with 4 mg/kg CRLX101 plus bevacizumab), 5 of 8 mice (treated with CRLX101 alone) and 6 of 9 mice (treated with 8 mg/kg CRLX101 plus bevacizumab) still alive and free of disease by the end of the experiment (7 months on-therapy and 2 months off-therapy). Discussion

We recently reported metronomic topotecan combined with pazopanib showed tumor growth delay of primary TNBC tumors and prolonged survival of mice with advanced metastatic disease (31). Nevertheless, topotecan as a single agent had no anti-tumor effect and resulted in increased HIF-Ια, and more importantly, increased expression of the ABCG2 transporter, thus requiring the concurrent administration of a TKI, either pazopanib or sunitinib, to overcome ABCG2-mediated resistance. Given this promising therapy of combining a topoisomerase-I inhibitor (topotecan) and an antiangiogenic drug (pazopanib) for treating TNBC in mice, here we evaluated CRLX101 and bevacizumab. There are several reasons why this alternative drug combination may be superior to that of topotecan and pazopanib. Unlike topotecan, single agent CRLX101 is potently effective in not only delaying tumor growth, but causing marked and sustained tumor regressions. Furthermore, all doses of CRLX101 whether alone or combined with bevacizumab durably suppressed HIF-Ια. Finally, one important concern of combining topotecan and pazopanib is the tolerability and toxicity of both topotecan and pazopanib, especially the combination, in the clinic (32-34). In contrast, current clinical data suggests that CRLX101 is tolerable in combination with standard doses of bevacizumab in patients (35). The combination of CRLX101 and bevacizumab would thus appear to be more promising considering its lesser toxicity and better preclinical efficacy - an improved therapeutic index compared to the pazopanib / topotecan combination.

Numerous marketed approvals of VEGF-pathway targeting antiangiogenic drugs have changed clinical practice for the treatment of a very broad spectrum of human cancers.

Nonetheless, the efficacy successes are usually limited, due to several possible factors (36).

Among these are reduced intratumoral delivery of concurrently administered drugs such as chemotherapy (37), as well as elevated hypoxia, and hence HIF-Ια, due to reduction in tumor blood vessel numbers and function (36). Elevated hypoxia and HIF-Ια can contribute to resistance, and may promote invasion and metastasis (18,38). Given these considerations, an ideal chemotherapy partner for combination with an antiangiogenic agent would be a highly potent agent that is able to improve tumor perfusion and reduce HIF-Ια, to improve intratumoral drug delivery and overcome hypoxia-induced adaptive resistance, respectively, without increasing tumor dissemination and/or metastases. Here we showed that CRLXlOl may be such a drug.

A number of strategies have been proposed, including vessel normalization (39) and

'vascular promotion' therapy (26,40), to improve intratumoral chemotherapy drug delivery. Another means of improving tumor perfusion is by reducing the compression (or solid stress) caused by the rapid growth of cancer cells within a confined space (26,27). Impaired perfusion, whether caused by antiangiogenic therapy or due to mechanical factors such as tumor cells compressing vessels, would be expected to reduce intratumoral delivery of chemotherapy drugs such as CRLXlOl, which relies on the EPR effect to passively accumulate in solid tumors (8,41). However, CRLXlOl maintained tumor perfusion despite concurrent antiangiogenic therapy. We report here that the potent anti-tumor activity of CRLXlOl alleviated the solid stress by rapidly targeting tumor cells, in effect leading to major tumor regressions and decompressing tumor blood vessels to improve tumor perfusion ('tumor priming' (42)). We and others have reported that metronomic dosing of a gemcitabine prodrug resulted in increased perfusion (43,44).

Paclitaxel-loaded tumor penetrating microparticles (TPMs) have also been used to enhance siRNA delivery into solid tumors (42). Clinically, a similar effect was observed where weekly paclitaxel reduced interstitial fluid pressure and improved tumor oxygenation (45). Interestingly, our work here with CRLXlOl, as well as the preclinical work with metronomic gemcitabine (43,44) , TPMs (42), and the clinical results with weekly paclitaxel (45), all involved administration of chemotherapy drugs in a metronomic or metronomic-like manner. This suggests chronic exposure of tumor cells to a cytotoxic drug, either by administering standard chemotherapy drugs in a frequent lower-dose fashion, or using drug formulations with intrinsically long half-lives such as CRLX101, may be required to maintain sufficient durable tumor cell kill to persistently reduce compression of tumoral vessels and thus potentially improve perfusion. This improvement in tumor perfusion significantly reduces tumor hypoxia, which also contributes to the ability of CRLX101 to effectively and durably suppress HIF-la. Even though HIF-la is elevated by tumor hypoxia, reduction in tumor hypoxia by improving tumor perfusion alone may not completely account for the HIF-la suppression by CRLX101. Rapisarda et al have shown in vitro that metronomic topotecan treatment was able to suppress HIF-la protein accumulation even when cells were maintained in normoxic conditions (46), suggesting HIF-la inhibition may occur even in the absence of elevated hypoxia. Taken together, we have shown in both orthotopic cell-line and patient-derived xenograft models that CRLX101 maintains or improves tumor perfusion, and is able to durably suppress HIF-la protein levels even in the presence of bevacizumab where tumors are more hypoxic, thus making CRLX101 an effective chemotherapy partner to pair with an antiangiogenic agent such as bevacizumab.

Early clinical evaluation of native CPT showed limited efficacy and serious toxicity that restricted further clinical evaluation. Nonetheless, a phase I study evaluating CPT in heavily pre- treated patients with different cancer types showed an objective response rate across all tumor types of 11%, including 8 patients with breast cancer, 2 of whom showed anti-tumor responses and 1 exhibited stable disease (47). However, given the associated toxicities, further clinical development of CPT stagnated until the development of the FDA-approved drugs, topotecan and irinotecan. Although both irinotecan and topotecan have been evaluated in phase II trials for metastatic breast cancer, with irinotecan showing variable response rates of 5-23% (12) and topotecan showing modest response rates of 6-10% (48,49), they have not been approved as treatments for that indication given the associated high grade toxicities experienced by patients for a modest benefit gain (33,34). Nonetheless, lessons learned from early trials using CPT, and more recent evaluation of irinotecan and topotecan, suggest CPT analogs are indeed active in the treatment of breast cancer, but further clinical development requires better drug solubility, improved lactone ring stabilization and less systemic toxicity. Like etirinotecan pegol, the improved formulation of irinotecan evaluated in the phase III BEACON trial (14), the CRLXlOl nanoparticle-CPT conjugate we have evaluated here was designed to have superior solubility and stabilization of the lactone ring, as well as favorable safety and pharmacokinetics in patients (15,17). CRLXlOl has been generally well tolerated to date at the maximum tolerated dose (50). Although no comparative study has been performed, current CRLXlOl tolerability data suggests that the hematological and gastrointestinal toxicities associated with CRLXlOl monotherapy may be less severe than the adverse events reported in the FDA labels of either irinotecan or topotecan.

In summary, given our preclinical results of CRLXlOl showing potent anti-tumor activity, particularly when treating advanced metastatic TNBC, and its complementary mode of action for combining with bevacizumab, combination therapy of CRLXlOl and bevacizumab may be a promising treatment strategy for breast cancer and other solid tumors.

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Animal Care

Female CB-17 SCID mice, 6-8 weeks old, were purchased from Charles River Canada and 8- week old female YFP-SCID mice were bred in-house from breeding pairs (i). All mice were housed in microisolator cages with vented racks and were manipulated using aseptic techniques. Procedures involving animals and their care were conducted in strict conformity with the animal care guidelines of Sunnybrook Health Science Centre and the Canadian Council of Animal Care. Caliper measurements were carried out twice a week to determine primary tumor growth, reported as tumor volume calculated using the formula width xlength/2. When average tumor volume was -200-250 mm , mice were randomized by tumor volume and treatments initiated. Mice were euthanized when showing more than 20% body weight loss, tumor volumes were 1500 mm , or when moribund.

Cell Line Authentication

MDA-MB-231/LM2-41ucl6+ cells were maintained in RPMI, supplemented with 5% FBS and were processed by Genetica DNA Laboratories (a LabCorp Specialty Testing Group; Burlington, NC) for authentication testing using analytical procedures for DNA extraction, polymerase chain reaction (PCR) and capillary electrophoresis on a 3130x1 genetic analyzer (Applied Biosystems). The thirteen core COD IS short tandem repeat (STR) loci plus PENTA E and PENTA D, and the gender-determining locus, amelogenin, were analyzed using the commercially available PowerPlex® 16HS amplification kit (Promega Corporation) and

GeneMapper ID v3.2.1 software (Applied Biosystems). Appropriate positive and negative controls were used concurrently throughout the analysis. Authentication of cell lines are confirmed by entering the STR DNA profile of each tested cell line into known repository cell line databases [i.e. ATCC, DSMZ, etc]; authentication is defined as having a percent match with the reference STR profile at or above 80% when using the ANSI/ ATCC guidelines (ASN-0002- 2011) OR having a "unique" STR DNA profile (no matches found) for "in-house" cell lines not distributed by any cell line repository.

Total Body Bioluminescence Imaging to Detect Metastases and Response to Therapy

Metastatic dissemination and growth of MDA-MB-231/LM2-4, which stably expresses firefly lucif erase, was monitored by weekly total body bioluminescence imaging. Mice were administered luciferin (150 mg/kg) and imaged in an IVIS200 Xenogen under isoflurane anesthesia, as previously described (2, 3). Bioluminescence images were analyzed using the Living Image software and bioluminescence is reported as photons / second.

Drug Dosages and Schedules

CRLX101 was supplied by Cerulean Pharma Inc. (Cambridge, Massachusetts, USA) and reconstituted into PBS as per manufacturer's instructions. Bevacizumab (Roche, Mississauga, Ontario, Canada) was purchased from the institutional pharmacy as a 25 mg/mL solution. All doses of CRLX101 were administered once a week by i.p. injection. Bevacizumab was administered twice a week by i.p. injection at 200 μg per mouse.

Immunohistochemistry

Primary tumors and lungs were excised, fixed in 10% formalin overnight and transferred to 70% ethanol. Tumor masses were paraffin-embedded and sectioned at 5 μηι thickness. Slides were dewaxed in xylene and hydrated in graded alcohol. Heat-induced antigen retrieval using sodium citrate-citric acid (pH 6 for carbonic anhydrase IX (CAIX), vimentin, cleaved caspase 3 and Ki67) or Tris-EDTA (pH 9 for CD31) and peroxidase quenching with 1% hydrogen peroxide were performed for all immunohistochemistry stainings. Blocking was done with Dako Protein Block (X0909) plus 20% normal donkey serum. Primary antibodies (goat anti-human CAIX, 1: 125, R&D AF2188; rabbit anti-cleaved caspase-3, 1 :500, Cell Signaling 5A1E; goat anti- PECAM-1 (M-20), 1: 100, Santa Cruz sc-1506; rabbit anti-Ki67, 1: 1000, Vector Laboratories VP-K451; mouse anti-human vimentin (clone V9), 1: 100, ThermoFisher 18-0052) were diluted in Dako Antibody Diluent (S3022) and slides were incubated overnight at 4°C. Dako LSAB+ System-HRP kit (K0690) and Liquid DAB+ Substrate Chromogen System (K3467) were used for detection. Tissue sections were counterstained with hematoxylin, dehydrated and mounted with Permount. Necrosis was assessed based on morphology of tumor sections stained with hematoxylin and eosin (H&E).

Tumor sections stained with cleaved caspase 3 and CD31 were visualized under an upright Leica DM LB2, with 10-20x objectives. Stainings were assessed on one section per tumor (n=5-6 mice per group) and images of the entire tumor area were captured for analysis using a Leica DFC300 FX camera (usually -5-30 fields per slide at 20x and 20-100 fields at lOx objective, depending on the size of the tumor). Images were analyzed and quantified using ImageJ (http://rsb.info.nih.gov/ij/). MVD reported as vessel count and cleaved caspase-3 reported as percentage positive pixel, normalized to total viable tumor area (n=5-6 tumors per group). Average tumor cell density was determined by counting the number of nuclei per field as a measure of cellular packing density (4, 5). Enough fields were imaged and analyzed to cover the entire viable tumor area. To evaluate the percentage of vessels with open lumen, five fields per tumor were randomly chosen, and the number of vessels with open lumen and the total number of vessels were manually counted (n=5-6 tumors per group).

Virtual Slide Scanning

Slides stained for H&E, Ki67, CAIX were scanned on Olympus VS120 Virtual Slide Microscope using a 20x objective (NA 0.85). The resulting images have a pixel resolution of 0.33micron/pixel. An image analysis software (cellSens) was then used to quantify and analyze different stained features. CAIX is reported as percentage positive pixel and Ki67 as cell count normalized to total viable tumor area (n=5-6 tumors per group).

Immunofluorescence Evaluation of Markers for Hypoxia and Perfusion

For tumor hypoxia analysis, mice received an i.v. injection of pimonidazole

hydrochloride (60 mg/kg, Chemicon International, Inc., Temecula, CA) 60 - 90 minutes before euthanasia, as described (6). Pimonidazole hydrochloride is a bioreductive chemical probe activation of which occurs at p0₂ levels <10 mm Hg. Mice also received an i.p. injection of Hoechst 33342 (40 mg/kg, Sigma- Aldrich, St. Louis, MO) 1 minute before euthanasia for blood vessel perfusion analysis. Tumors were excised and immediately embedded in OCT (Tissue-Tek optimum cutting temperature compound, Miles Inc., Elkhart, IN) for cryosectioning (6). Tumor cryosections (5-10 μπι thick) were used for subsequent immunofluorescence staining for rat anti- mouse CD31 (1 : 100; PharMingen, San Diego, CA) and pimonidazole (Hypoxyprobe- 1 mouse monoclonal antibody, 1 :200, Chemicon International). A cy3-conjugated donkey anti-rat antibody (1 :200) and FITC-conjugated anti-mouse antibody (1 :200, both from Jackson

ImmunoResearch Laboratories, Inc., West Grove, PA) were used for CD31 and pimonidazole staining, respectively.

Tumor sections were visualized under a Carl Zeiss Axioplan2 microscope with 10-20x objectives. Stainings were assessed on one section per tumor (n=4-6 mice per group) and images of the entire tumor area were captured for analysis using a Carl Zeiss AxioCam MRc (usually -10-30 fields per slide, depending on the size of the tumor). Images were analyzed and quantified using ImageJ (http://rsb.info.nih.gov/ij/). All stainings were normalized to total tumor area and averaged (n=4-6 tumors per group).

Western Blot

Tumors were collected and snap frozen on dry ice. Tumors were thawed on ice in Cell Extraction Buffer (50mM Tris, 300mM NaCl, 10% glycerol, 3mM EDTA, ImM MgC12, 20mM B-glycerol, 25mM NaF, 1% Triton, ImM PMSF, in purified water, supplemented with a protease inhibitor cocktail (Roche). Protein concentrations were measured using a BCA protein assay (Thermo Fisher). Samples were denatured using 2X sample buffer consisting of Bolt LDS Sample buffer, Bolt reducing agent (Life Technologies) in water and then heated to 95°C for 5 min. Protein samples were loaded at 20 μg/lane on to 4- 12% Bolt Bis-Tris gel in IX MES SDS Running buffer (Life Technologies) and separated at 165V using a Bolt apparatus (Life

Technologies). Gels were soaked in 20% ethanol then dry transferred on to PVDF membrane using an iBlot2 apparatus (Life Technologies) at 20V. Membranes were blocked for 1 hr at room temperature with blocking buffer (LiCor, #927-40000). Primary antibodies: HIF-la (1 :2,000, Novus Biologies, #NB 100-479) and actin (1 : 1000, Santa Cruz Biologicals, SC-8432) were diluted in blocking buffer containing 0.2% Tween. Membranes were incubated overnight at 4°C. Blots were then washed in PBS containing 0.1% Tween at room temperature. Secondary antibodies (Licor): goat anti-rabbit-800CW, goat anti-mouse-800CW, goat anti-rabbit 680CW and goat anti-mouse 680 were used at 1: 10,000 dilution, incubated in the dark for 35 minutes at room temperature. Blots were washed in PBS containing 0.1% Tween, followed by a PBS wash. Blots were dried and scanned using Odyssey CLx imaging system (Licor). Image analysis was completed using Image Studio Software (Licor). HIF levels were normalized to actin levels and reported as percentage difference compared to vehicle.

LC-MS/MS Analysis of Intra-Tumoral Accumulation of CPT

Collected tumors were homogenized in cell extraction buffer containing esterase inhibitor cocktail at a target concentration of 100 mg/ml. The homogenates were analyzed for released and total camptothecin using protein precipitation with acetonitrile followed by high

performance liquid chromatography- tandem mass spectrometry (LC-MS/MS). Released camptothecin was quantified using a calibration curve of camptothecin over a concentration range of 10-1000 ng/ml. Total camptothecin was quantified using a calibration curve of hydrolyzed CRLX101, which results in the liberation of camptothecin from the polymer carrier, over a concentration range of 50-2500 ng/ml. Both assays utilized 7-ethylcamptothecin (7E- CPT) as an internal-standard.

To process samples to determine released CPT, 10 μΐ of 50% formic acid was added to 50 μΐ of each sample, followed by 200 μΐ of 200 ng/ml 7E-CPT stock in 0.1% formic acid in acetonitrile. To process samples to determine total CPT concentrations, 10 μΐ of 2M NaOH was added to 50 μΐ of each sample. After hydrolysis, 10 μΐ of 50% formic acid was added followed by 200 μΐ of 200ng/ml 7E-CPT in 0.1% formic acid in acetonitrile. All samples were then vortexed and placed on a rotary shaker for 10 minutes to ensure conversion of camptothecin to the lactone form. The samples were centrifuged at 4°C for 10 minutes @ 2000 G and 100 μΐ of the clear supernatant was transferred to a fresh 96 well plate for analysis.

An Agilent 1100 HPLC system coupled to an Agilent 6410 triple-quadrupole mass spectrometer was used for the analysis. For the separation a Waters XBridge Phenyl, 3.5 um particle diameter, 2.1 x 50 mm analytical column was used with mobile phases of 10 mM ammonium acetate in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). At a flow rate of 0.7 ml/min a gradient from 2% to 80% mobile phase B was established between 0 and 2.3 minutes and the organic phase was held at 80% for another 0.7min, back to initial conditions with a total analysis time of 4 minutes. A 10 μΐ injection was made. The multiple reaction monitoring (MRM) mode was used to quantify camptothecin and m/z transition were recorded: 349.3->305.1 for camptothecin and 377.3-> 333.1 for 7E-CPT. Quantitation was performed using a standard curve derived from the AUC ratio of analytednternal-standard plotted against a series of standard concentrations.

Contrast-Enhanced Ultrasound and Photoacoustic Imaging

Mice bearing either MDA-MB-231/LM2-4 or HCI-002 primary tumors were randomized when tumor averages reached -500 mm and imaged prior to therapy to determine baseline pretreatment measurements. Mice from all treatment groups were treated for two weeks and imaging was done once weekly. In vivo imaging was conducted using a commercially available high-frequency laser integrated ultrasound system, the Vevo^®LAZR (VisualSonics, Toronto, Canada), which allowed for both photoacoustic and contrast enhanced imaging (7). Prior to imaging, depilatory cream was applied to the region of interest to remove any hair that can come in contact with the ultrasound probe. Mice were then anesthetized with 2% isoflurane, used in combination with medical air, delivered at lL/min. A 27G butterfly catheter was inserted into one of the lateral tail veins for contrast agent injection. All animals were imaged using the LZ250 probe, an ultrasound transducer equipped with fiber optical bundles for light delivery from a tunable laser (680-950nm). The probe was placed on top of the tumor, coupled by ultrasound gel. Imaging region selection was manually performed for each tumor. Contrast data was quantified with VevoCQ using time intensity curves (TICs) generated from the wash-in of microbubbles (Figs. 1 lA-1 IB). Two parameters are taken from the TIC: peak enhancement (PE) and wash-in rate (WiR).

Dual- wavelength PA imaging was used for real-time oxygen saturation detection, whereby each image is constructed from data collected at two different wavelengths (750nm and 850nm). Contrast enhanced images were acquired after a bolus injection of 50 ί of

MicroMarker (VisualSonics, Toronto, Canada) ultrasound contrast agents. All 3D data collection was completed using an external stepper motor at step size of 51 πι. All data analysis was done offline using the Vevo^®LAB software. PA images for tumor oxygen saturation calculation were collected as a 3D stack, containing 250 to 300 image frames. A region of interest (ROI) encompassing the tumor was drawn for each frame to determine the overall averaged oxygen saturation for the whole tumor.

Statistical Analysis Results were reported as the mean ± SD or SEM, as indicated. Tumor growth curves were reported as the mean ± SD. Survival curves were plotted by the method of Kaplan and Meier and tested for survival differences with the log-rank test. Statistical significance was assessed by one-way analysis of variance (ANOVA, Kruskal-Wallis test with Dunn's post-hoc) or t-test (Mann- Whitney, two-tailed) using Graph-Pad Prism 4 (p<0.05 was used as the threshold of statistical significance).

Equivalents and Scope

In the claims articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain

embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms "comprising" and "containing" are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the

specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

Claims

Claims

1. A method of treating post-surgical, advanced, metastatic triple-negative breast cancer (TNBC) in a subject, e.g., a human, the method comprising administering to the subject, e.g., human, a cyclodextrin-containing polymer camptothecin conjugate, e.g., CRLXlOl, in combination with bevacizumab, to thereby treat the subject, e.g., human.

2. The method of claim 1, wherein the cyclodextrin-containing polymer camptothecin conjugate is administered before the bevacizumab.

3. The method of claim 1, wherein the bevacizumab is administered before the

cyclodextrin-containing polymer camptothecin conjugate.

4. The method of claim 1, wherein the bevacizumab and the cyclodextrin-containing polymer camptothecin conjugate are administered concurrently.

5. The method of claim 1, wherein the cyclodextrin-containing polymer camptothecin conjugate is administered by injection.

6. The method of claim 1, wherein the bevacizumab is administered by injection.

7. The method of claim 1, wherein the cyclodextrin-containing polymer camptothecin conjugate is administered by intraperitoneal administration (e.g., intraperitoneal injection).

8. The method of claim 1, wherein the bevacizumab is administered by intraperitoneal administration (e.g., intraperitoneal injection).

9. The method of claim 1, wherein the cyclodextrin-containing polymer camptothecin conjugate is administered at least once a month, e.g., at least every two weeks, e.g., at least once a week.

10. The method of claim 1, wherein the bevacizumab is administered at least once a month, e.g., at least every two weeks, e.g., at least once a week, e.g., at least twice weekly.

11. The method of claim 1, wherein the cyclodextrin-containing polymer camptothecin conjugate is CRLX 101.

12. The method of claim 1, wherein the cyclodextrin-containing polymer camptothecin conjugate is administered prior to surgery, after surgery, or both before and after surgery to remove the cancer, e.g. , to remove a primary tumor and/or a metastases.

13. A method of treating post-surgical, advanced metastatic triple-negative breast cancer (TNBC) in a human subject, the method comprising administering to the subject CRLXlOl in combination with bevacizumab to thereby treat the subject.