WO2017177055A1

WO2017177055A1 - Cyclodextrin-based polymers for therapeutic delivery

Info

Publication number: WO2017177055A1
Application number: PCT/US2017/026430
Authority: WO
Inventors: Liang Zhao; Roy l. CASE; Derek Gregory Van Der Poll; Tiffany HALO; Scott Eliasof; Chester A. Metcalf, Iii; Lata Jayaraman; Ellen ROHDE; Christian Peters
Original assignee: Liang Zhao; Case Roy L; Derek Gregory Van Der Poll; Halo Tiffany; Scott Eliasof; Metcalf Chester A Iii; Lata Jayaraman; Rohde Ellen; Christian Peters
Priority date: 2016-04-08
Filing date: 2017-04-06
Publication date: 2017-10-12

Abstract

Methods and compositions relating to CDP-therapeutic agent antibody conjugates are described herein.

Description

CYCLODEXTRIN-BASED POLYMERS FOR THERAPEUTIC DELIVERY

Claim of Priority

This application claims priority to U.S.S.N. 62/319,922, filed April 8, 2016 and U.S.S.N. 62/348,613 filed June 10, 2016, the contents of each of which are incorporated herein by reference in their entirety.

Background

Drug delivery of some small molecule therapeutic agents has been problematic due to their poor pharmacological profiles. These therapeutic agents often have low aqueous solubility, their bioactive forms exist in equilibrium with an inactive form, or high systemic concentrations of the agents lead to toxic side-effects.

Summary

In one aspect, the disclosure features a cyclodextrin-containing polymer (CDP)- therapeutic agent antibody conjugate, e.g. , a CDP-therapeutic agent monoclonal antibody conjugate, e.g. , a CDP-camptothecin trastuzumab (Herceptin) conjugate, e.g. , a CDP- camptothecin trastuzumab conjugate described herein, or a nanoparticle comprising a CDP-therapeutic agent antibody conjugate, e.g. , a CDP-therapeutic agent monoclonal antibody conjugate, e.g. , a CDP-camptothecin trastuzumab (Herceptin) conjugate, e.g. , a CDP-camptothecin trastuzumab conjugate described herein.

In an embodiment, CDP is not biodegradable.

In an embodiment, CDP is biocompatible.

In an embodiment, the CDP-therapeutic agent antibody conjugate, e.g. , a CDP- therapeutic agent monoclonal antibody conjugate, e.g. , a CDP-camptothecin trastuzumab (Herceptin) conjugate, e.g. , a CDP-camptothecin trastuzumab conjugate described herein, includes an inclusion complex between a therapeutic agent, e.g. , camptothecin, and an antibody, e.g. , a monoclonal antibody, e.g. , trastuzumab (Herceptin), attached or conjugated to the CDP, e.g. , via a covalent linkage or via a linker such as a linker described herein, and another molecule in the CDP. In an embodiment, the CDP-therapeutic agent antibody conjugate, e.g. , a CDP- therapeutic agent monoclonal antibody conjugate, e.g. , a CDP-camptothecin trastuzumab (Herceptin) conjugate, e.g. , a CDP-camptothecin trastuzumab conjugate described herein, forms a nanoparticle. In an embodiment, the CDP-therapeutic agent antibody conjugate, e.g. , a CDP-therapeutic agent monoclonal antibody conjugate, e.g. , a CDP-camptothecin trastuzumab (Herceptin) conjugate, e.g. , a CDP-camptothecin trastuzumab conjugate described herein, including an inclusion complex forms a nanoparticle. The nanoparticle ranges in size from 10 to 300 nm in diameter, e.g. , 10 to 280, 20 to 280, 30 to 250, 30 to 200, 20 to 150, 30 to 100, 20 to 80, 10 to 80, 10 to 70, 20 to 60 or 20 to 50 nm 10 to 70, 10 to 60 or 10 to 50 nm. In an embodiment, the nanoparticle is 20 to 60 nm in diameter.

In an embodiment, the composition comprises a population or a plurality of nanoparticles with an average diameter from 10 to 300 nm, e.g. , 20 to 280, 15 to 250, 15 to 200, 20 to 150, 15 to 100, 20 to 80, 15 to 80, 15 to 70, 15 to 60, 15 to 50, or 20 to 50 nm. In an embodiment, the average nanoparticle diameter is from 15 to 60 nm (e.g. , 20- 60). In an embodiment, the surface charge of the molecule is neutral, or slightly negative. In an embodiment, the zeta potential of the particle surface is from about -80 mV to about 50 mV, about -20 mV to about 20 mV, about -20 mV to about -10 mV, or about - 10 mV to about 0.

In an embodiment, the CDP-therapeutic agent antibody conjugate, e.g. , a CDP- therapeutic agent monoclonal antibody conjugate, e.g. , a CDP-camptothecin trastuzumab (Herceptin) conjugate, e.g. , a CDP-camptothecin trastuzumab conjugate described herein, complex forms a particle or nanoparticle having a conjugate number described herein. By way of example, a CDP-therapeutic agent antibody conjugate, e.g. , a CDP-therapeutic agent monoclonal antibody conjugate, e.g. , a CDP-camptothecin trastuzumab (Herceptin) conjugate, e.g. , a CDP-camptothecin trastuzumab conjugate described herein, forms, or is provided in, a particle or nanoparticle having a conjugate number of: 1 or 2 to 25; 1 or 2 to 20; 1 or 2 to 15; 1 or 2 to 10; 1 to 3; 1 to 4; 1 to 5; 1 to 6; 1 to 7; 1 to 10; 2 to 3; 2 to 4; 2 to 5; 2 to 6; 2 to 7; 2 to 10; 3 to 4; 3 to 5; 3 to 6; 3 to 7; 3 to 10; 5 to 10; 10 to 15; 15-20; 20-25; 1 to 40; 1 to 30; 1 to 20; 1 to 15; 10 to 40; 10 to 30; 10 to 20; 10 to 15; 20 to 40; 20 to 30; or 20 to 25; 1-100; 25 to 100; 50 to 100; 75- 100; 25 to 75, 25 to 50, or 50 to 75; 25 to 40; 25 to 50; 30 to 50; 30 to 40; or 30 to 75. In an embodiment the conjugate number is 2 to 4 or 2 to 5.

In an embodiment the conjugate number is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In an embodiment the nanoparticle forms, or is provided in, a preparation of nanoparticles, e.g. , a pharmaceutical preparation, wherein at least 40, 50, 60, 70, 80, 90 or 95% of the particles in the preparation have a conjugate number provided herein. In an embodiment the nanoparticle forms, or is provided in, a preparation of nanoparticles, e.g. , a pharmaceutical preparation, wherein at least 60% of the particles in the preparation have a conjugate number of 1-5 or 2-5.

In an embodiment, the CDP-therapeutic agent antibody conjugate, e.g. , a CDP- therapeutic agent monoclonal antibody conjugate, e.g. , a CDP-camptothecin trastuzumab (Herceptin) conjugate, e.g. , a CDP-camptothecin trastuzumab conjugate described herein, is administered as a nanoparticle or preparation of nanoparticles, e.g. , a pharmaceutical preparation, wherein at least 60% of the particles in the preparation have a conjugate number of 1 or 2 to 25; 1 or 2 to 20; 1 or 2 to 15; 1 or 2 to 10; 1 to 3; 1 to 4; 1 to 5; 1 to 6; 1 to 7; 1 to 10; 2 to 3; 2 to 4; 2 to 5; 2 to 6; 2 to 7; 2 to 10; 3 to 4; 3 to 5; 3 to 6; 3 to 7; 3 to 10; 5 to 10; 10 to 15; 15-20; 20-25; 1 to 40; 1 to 30; 1 to 20; 1 to 15; 10 to 40; 10 to 30; 10 to 20; 10 to 15; 20 to 40; 20 to 30; or 20 to 25; 1- 100; 25 to 100; 50 to 100; 75- 100; 25 to 75, 25 to 50, or 50 to 75; 25 to 40; 25 to 50; 30 to 50; 30 to 40; or 30 to 75.

In an embodiment, the therapeutic agent, e.g. , camptothecin, and an antibody, e.g. , a monoclonal antibody, e.g. , trastuzumab (Herceptin), conjugated to the CDP is more soluble when conjugated to the CDP, than when not conjugated to the CDP.

In an embodiment, the composition comprises a population, mixture or plurality of CDP-therapeutic agent antibody conjugates, e.g. , CDP-therapeutic agent monoclonal antibody conjugates, e.g. , CDP-camptothecin trastuzumab (Herceptin) conjugates, e.g. , CDP-camptothecin trastuzumab conjugates described herein. In an embodiment, the population, mixture or plurality of CDP-therapeutic agent antibody conjugates comprises a plurality of different therapeutic agents and antibodies conjugated to a CDP (e.g. , two different therapeutic agents and two different antibodies are in the composition such that two different therapeutic agents and two different antibodies are attached to a single CDP; or a first therapeutic agent and antibody is attached to a first CDP and a second therapeutic agent and antibody are attached to a second CDP and both CDP-therapeutic agent antibody conjugates are present in the composition).

In an embodiment, the therapeutic agent, e.g. , camptothecin, is attached to the CDP through a hydroxyl group of the therapeutic agent, e.g. , camptothecin. In some embodiment, the therapeutic agent, e.g. , camptothecin, is attached to the CDP through the primary hydroxyl group of a therapeutic agent, e.g. , camptothecin.

In an embodiment, the antibody, e.g. , monoclonal antibody, e.g. trastuzumab (Herceptin) is attached to the CDP through the nitrogen of the the antibody, e.g. , monoclonal antibody, e.g. trastuzumab (Herceptin).

In an embodiment, the CDP-therapeutic agent antibody conjugate, e.g. , a CDP- therapeutic agent monoclonal antibody conjugate, e.g. , a CDP-camptothecin trastuzumab (Herceptin) conjugate, e.g. , a CDP-camptothecin trastuzumab conjugate described herein, comprises a therapeutic agent, e.g. , camptothecin, coupled, e.g. , via a linker such as a linker described herein, to a CDP described herein, and an antibody, e.g. , a monoclonal antibody, e.g. trastuzumab (Herceptin), coupled, e.g. , via a linker such as a linker described herein, to the same CDP described herein. In an embodiment, camptothecin and trastuzumab (Herceptin) are each coupled, e.g. , via a linker such as a linker described herein, to a CDP described herein. In an embodiment, the CDP-therapeutic agent antibody conjugate is a CDP-therapeutic agent antibody conjugate disclosed herein. Examplary CDP-therapeutic agent antibody conjugates are depicted in FIG. 1.

In another aspect, the disclosure features a method of treating a disorder, e.g. , a cancer, in a subject, e.g. , a human, the method comprising: administering a composition that comprises a CDP-therapeutic agent antibody conjugate, e.g. , a CDP-therapeutic agent monoclonal antibody conjugate, e.g. , a CDP-camptothecin trastuzumab (Herceptin) conjugate, e.g. , a CDP-camptothecin trastuzumab conjugate described herein, to a subject in an amount effective to treat the disorder, e.g. , cancer, in the subject, to thereby treat the disorder, e.g. , cancer.

In an embodiment, the cancer is HER2 overexpressing breast cancer, e.g. , metastatic HER2 overexpressing breast cancer. In an embodiment, the cancer is HER2-overexpressing metastaic gastric or gastroesophageal junction adenocarcinoma.

In an embodiment, the cancer is selected from ovarian cancer, stomach cancer, uterine cancer, uterine serous carcinoma (i.e. , uterine papillary serous carcinoma or uterine serous adenocarcinoma), and non-small cell lung cancer.

In an embodiment, the CDP-therapeutic agent antibody conjugate, e.g. , a CDP- therapeutic agent monoclonal antibody conjugate, e.g. , a CDP-camptothecin trastuzumab (Herceptin) conjugate, e.g. , a CDP-camptothecin trastuzumab conjugate described herein, is administered by subcutaneous administration. In an embodiment, the CDP-therapeutic agent antibody conjugate, e.g. , a CDP-therapeutic agent monoclonal antibody conjugate, e.g. , a CDP-camptothecin trastuzumab (Herceptin) conjugate, e.g. , a CDP-camptothecin trastuzumab conjugate described herein, is administered by intravenous administration.

In another aspect, the disclosure features a method of making a CDP-therapeutic agent antibody conjugate, e.g. , a CDP-therapeutic agent monoclonal antibody conjugate, e.g. , a CDP-camptothecin trastuzumab (Herceptin) conjugate, e.g. , a CDP-camptothecin trastuzumab conjugate described herein. In an embodiment, the method comprises making a CDP-therapeutic agent antibody conjugate, e.g. , a CDP-therapeutic agent monoclonal antibody conjugate, e.g. , a CDP-camptothecin trastuzumab (Herceptin) conjugate, e.g. , a CDP-camptothecin trastuzumab conjugate described herein, by conjugating a plurality of therapeutic agents and a pluraility of antibodies to the CDP. In an embodiment, the method comprises making a CDP-therapeutic agent antibody conjugate, e.g. , a CDP-therapeutic agent monoclonal antibody conjugate, e.g. , a CDP- camptothecin trastuzumab (Herceptin) conjugate, e.g. , a CDP-camptothecin trastuzumab conjugate described herein, by conjugating a plurality of antibodies, e.g. , monoclonal antibodies, e.g. , trastuzumab (Herceptin), to a CDP-therapeutic agent conjugate, e.g. , a CDP-camptothecin conjugate modified with a thiol linker, e.g. , a linker comprising a polyethylene glycol and a thiol group. In an embodiment, less than 100% of the available positions on the CDP are reacted with a therapeutic agent and an antibody. Exemplary methods are described herein. In one aspect, the disclosure features a method of making a nanoparticle comprising a CDP-therapeutic agent antibody conjugate, e.g. , a CDP-therapeutic agent monoclonal antibody conjugate, e.g. , a CDP-camptothecin trastuzumab (Herceptin) conjugate, e.g. , a CDP-camptothecin trastuzumab conjugate described herein. In embodiments, a composition comprising a CDP-therapeutic agent antibody conjugate, e.g. , a CDP-therapeutic agent monoclonal antibody conjugate, e.g. , a CDP-camptothecin trastuzumab (Herceptin) conjugate, e.g. , a CDP-camptothecin trastuzumab conjugate described herein, (e.g. , a reaction mixture) is contacted with an antisolvent (e.g. , a solvent in which the CDP-therapeutic agent antibody conjugate is not soluble), thereby producing a nanoparticle comprising a CDP-therapeutic agent antibody conjugate. In an

embodiment, the method further comprises filtering the nanoparticle.

In one aspect, the disclosure features a method of formulating a CDP-therapeutic agent antibody conjugate or a nanoparticle comprising a CDP-therapeutic agent antibody conjugate into a composition such as a pharmaceutical composition described

herein. The method comprises combining a CDP-therapeutic agent antibody conjugate or a nanoparticle comprising a CDP-therapeutic agent antibody conjugate with a

pharmaceutically acceptable excipient. In an embodiment, the composition is formulated for intravenous or subcutaneous administration.

In another aspect, the disclosure features, a method of evaluating a particle or a preparation of particles, wherein said particles, comprise one or a plurality of CDP- therapeutic agent antibody conjugate molecules, e.g. , CDP-therapeutic agent antibody conjugates, e.g. , CDP-therapeutic agent antibody conjugates described herein. The method comprises:

providing a sample comprising one or a plurality of said particles;

determining a value for the number of CDP-therapeutic agent antibody conjugate molecules in a particle in said sample (the conjugate number),

thereby evaluating a preparation of particles.

In an embodiment the method comprises one or both of: a) comparing said determined value with a reference value, e.g. , a range of values, or

b) responsive to said determination, classifying said particles,

In an embodiment the particle is a nanoparticle.

In an embodiment the method further comprises comparing said determined value with a reference standard. In an embodiment the reference value can be selected from a value, e.g. , a range, provided herein, e.g. , 1 or 2 to 8, 1 or 2 to 7, 1 or 2 to 6, 1 or 2 to 5, or 2-4.

In an embodiment the reference value can be selected from a value, e.g. , a range, provided herein, e.g. , 1 or 2 to 25; 1 or 2 to 20; 1 or 2 to 15; 1 or 2 to 10; 1 to 3; 1 to 4; 1 to 5; 1 to 6; 1 to 7; 1 to 10; 2 to 3; 2 to 4; 2 to 5; 2 to 6; 2 to 7; 2 to 10; 3 to 4; 3 to 5; 3 to 6; 3 to 7; 3 to 10; 5 to 10; 10 to 15; 15-20; 20-25; 1 to 40; 1 to 30; 1 to 20; 1 to 15; 10 to 40; 10 to 30; 10 to 20; 10 to 15; 20 to 40; 20 to 30; or 20 to 25; 1- 100; 25 to 100; 50 to 100; 75- 100; 25 to 75, 25 to 50, or 50 to 75; 25 to 40; 25 to 50; 30 to 50; 30 to 40; or 30 to 75.

In an embodiment, responsive to said comparison, a decision or step is taken, e.g. , a production parameter in a process for making a particle is altered, the sample is classified, selected, accepted or discarded, released or withheld, processed into a drug product, shipped, moved to a different location, formulated, e.g. , formulated with another substance, e.g. , an excipient, labeled, packaged, released into commerce, or sold or offered for sale.

In an embodiment said CDP-therapeutic agent antibody conjugate is selected from those disclosed in herein.

In an embodiment said particle is selected from those disclosed in herein.

In an embodiment, the determined value for conjugate number is compared with a reference, and responsive to said comparison said particle or preparation of particles is classified, e.g. , as suitable for use in human subjects, not suitable for use in human subjects, suitable for sale, meeting a release specification, or not meeting a release specification. In another aspect, the disclosure features, a particle, e.g. , a nanoparticle, comprising one or more CDP-therapeutic agent antibody conjugates described herein, having a conjugate number of: 1 or 2 to 25; 1 or 2 to 20; 1 or 2 to 15; 1 or 2 to 10; 1 to 3; 1 to 4; 1 to 5; 1 to 6; 1 to 7; 1 to 10; 2 to 3; 2 to 4; 2 to 5; 2 to 6; 2 to 7; 2 to 10; 3 to 4; 3 to 5; 3 to 6; 3 to 7; 3 to 10; 5 to 10; 10 to 15; 15-20; 20-25; 1 to 40; 1 to 30; 1 to 20; 1 to 15; 10 to 40; 10 to 30; 10 to 20; 10 to 15; 20 to 40; 20 to 30; or 20 to 25; 1-100; 25 to 100; 50 to 100; 75-100; 25 to 75, 25 to 50, or 50 to 75; 25 to 40; 25 to 50; 30 to 50; 30 to 40; or 30 to 75.

The details of one or more embodiments of the disclosure are set forth in the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and the drawings, and from the claims.

Brief Description of the Figures

FIG. 1 depicts an examplary CDP-therapeutic agent antibody conjugate.

FIG. 2 depicts a line graph showing a calculated strand dependence on particle size.

FIG. 3 depicts a schematic representation of the conjugation reaction of a nanoparticle comprising a CDP-camptothecin conjugate modified with a PEG thiol (sulfhydryl) linker ("Thiol Modified NDC") and a maleimide modified trastuzumab (Herceptin) antibody to prepare nanoparticles comprising CDP-camptothecin

trastuzumab (Herceptin) conjugates ("ANDCs").

FIG. 4 depicts a line graph showing the % release of camptothecin (CPT) from the CDP-camptothecin modified trastuzumab (Herceptin) conjugate ("ANDC") under physiological conditions.

FIGs. 5A-5E depict HPLC traces showing the HER2 reference standard (FIG.

5A); the Herceptin antibody at 280 nm (FIG. 5B); the mixture of Herceptin and HER2 at 280 nm (FIG. 5C); ANDC at 280-305 nm (FIG. 5D); and the mixture of ANDC and HER2 at 650 nm (FIG. 5E).

FIG. 6 depicts a line graph of un-complexed Herceptin. FIG. 7 depicts a line graph of percent of cell viability vs. the IC₅₀ value for the

ANDC.

FIG. 8 depicts images of rhodamine-labelled ANDC in SK-BR-3 (HER2⁺⁺⁺) imaged using a Leica SP5 inverted confocal scanning microscope using a Zeiss 63x Plan Apo oil immersion objective.

FIG. 9 depicts a line graph of the plasma pharmacokinetic data performed in female Ncr nude mice for the ANDC.

FIGs. 10A and 10B depict line graphs of the plasma and tumor pharmacokinetic data as single IV treatments in HCT-116 tumor-bearing female Ncr nude mice for CRLXlOl and ANDC, respectively.

FIG. 11 depicts a line graph of the percent of initial body weights of HCT-116 tumor-bearing mice administered CRLXlOl or ANDC at 8 mg/kg as single IV treatment.

FIGs. 12A and 12B depict line graphs of a pharmacokinetic data comparing CRLXlOl (8 mg/kg) (FIG. 12A) and ANDC (8 mg/kg CPT + 6.5 mg/kg Herceptin combined) (FIG. 12B) as single IV treatments in SK-BR-3 tumor-bearing female Ncr nude mice.

FIG. 13 depicts a line graph of the percent of initial body weights of SK-BR-3 tumor-bearing mice administered CRLXlOl or ANDC at 8 mg/kg as single IV treatment.

FIG. 14 depicts a line graph of the percent of initial body weights of ANDC vs. CRLXlOl in non-tumor bearing nude mide.

FIG. 15 depicts a line graph of the percent of initial body weights of Ncr nude mice given ANDC at 10 mg/kg, 12 mg/kg, and 14 mg/kg.

FIG. 16 depicts a line graph of the percent of initial body weights of Ncr nude mice given ANDC at 18 mg/kg and 22 mg/kg.

FIG. 17 depicts a line graph of the tumor volumes in SK-BR-3 (HER2⁺⁺⁺) tumor- bearing mice from an efficacy study performed comparing ANDC, CRLX01 and

Herceptin.

FIG. 18 depicts a line graph of percent of initial body weights of SK-BR-3 tumor- bearing mice given CRLXlOl or ANDC. Detailed Description

The disclosure relates to novel compositions of therapeutic cyclodextrin- containing polymers (CDPs) conjugated to a therapeutic agent and an antibody, particles containing therapeutic cyclodextrin-containing polymers conjugated to a therapeutic agent and an antibody, compositions and mixtures comprising cyclodextrin-containing polymers, and methods of use thereof. In certain embodiments, these cyclodextrin- containing polymers improve therapeutic agent and antibody stability and/or therapeutic agent and antibody solubility, and/or reduce therapeutic agent and antibody toxicity, and/or improve efficacy of the therapeutic agent and antibody when used in vivo.

By selecting from a variety of linker groups used to link a therapeutic agent and an antibody to a CDP, the rate of therapeutic agent and antibody release from the CDP can be attenuated for controlled delivery. The disclosure also relates to methods of treating subjects, e.g., humans, with a CDP-therapeutic agent antibody conjugate described herein.

More generally, the disclosure provides water-soluble, biocompatible polymer conjugates comprising a water-soluble, biocompatible cyclodextrin containing polymer covalently attached to a therapeutic agent and an antibody through attachments that are cleaved under biological conditions to release the therapeutic agent and the antibody.

Polymeric conjugates featured in the disclosure may be useful to improve solubility and/or stability of a bioactive/therapeutic agent, such as a therapeutic agent and an antibody, 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. Poorly soluble and/or toxic compounds may benefit particularly from incorporation into polymeric compounds of the disclosure.

An "effective amount" or "an amount effective" refers to an amount of the CDP- therapeutic agent antibody conjugate which is effective, upon single or multiple dose administrations to a subject, in treating a cell, or curing, alleviating, relieving or improving a symptom of a disorder. An effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.

"Pharmaceutically acceptable carrier or adjuvant," as used herein, refers to a carrier or adjuvant that may be administered to a patient, together with a CDP-therapeutic agent antibody conjugate described herein, and which does not destroy the

pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the particle. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, mannitol and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in

pharmaceutical compositions.

As used herein the term "low aqueous solubility" refers to water insoluble compounds having poor solubility in water, that is <5 mg/ml at physiological pH (6.5- 7.4). Preferably, their water solubility is <1 mg/ml, more preferably <0.1 mg/ml. It is desirable that the drug is stable in water as a dispersion; otherwise a lyophilized or spray- dried solid form may be desirable.

As used herein, the term "prevent" or "preventing" as used in the context of the administration of an agent to a subject, refers to subjecting the subject to a regimen, e.g. , the administration of a CDP-therapeutic agent antibody conjugate such that the onset of at least one symptom of the disorder is delayed as compared to what would be seen in the absence of the regimen.

As used herein, the term "subject" is intended to include human and non-human animals. Exemplary human subjects include a human patient having a disorder, e.g. , a disorder described herein, or a normal subject. The term "non-human animals" includes all vertebrates, e.g. , non-mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates, domesticated and/or agriculturally useful animals, e.g. , sheep, dog, cat, cow, pig, etc.

As used herein, the term "treat" or "treating" a subject having a disorder refers to subjecting the subject to a regimen, e.g. , the administration of a CDP-therapeutic agent antibody conjugate such that at least one symptom of the disorder is cured, healed, alleviated, relieved, altered, remedied, ameliorated, or improved. Treating includes administering an amount effective to alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder or the symptoms of the disorder. The treatment may inhibit deterioration or worsening of a symptom of a disorder.

The term "alkenyl" refers to an aliphatic group containing at least one double bond.

The terms "alkoxyl" or "alkoxy" refers to an alkyl group, as defined below, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An "ether" is two hydrocarbons covalently linked by an oxygen.

The term "alkyl" refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g. , C1-C₃₀ for straight chains, C₃-C₃₀ for branched chains), and more preferably 20 or fewer, and most preferably 10 or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.

The term "alkynyl" refers to an aliphatic group containing at least one triple bond. The term "aralkyl" or "arylalkyl" refers to an alkyl group substituted with an aryl group (e.g. , a phenyl or naphthyl).

The term "aryl" includes 5- 14 membered single-ring or bicyclic aromatic groups, for example, benzene, naphthalene, and the like. The aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, polycyclyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF₃, -CN, or the like. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic, e.g. , the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls. Each ring can contain, e.g. , 5-7 members. The term "arylene" refers to a divalent aryl, as defined herein.

The term "arylalkenyl" refers to an alkenyl group substituted with an aryl group.

The terms "halo" and "halogen" means halogen and includes chloro, fluoro, bromo, and iodo.

The terms "hetaralkyl", "heteroaralkyl" or "hetero arylalkyl" refers to an alkyl group substituted with a heteroaryl group.

The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8- 12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g. , carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. Examples of heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the like. The term "heteroarylene" refers to a divalent heteroaryl, as defined herein.

The term "heteroarylalkenyl" refers to an alkenyl group substituted with a heteroaryl group. The terms "heterocyclyl" or "heterocyclic group" refer to 3- to 14-membered non- aromatic ring structures (e.g., 3- to 14-membered rings, more preferably 3- to 7- membered rings), whose ring structures include one to four heteroatoms independently selected from O, N and S. The heterocyclyl or heterocyclic groups can contain fused or spiro rings. Heterocycles can also be polycycles, with each group having, e.g., 5-7 ring members. The term "heterocyclyl" or "heterocyclic group" includes saturated and partially saturated heterocyclyl structures.

The term "hydrocarbyl" refers to a monovalent hydrocarbon radical comprised of carbon chains or rings to which hydrogen atoms are attached. The term includes alkyl, cycloalkyl, alkenyl, alkynyl and aryl groups, groups which have a mixture of saturated and unsaturated bonds, carbocyclic rings and includes combinations of such groups. Hydrocarbyl may refer to straight chain, branched-chain, cyclic structures or

combinations thereof.

The term "hydrocarbylene" refers to a divalent hydrocarbyl radical.

CDP- Therapeutic Agent Antibody Conjugates

Described herein are cyclodextrin containing polymer ("CDP")-therapeutic agent antibody conjugates, wherein one or more therapeutic agents, and one or more antibodies, are covalently attached to the CDP (e.g. , either directly or through a linker). The CDP- therapeutic agent antibody 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, the contents of which are incorporated by reference in their entirety.

Accordingly, In an embodiment the CDP-therapeutic agent antibody conjugate is represented by the following formula:

wherein each L is independently a linker or absent, each D is independently a therapeutic agent, e.g. , camptothecin, an antibody, e.g. , trastuzumab (Herceptin), or L comprises a polyethylene glycol thiol linker, or a polyethylene disulfide linker and D is absent, or -L-D is OH; wherein the group ^ has a Mw of about 2 to about 5 kDa (e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da)); and

n is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, provided that at least one L comprises a polyethylene glycol thiol (i.e. , sulfhydryl) linker, or a polyethylene glycol disulfide linker and D is absent.

In an embodiment, L is glycine.

In an embodiment, the conjugate is a component of a nanoparticle, e.g. , a nanoparticle described herein.

In an embodiment, the CDP-therapeutic agent antibody conjugate is represented by the following formula:

wherein each -L-D is a linker-therapeutic agent, e.g. , a linker-camptothecin, e.g. , a gly-camptothecin, or -L-D is OH; wherein the group ^ has a Mw of about 2 to about 5 kDa (e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da)); n is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; and p is about 45 to about 250, e.g. , about 50 to about 200, about 100 to about 150, e.g. , about 114.

In an embodiment, L is glycine.

wherein each -L-D is a linker-therapeutic agent, e.g. , a linker-camptothecin, e.g. , a gly-camptothecin, or -L-D is OH; wherein the group ^ has a Mw of about 2 to about 5 kDa (e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da));

n is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; and p is about 45 to about 250, e.g. , about 50 to about 200, about 100 to about 150, e.g. , about 114.

In an embodiment, L is glycine.

In an embodiment, the conjugate is a component of a nanoparticle, e.g. , a nanoparticle described herein. In an embodiment, the CDP-therapeutic agent antibody conjugate is represented by the following

i

wherein each -L-D is a linker-therapeutic agent, e.g. , a linker-camptothecin, e.g. , a gly-camptothecin, or -L-D is OH; wherein the group has a Mw of about 2 to about 5 kDa (e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, {e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da));

In an embodiment, L is glycine.

wherein the group has a Mw of about 2 to about 5 kDa (e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da));

wherein the group ^ has a Mw of about 2 to about 5 kDa (e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da));

In an embodiment, the CDP-therapeutic agent antibody conjugate is represented by the following

H

i wherein the group ^m has a Mw of about 2 to about 5 kDa (e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da));

wherein the group ^m has a Mw of about 2 to about 5 kDa (e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da));

In an embodiment, the conjugate is a component of a nanoparticle, e.g. , a nanoparticle described herein. In an embodiment, the CDP-therapeutic agent antibody conjugate is represented by the following formula:

Herceptin wherein the group has a Mw of about 2 to about 5 kDa (e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da));

In an embodiment, the CDP-therapeutic agent antibody conjugate comprises a subunit of the following formula:

wherein each L is independently a linker or absent, each D is independently a therapeutic agent, e.g. , camptothecin, or an antibody, e.g. , trastuzumab (Herceptin), or L comprises a polyethylene glycol thiol (i.e. , sulfhydryl) linker, or a polyethylene disulfide linker and D is absent, or -L-D is OH; wherein the group ^m has a Mw of about 2 to about 5 kDa (e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da)); provided that at least one L comprises a polyethylene glycol sulfide linker, or a polyethylene glycol disulfide linker and D is absent.

wherein each L is independently a linker or absent, each D is independently a therapeutic agent, e.g. , camptothecin, or absent, or -L-D is OH;

p is about 45 to about 250, e.g. , about 50 to about 200, about 100 to about 150, e.g. , about 114; and wherein the group ^ has a Mw of about 2 to about 5 kDa (e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da)).

In an embodiment, the CDP-therapeutic agent antibody conjugate comprises subunit of the follo ing formula:

ercep n wherein each L is independently a linker or absent, each D is independently a therapeutic agent, e.g. , camptothecin, or absent, or -L-D is OH;

p is about 45 to about 250, e.g. , about 50 to about 200, about 100 to about 150, e.g. , about 114; and wherein the group ^ ^"^^^ has a Mw of about 2 to about 5 kDa (e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da)).

wherein p is about 45 to about 250, e.g. , about 50 to about 200, about 100 to about 150, e.g. , about 114; and the group ^ ¾_{as a} jy[_{w Q}f about 2 to about 5 kDa (e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da)).

e i

wherein p is about 45 to about 250, e.g. , about 50 to about 200, about 100 to about 150, e.g. , about 114; and the group ^ ¾_{AS A} M_{w Q}f about 2 to about 5 kDa (e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da)). In an embodiment, one or more of the therapeutic agents and one or more of the antibodies in the CDP-therapeutic agent antibody conjugate can be replaced with another therapeutic agent, e.g. , another anticancer agent or anti-inflammatory agent, or another antibody. Nanoparticles Comprising CDP-Therapeutic Agent Antibody Conjugates

The CDP-therapeutic agent anibody conjugates described herein are components of the nanoparticles described herein.

In one aspect, a composition comprising a CDP-therapeutic agent antibody conjugate, e.g. , a CDP-therapeutic agent monoclonal antibody conjugate, e.g. , a CDP- camptothecin trastuzumab (Herceptin) conjugate, e.g. , a CDP-camptothecin trastuzumab conjugate described herein, (e.g. , a reaction mixture) is contacted with an antisolvent (e.g. , a solvent in which the CDP-therapeutic agent antibody conjugate is not soluble), e.g. , acetone, isopropanol, or a mixture thereof, thereby producing nanoparticles comprising the CDP-therapeutic agent antibody conjugates described herein. In an embodiment, the nanoparticles comprising the CDP-therapeutic agent antibody conjugates described herein can be filtered.

Cyclodextrins

In certain embodiments, the cyclodextrin moieties make up at least about 2%, 5% or 10% by weight, up to 20%, 30%, 50% or even 80% of the CDP by weight. In certain embodiments, the therapeutic agent, e.g. , camptothecin, make up at least about 1%, 5%, 10% or 15%, 20%, 25%, 30% or even 35% of the CDP by weight. Number-average molecular weight (M_n) may also vary widely, but generally fall in the range of about 1,000 to about 500,000 daltons, preferably from about 5000 to about 200,000 daltons and, even more preferably, from about 10,000 to about 100,000. Most preferably, M_n varies between about 12,000 and 65,000 daltons. In certain other embodiments, M_n varies between about 3000 and 150,000 daltons. Within a given sample of a subject polymer, a wide range of molecular weights may be present. For example, molecules within the sample may have molecular weights that differ by a factor of 2, 5, 10, 20, 50, 100, or more, or that differ from the average molecular weight by a factor of 2, 5, 10, 20, 50, 100, or more. Exemplary cyclodextrin moieties include cyclic structures consisting essentially of from 7 to 9 saccharide moieties, such as cyclodextrin and oxidized cyclodextrin. A cyclodextrin moiety optionally comprises a linker moiety that forms a covalent linkage between the cyclic structure and the polymer backbone, preferably having from 1 to 20 atoms in the chain, such as alkyl chains, including dicarboxylic acid derivatives (such as glutaric acid derivatives, succinic acid derivatives, and the like), and heteroalkyl chains, such as oligoethylene glycol chains.

Cyclodextrins are cyclic polysaccharides containing naturally occurring D-(+)- glucopyranose units in an a-(l,4) linkage. The most common cyclodextrins are alpha ((a)-cyclodextrins, beta (P)-cyclodextrins and gamma (y)-cyclodextrins which contain, respectively six, seven, or eight glucopyranose units. Structurally, the cyclic nature of a cyclodextrin forms a torus or donut-like shape having an inner apolar or hydrophobic cavity, the secondary hydroxyl groups situated on one side of the cyclodextrin torus and the primary hydroxyl groups situated on the other. Thus, using (P)-cyclodextrin as an example, a cyclodextrin is often represented schematically as follows.

The side on which the secondary hydroxyl groups are located has a wider diameter than the side on which the primary hydroxyl groups are located. The disclosure contemplates covalent linkages to cyclodextrin moieties on the primary and/or secondary hydroxyl groups. The hydrophobic nature of the cyclodextrin inner cavity allows for host-guest inclusion complexes of a variety of compounds, e.g., adamantane.

(Comprehensive Supramolecular Chemistry, Volume 3, J.L. Atwood et al., eds., Pergamon Press (1996); T. Cserhati, Analytical Biochemistry, 225:328-332(1995);

Husain et al., Applied Spectroscopy, 46:652-658 (1992); FR 2 665 169). Additional methods for modifying polymers are disclosed in Suh, J. and Noh, Y., Bioorg. Med. Chem. Lett. 1998, 8, 1327-1330.

Comonomers

In addition to a cyclodextrin moiety, the CDP can also include a comonomer, for example, a comonomer described herein. In an embodiment, a comonomer of the CDP- therapeutic agent antibody conjugatecomprises a moiety selected from the group consisting of: an alkylene chain, polysuccinic anhydride, poly-L-glutamic acid, poly(ethyleneimine), an oligosaccharide, and an amino acid chain. In an embodiment, a CDP-therapeutic agent antibody conjugate comonomer comprises a polyethylene glycol chain. In an embodiment, a comonomer comprises a moiety selected from: polyglycolic acid and polylactic acid chain. In an embodiment, a comonomer comprises a

hydrocarbylene group wherein one or more methylene groups is optionally replaced by a group Y (provided that none of the Y groups are adjacent to each other), wherein each Y, independently for each occurrence, is selected from, substituted or unsubstituted aryl, heteroaryl, cycloalkyl, heterocyclyl, or -0-, C(=X) (wherein X is NRi, O or S), -OC(O)-, -C(=0)0, -NRi-, -NRiCO-, -C(0)NR , -S(0)„- (wherein n is 0, 1, or 2), -OC(0)-NRi, - NRi-C(0)-NRi-, -NRi l-C(NRi)-NRi-, and -B(ORi)-; and R_ls independently for each occurrence, represents H or a lower alkyl.

Antibodies

The term "antibody" refers to (a) immunoglobulin polypeptides and

immunologically active portions of immunoglobulin polypeptides, i.e., polypeptides of the immunoglobulin family, or fragments thereof, that contain an antigen binding site that immunospecifically binds to a specific antigen and an Fc domain, or (b) conservatively substituted derivatives of such immunoglobulin polypeptides or fragments that immunospecifically bind to the antigen. Antibodies are generally described in, for example, Harlow & Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988). In an embodiment, the antibody molecule is a full length antibody, e.g. , trastuzumab (Herceptin).

Therapeutic Agents

The therapeutic agents may be a small molecule, organometallic compound, nucleic acid, protein, peptide, metal, isotopically labeled chemical compound, drug, vaccine, or immunological agent. In an embodiment, the therapeutic agent is

camptothecin, or a derivative thereof.

In an embodiment, the therapeutic agent is a compound with pharmaceutical activity. In another embodiment, the therapeutic agent is a clinically used or investigated drug. In another embodiment, the agent has been approved by the U. S. Food and Drug Administration for use in humans or other animals. In an embodiment, the therapeutic agent is an antibiotic, anti- viral agent, anesthetic, steroidal agent, anti-cancer agent, antiinflammatory agent (e.g. , a non-steroidal anti-inflammatory agent), anti-neoplastic agent, antigen, vaccine, antibody, decongestant, antihypertensive, sedative, birth control agent, progestational agent, anti-cholinergic, analgesic, anti-depressant, anti-psychotic, p- adrenergic blocking agent, diuretic, cardiovascular active agent, vasoactive agent, nutritional agent, vitamin (e.g. , riboflavin, nicotinic acid, pyridoxine, pantothenic acid, biotin, choline, inositol, carnitine, vitamin C, vitamin A, vitamin E, vitamin K), or a gene therapy agent (e.g. , DNA-protein conjugates, anti-sense agents).

The therapeutic agent, e.g. , camptothecin, may be present in varying amounts of a polymer-therapeutic agent conjugate, particle or composition described herein. When present in a particle, the therapeutic agent may be present in an amount, e.g. , from about 1 to about 30% by weight (e.g. , from about 2 to about 30% by weight, from about 4 to about 25 % by weight, or from about 5 to about 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% by weight). Exemplary CDP-therapeutic Agent Antibody Conjugates

CDP-therapeutic agent antibody conjugates can be made using many different combinations of components described herein. For example, various combinations of cyclodextrins (e.g. , beta-cyclodextrin), comonomers (e.g. , PEG containing comonomers), linkers linking the cyclodextrins and comonomers, and/or linkers tethering the therapeutic agent and antibody to the CDP are described herein.

An exemplary cyclodextrin containing polymer (CDP) is shown below:

wherein the group has a Mw of about 2 to about 5 kDa (e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da)) and n is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Note that the therapeutic agent is conjugated to the CDP through the carboxylic acid moieties of the polymer as provided above. Full loading of the therapeutic agent onto the CDP is not required. In an embodiment, at least one, e.g. , at least 2, 3, 4, 5, 6 or 7, of the carboxylic acid moieties remains unreacted with the therapeutic agent after conjugation (e.g. , a plurality of the carboxylic acid moieties remain unreacted).

CDP-Therapeutic Agent Antibody Conjugate Characteristics

In an embodiment, the CDP and/or CDP-therapeutic agent antibody conjugates as described herein have polydispersities less than about 3, or even less than about 2. One embodiment of the disclosure provides an improved delivery of certain therapeutic agents by covalently conjugating them to a CDP. Such conjugation improves the aqueous solubility and hence the bioavailability of the therapeutic agent.

Accordingly, in an embodiment of the disclosure, the therapeutic agent is a hydrophobic compound with a log P >0.4, >0.6, >0.8, >1, >2, >3, >4, or even >5. In other

embodiments, a therapeutic agent may be attached to another compound, such as an amino acid, prior to covalently attaching the conjugate onto the CDP.

The CDP-therapeutic agent antibody conjugates described herein preferably have molecular weights in the range of 10,000 to 500,000; 30,000 to 200,000; or even 70,000 to 150,000 amu. In certain embodiments as disclosed herein, the compound has a number average (M_n) molecular weight between 1,000 to 500,000 amu, or between 5,000 to 200,000 amu, or between 10,000 to 100,000 amu. One method to determine molecular weight is by gel permeation chromatography ("GPC"), e.g. , mixed bed columns, CH₂CI₂ solvent, light scattering detector, and off-line dn/dc. Other methods are known in the art.

In certain embodiments as disclosed herein, the CDP-therapeutic agent antibody conjugate is biodegradable or bioerodable.

In certain embodiments as disclosed herein, the therapeutic agent thereof makes up at least 3% (e.g. , at least about 5%, 10%, 15%, or 20%) by weight of the compound. In certain embodiments, the therapeutic agent makes up at least 15% or 20% by weight of the compound (e.g. , from 17-21% by weight).

In other embodiments, the CDP-therapeutic agent antibody conjugate may be a flexible or flowable material. When the CDP used is itself flowable, the CDP

composition of the disclosure, even when viscous, need not include a biocompatible solvent to be flowable, although trace or residual amounts of biocompatible solvents may still be present.

When a solvent is used to facilitate mixing or to maintain the flowability of the CDP-therapeutic agent antibody conjugate, it should be non-toxic, otherwise

biocompatible, and should be used in relatively small amounts. Examples of suitable biocompatible solvents, when used, include N-methyl-2-pyrrolidone, 2-pyrrolidone, ethanol, propylene glycol, acetone, methyl acetate, ethyl acetate, methyl ethyl ketone, dimethylformamide, dimethylsulfoxide, tetrahydrofuran, caprolactam, oleic acid, or 1- dodecylazacylcoheptanone. Preferred solvents include N-methylpyrrolidone, 2- pyrrolidone, dimethylsulfoxide, and acetone because of their solvating ability and their biocompatibility.

In certain embodiments, the CDP-therapeutic agent antibody conjugates are soluble in one or more common organic solvents for ease of fabrication and processing. Common organic solvents include such solvents as chloroform, dichloromethane, dichloroethane, 2-butanone, butyl acetate, ethyl butyrate, acetone, ethyl acetate, dimethylacetamide, N-methylpyrrolidone, dimethylformamide, and dimethylsulfoxide.

In certain embodiments, the CDP-therapeutic agent antibody conjugates described herein, upon contact with body fluids, undergo gradual degradation. The life of a biodegradable polymer in vivo depends upon, among other things, its molecular weight, crystallinity, biostability, and the degree of crosslinking. In general, the greater the molecular weight, the higher the degree of crystallinity, and the greater the biostability, the slower biodegradation will be.

If a subject composition is formulated with a therapeutic agent or other material, release of the therapeutic agent or other material for a sustained or extended period as compared to the release from an isotonic saline solution generally results. Such release profile may result in prolonged delivery (over, say 1 to about 2,000 hours, or alternatively about 2 to about 800 hours) of effective amounts (e.g., about 0.0001 mg/kg/hour to about 10 mg/kg/hour, e.g., 0.001 mg/kg/hour, 0.01 mg/kg/hour, 0.1 mg/kg/hour, 1.0

mg/kg/hour) of the therapeutic agent or any other material associated with the polymer.

A variety of factors may affect the desired rate of hydrolysis of CDP-therapeutic agent antibody conjugates, the desired softness and flexibility of the resulting solid matrix, rate and extent of bioactive material release. Some of such factors include the selection/identity of the various subunits, the enantiomeric or diastereomeric purity of the monomeric subunits, homogeneity of subunits found in the polymer, and the length of the polymer. For instance, the disclosure contemplates heteropolymers with varying linkages, and/or the inclusion of other monomeric elements in the polymer, in order to control, for example, the rate of biodegradation of the matrix.

To illustrate further, a wide range of degradation rates may be obtained by adjusting the hydrophobicities of the backbones or side chains of the polymers while still maintaining sufficient biodegradability for the use intended for any such polymer. Such a result may be achieved by varying the various functional groups of the polymer. For example, the combination of a hydrophobic backbone and a hydrophilic linkage produces heterogeneous degradation because cleavage is encouraged whereas water penetration is resisted.

One protocol generally accepted in the field that may be used to determine the release rate of a therapeutic agent such as a therapeutic agent or other material loaded in the CDP-therapeutic agent antibody conjugates of the disclosure involves degradation of any such matrix in a 0.1 M PBS solution (pH 7.4) at 37 °C, an assay known in the art. For purposes of the disclosure, the term "PBS protocol" is used herein to refer to such protocol.

In certain instances, the release rates of different CDP-therapeutic agent antibody conjugates of the disclosure may be compared by subjecting them to such a protocol. In certain instances, it may be necessary to process polymeric systems in the same fashion to allow direct and relatively accurate comparisons of different systems to be made. For example, the disclosure teaches several different methods of formulating the CDP- therapeutic agent antibody conjugates. Such comparisons may indicate that any one CDP-therapeutic agent antibody conjugate releases incorporated material at a rate from about 2 or less to about 1000 or more times faster than another polymeric system.

Alternatively, a comparison may reveal a rate difference of about 3, 5, 7, 10, 25,

50, 100, 250, 500 or 750 times. Even higher rate differences are contemplated by the disclosure and release rate protocols.

In certain embodiments, when formulated in a certain manner, the release rate for CDP-therapeutic agent antibody conjugates of the disclosure may present as mono- or bi- phasic.

Release of any material incorporated into the polymer matrix, which is often provided as a microsphere, may be characterized in certain instances by an initial increased release rate, which may release from about 5 to about 50% or more of any incorporated material, or alternatively about 10, 15, 20, 25, 30 or 40%, followed by a release rate of lesser magnitude. The release rate of any incorporated material may also be characterized by the amount of such material released per day per mg of polymer matrix. For example, in certain embodiments, the release rate may vary from about 1 ng or less of any

incorporated material per day per mg of polymeric system to about 500 or more ng/day/mg. Alternatively, the release rate may be about 0.05, 0.5, 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 ng/day/mg. In still other

embodiments, the release rate of any incorporated material may be 10,000 ng/day/mg, or even higher. In certain instances, materials incorporated and characterized by such release rate protocols may include therapeutic agents, fillers, and other substances.

In another aspect, the rate of release of any material from any CDP-therapeutic agent antibody conjugate of the disclosure may be presented as the half-life of such material in the matrix.

In addition to the embodiment involving protocols for in vitro determination of release rates, in vivo protocols, whereby in certain instances release rates for polymeric systems may be determined in vivo, are also contemplated by the disclosure. Other assays useful for determining the release of any material from the polymers of the present system are known in the art.

Physical Structures of the CDP-Therapeutic Agent Antibody Conjugates

The CDP-therapeutic agent antibody conjugates described herein may be formed in a variety of shapes. For example, in certain embodiments, the CDP-therapeutic agent antibody conjugates may be presented in the form of a nanoparticle. In an embodiment, the CDP-therapeutic agent antibody conjugate self assembles into a nanoparticle. In an embodiment, the CDP-therapeutic agent antibody conjugate self assembles into a nanoparticle in an aqueous solution, e.g., water.

In addition to intracellular delivery of a therapeutic agent, it also possible that nanoparticles of the CDP-therapeutic agent antibody conjugates may undergo

endocytosis, thereby obtaining access to the cell. The frequency of such an endocytosis process will likely depend on the size of any nanoparticle. In an embodiment, the surface charge of the molecule is neutral, or slightly negative. In an embodiment, the zeta potential of the particle surface is from about -80 mV to about 50 mV. Particles: Conjugate Number

Conjugate number, as used herein, is the number of cyclodextrin containing polymer ("CDP") therapeutic agent conjugate molecules, present in a particle or nanoparticle. For purposes of determining conjugate number, a particle or nanoparticle is an entity having one, or typically, more than one CDP-therapeutic agent antibody conjugate molecules, which, at the concentration suitable for administration to humans, behaves as a single unit in any of water, e.g. , water at neutral pH, PBS, e.g. , PBS at pH 7.4, or in a formulation in which it will be administered to patients. For purposes of calculating conjugate number, a CDP-therapeutic agent antibody conjugate molecule is a single CDP polymer with its covalently linked therapeutic agent.

Methods disclosed herein provide for evaluating a particle, e.g. , a nanoparticle, or preparation of particles, e.g. , nanoparticles, wherein said particles, e.g. , nanoparticles, comprise a CDP-therapeutic agent antibody conjugate. Generally, the method comprises providing a sample comprising a plurality of said particles, e.g. , nanoparticles, determining a value for the number of CDP-therapeutic agent antibody conjugates in a particle, e.g. , nanoparticle, in the sample, to thereby evaluate a preparation of particles, e.g. , nanoparticles.

Typically the value for a particle will be a function of the values obtained for a plurality of particles, e.g. , the value will be the average of values determined for a plurality of particles.

In embodiments the method further comprises comparing the determined value with a reference value. The comparison can be used in a number of ways. By way of example, in response to a comparison or determination made in the method, a decision or step is taken, e.g. , a production parameter in a process for making a particle is altered, the sample is classified, selected, accepted or discarded, released or withheld, processed into a drug product, shipped, moved to a different location, formulated, e.g. , formulated with another substance, e.g. , an excipient, labeled, packaged, released into commerce, or sold or offered for sale. E.g. , based on the result of the determination, or upon comparison to a reference standard, the batch from which the sample is taken can be processed, e.g. , as just described.

As discussed above, conjugate number is defined as the number of CDP- therapeutic agent antibody conjugate molecules that self- assemble into a particle or nanoparticle, thus

Cj = [CDP-therapeutic agent antibody conjugate]/P (or NP)

where Cj is conjugate number, [CDP-therapeutic agent antibody conjugate]/ is the number of CDP-therapeutic agent antibody conjugate molecules, and P (or NP) is a single particle (or nanoparticle).

In order to arrive and conjugate number one determines the size of a particle, e.g. , by dynamic light scattering. The size should be viscosity-adjusted size. The

hydrodynamic volume of a CDP-therapeutic agent antibody conjugate, or a molecule of similar molecular weight, is determined, to provide an expected hydrodynamic volume. Comparison of the expected hydrodynamic volume for the CDP-therapeutic agent antibody conjugate with the volume for a particle of determined size provides conjugate number.

The determination of conjugate number is demonstrated with CRLX101, in which camptothecin is coupled to the CDP backbone. In the case of CRLX101, a number of fundamental assumptions are made in postulating nanoparticle characteristics. First, macromolecular volume estimates are based on work done with bovine serum albumin (BSA), a biological macromolecule of similar size to CRLX101 (BSA MS=67kDa, 101 MW=66.5kDa). It has been demonstrated that a single strand of BSA has a

hydrodynamic diameter of 9.5 nm. Simple volume calculations yield a volume of 3589 nm . Extending this to CRLX101 with an average 30 nm particle, gives a volume of

33,485 nm . With a particle size of 5-40 nm the conjugate number is 1-30. FIG. 2 shows a calculated strand dependence on particle size.

Polymer Polydispersity. CRLX101 molecules fall within a range of molecular weights, with molecules of varying weight providing varying contributions to the particle diameter and conjugate number. Particles could form which are made up of strands that are larger and smaller than the average. Strands may also associate to a maximum size which could be shear-limited.

Particle Shape. Particle shape is assumed to be roughly spherical, and driven by either (or both) the hydrophobic region created by the CDP-therapeutic agent antibody conjugate, or by guest-host complexation with pendant therapeutic agent molecules making inclusion complexes with CDs from adjacent strands. One critical point of note is that as a drug product, the NPs are in a somewhat controlled environment as they are characterized. Upon administration, myriad possibilities exist for interaction with endogenous substances: inclusion complexes of circulating small molecules, metal ion complexation with the PEG subunits, etc. Any one of these are all of them in concert could dramatically alter the NP structure and function.

CDPs, methods of making same, and methods of conjugating Therapeutic Agents and Antibodies to CDPs

The CDP-therapeutic agent antibody conjugates described herein can be prepared by covalently attaching one or more therapeutic agent(s) and one or more antibodies to a CDP prepared by the methods disclosed in U.S. Patent No. 7,270,808, which is incorporated herein in its entirety.

In an embodiment, the cyclodextrin moiety comprises an alpha, beta, or gamma cyclodextrin moiety.

In an embodiment, the CDP is suitable for the attachment of sufficient therapeutic agent such that up to at least 3%, 5%, 10%, 15%, 20%, 25%, 30%, or even 35% by weight of the CDP, when conjugated, is therapeutic agent.

In an embodiment, the CDP has a molecular weight of 10,000-500,000. In an embodiment, the cyclodextrin moieties make up at least about 2%, 5%, 10%, 20%, 30%, 50% or 80% of the CDP by weight.

In an embodiment, the CDP comprises a comonomer selected from the group consisting of: an alkylene chain, polysuccinic anhydride, poly-L-glutamic acid, poly(ethyleneimine), an oligosaccharide, and an amino acid chain. In an embodiment, a comonomer comprises a polyethylene glycol chain. In an embodiment, the CDP comprises a comonomer selected from the group consisting of: polyglycolic acid and polylactic acid chain, the CDP comprises a comonomer selected from the group consisting of a comonomer comprises a hydrocarbylene group wherein one or more methylene groups is optionally replaced by a group Y (provided that none of the Y groups are adjacent to each other), wherein each Y, independently for each occurrence, is selected from, substituted or unsubstituted aryl, heteroaryl, cycloalkyl, heterocyclyl, or - 0-, C(=X) (wherein X is NRi, O or S), -OC(O)-, -C(=0)0, -NR , -NRiCO-, -C(0)NR , -S(0)„- (wherein n is 0, 1, or 2), -OC(0)-NRi, -NRi-C(0)-NR , -NRi-C(NRi)-NRr, and -B(ORi)-; and Ri, independently for each occurrence, represents H or a lower alkyl.

In an embodiment, a CDP of the following formula can be made by the scheme below:

providing a compound of formula A and formula B:

Formula A Formula B wherein LG is a leaving group;

and contacting the compounds under conditions that allow for the formation of a covalent bond between the compounds of formula A and B, to form a polymer of the following formula:

wherein the group ' has a Mw of about 2 to about 5 kDa (e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da)) and n is at least four.

In an embodiment, Formula B is

In an embodiment, the group ^m has a Mw of about 2 to about 5 kDa

(e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da)) and the Mw of the compound is from 27kDa to 99.6kDa.

In an embodiment, the compounds of formula A and formula B are contacted in the presence of a base. In an embodiment, the base is an amine containing base. In an embodiment, the base is DEA.

wherein R is of the form:

;

comprising the steps of:

reacting a compound of the formula below:

with a compound of the formula below:

wherein the group

has a Mw of about 2 to about 5 kDa (e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da)) and n is at least four, in the presence of a non-nucleo hilic organic base in a solvent.

In an embodiment, the solvent is a polar aprotic solvent. In an embodiment, the solvent is DMSO.

In an embodiment, the method also includes the steps of dialysis; and

lyophylization.

In an embodiment, a CDP provided below can be made by the following scheme:

wherein R is of the form:

comprising the steps of:

reacting a compound of the formula below:

with a compound of the formula below:

wherein the group

has a Mw of about 2 to about 5 kDa (e.g., from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da)) and n is at least four, or with a compound provided below:

wherein the group has a Mw of about 2 to about 5 kDa

(e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da));

in the presence of a non-nucleophilic organic base in DMSO;

and dialyzing and lyophilizing the following polymer

A linear CDP may be characterized by any means known in the art. Such characterization methods or techniques include, but are not limited to, gel permeation chromatography (GPC), matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF Mass spec), 1H and ¹³C NMR, light scattering and titration.

One aspect of the disclosure contemplates attaching a therapeutic agent, e.g. , camptothecin, and an antibody to a CDP for delivery of a therapeutic agent, e.g. , camptothecin and an antibody. In certain embodiments, the therapeutic agent, e.g. , camptothecin, and the antibody are covalently linked via a biohydrolyzable bond, for example, an ester, amide, carbamates, or carbonate.

In an embodiment, the therapeutic agent is attached via a linker. In an embodiment, the therapeutic agent is attached to the water soluble linear polymer through an attachment that is cleaved under biological conditions to release the therapeutic agent. In an embodiment, the therapeutic agent is attached to the water soluble linear polymer at a cyclodextrin moiety or a comonomer. In an embodiment, the therapeutic agent is attached to the water soluble linear polymer via an optional linker to a cyclodextrin moiety or a comonomer.

In an embodiment, the cyclodextrin moieties comprise linkers to which therapeutic agents are linked. In an embodiment, the cyclodextrin moieties comprise linkers to which therapeutic agents are linked via a second linker.

In an embodiment, the CDP is made by a process comprising: providing cyclodextrin moiety precursors, providing comonomer precursors, and copolymerizing said cyclodextrin moiety precursors and comonomer precursors to thereby make a CDP comprising cyclodextrin moieties and comonomers. In an embodiment, the CDP is conjugated with a therapeutic agent, e.g. , camptothecin, and an antibody to provide a CDP-therapeutic agent antibody conjugate.

In an embodiment, the method includes providing cyclodextrin moiety precursors modified to bear one reactive site at each of exactly two positions, and reacting the cyclodextrin moiety precursors with comonomer precursors 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 comonomers and the cyclodextrin moieties, whereby a CDP comprising alternating units of a cyclodextrin moiety and a comonomer is produced.

In an embodiment, the therapeutic agent, e.g. , camptothecin, and antibody are attached to the CDP via a linker. In an embodiment, the linker is cleaved under biological conditions.

In an embodiment, the comonomer precursor is a compound containing at least two functional groups through which reaction and thus linkage of the cyclodextrin moieties is achieved. In an embodiment, the functional groups, which may be the same or different, terminal or internal, of each comonomer precursor comprise an amino, acid, imidazole, hydroxyl, thio, acyl halide, -HC=CH-, C≡C group, or derivative thereof. In an embodiment, the two functional groups are the same and are located at termini of the comonomer precursor. In an embodiment, a comonomer contains one or more pendant groups with at least one functional group through which reaction and thus linkage of a therapeutic agent is achieved. In an embodiment, 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 an embodiment, the pendant group is a substituted or unsubstituted branched, cyclic or straight chain CI -C IO alkyl, or arylalkyl optionally containing one or more heteroatoms within the chain or ring.

In an embodiment, the therapeutic agent, e.g. , camptothecin, is poorly soluble in water.

In an embodiment, the solubility of the therapeutic agent, e.g. , camptothecin, is <5 mg/ml at physiological pH.

In an embodiment, the therapeutic agent, e.g. , camptothecin, is a hydrophobic compound with a log P>0.4, >0.6, >0.8, >1, >2, >3, >4, or >5. In an embodiment, the therapeutic agent, e.g. , camptothecin, is hydrophobic and is attached via a second compound.

In an embodiment, administration of the CDP-therapeutic agent antibody conjugate to a subject results in release of the therapeutic agent, e.g. , camptothecin, over a period of at least 6 hours. In an embodiment, administration of the CDP-therapeutic agent antibody conjugate to a subject results in release of the therapeutic agent, e.g. , camptothecin, over a period of 6 hours to a month. In an embodiment, upon

administration of the CDP-therapeutic agent antibody conjugate to a subject the rate of therapeutic agent, e.g. , camptothecin, release is dependent primarily upon the rate of hydrolysis as opposed to enzymatic cleavage.

In an embodiment, the CDP-therapeutic agent antibody conjugate has a molecular weight of 10,000-500,000.

In an embodiment, the cyclodextrin moieties make up at least about 2%, 5%, 10%, 20%, 30%, 50% or 80% of the polymer by weight.

In an embodiment, a the CDP includes a comonomer selected from the group consisting of: an alkylene chain, polysuccinic anhydride, poly-L-glutamic acid, poly(ethyleneimine), an oligosaccharide, and an amino acid chain. In an embodiment, a comonomer comprises a polyethylene glycol chain. In an embodiment, a comonomer comprises a polyglycolic acid or polylactic acid chain. In an embodiment, a comonomer comprises a hydrocarbylene group wherein one or more methylene groups is optionally replaced by a group Y (provided that none of the Y groups are adjacent to each other), wherein each Y, independently for each occurrence, is selected from, substituted or unsubstituted aryl, heteroaryl, cycloalkyl, heterocyclyl, or -0-, C(=X) (wherein X is NRi, O or S), -OC(O)-, -C(=0)0, -NR , -NRiCO-, -C(0)NR , -S(0)„- (wherein n is 0, 1, or 2), -OC(0)-NRi, -NRi-C(0)-NRi-, -NRi-C(NRi)-NRi-, and -B(ORi)-; and R_ls independently for each occurrence, represents H or a lower alkyl.

In an embodimen -polymer con ugate of the following formula

can be made as follows:

providing a polymer of the formula below:

and coupling the polymer with a plurality of D moieties, wherein each D is independently absent or independently a therapeutic agent or antibody, to provide:

wherein the comonomer has a Mw of about 2 to about 5 kDa (e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa +

10%, e.g. , about 3060 Da to about 3740 Da)) and n is at least 4, 5, 6, 7, 8, 9, 10, 11, 12,

13, 14, 15, 16, 17, 18, 19 or 20.

In an embodiment, one or more of the therapeutic agent moieties or antibody moieties in the CDP-therapeutic agent antibody conjugate can be replaced with another therapeutic agent. In an embodiment, a CDP-therapeutic agent antibody conjugate of the following formula:

can be made as follows:

providing a polymer of the formula below:

and coupling the polymer with a plurality of D moieties, wherein each D is independently abse vide:

wherein the group

has a Mw of about 2 to about 5 kDa (e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da)) and n is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

In an embodiment, one or more of the therapeutic agent, e.g. , camptothecin, moieties in the CDP-therapeutic agent antibody conjugate can be replaced with another therapeutic agent.

The reaction scheme as provided above includes embodiments where D is absent in one or more positions as provided above. This can be achieved, for example, when less than 100% yield is achieved when coupling the therapeutic agent, e.g. , camptothecin, to the polymer (e.g. , 80-90%) and/or when less than an equivalent amount of therapeutic agent, e.g. , camptothecin, is used in the reaction. Accordingly, the loading of the therapeutic agent, e.g. , camptothecin, by weight of the polymer, can vary, for example, the loading of the Therapeutic agent, e.g. , camptothecin, can be at least about 3% by weight, e.g. , at least about 5%, at least about 8%, at least about 10%, at least about 13%, at least about 15%, or at least about 20%. In an embodiment, a CDP-therapeutic agent antibody conjugate of the following formula:

be made as follows:

providing a polymer below:

and coupling the polymer with a plurality of L-D moieties, wherein L is a linker or absent and D is a therapeutic agent, e.g. , camptothecin, or an antibody, or -L-D is OH, to provide:

wherein the group

In an embodiment, a CDP-therapeutic agent antibody conjugate of the following formula:

be made as follows:

providing a polymer below:

and coupling the polymer with a plurality of L-D moieties, wherein -L-D is gly- therapeutic agent, e.g. , gly-camptothecin, or -L-D comprises a polyethylene glycol thiol linker, or a polyethylene disulfide linker and D is absent, or -L-D is OH;

to provide:

wherein each L is independently a linker or absent, each D is independently a therapeutic agent, e.g. , camptothecin, an antibody, e.g. , trastuzumab (Herceptin), or L comprises a polyethylene glycol thiol linker, or a polyethylene disulfide linker and D is absent, or -L-D is OH; wherein the group ^ ^"^^^ has a Mw of about 2 to about 5 kDa (e.g. , from about 2 to about 4.5 kDa, from about 3 to about 4 kDa, or less than about 4 kDa, (e.g. , about 3.4 kDa + 10%, e.g. , about 3060 Da to about 3740 Da)); and

n is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, provided that at least one L comprises a polyethylene glycol thiol linker, or a polyethylene glycol disulfide linker and D is absent.

In an embodiment, a maleimide modified antibody, e.g. , a maleimide modified Herceptin, can be coupled, e.g. , covalently attached, to a polyethylene glycol thiol linker through the maleimide on the maleimide modified antibody, e.g. , a maleimide modified Herceptin.

The reaction schemes as provided above includes embodiments where L-D is absent in one or more positions as provided above.

The reaction scheme as provided above includes embodiments where L-D is absent in one or more positions as provided above. This can be achieved, for example, when less than 100% yield is achieved when coupling the therapeutic agent- linker to the polymer (e.g. , 80-90%) and/or when less than an equivalent amount of therapeutic agent- linker is used in the reaction. Accordingly, the loading of the therapeutic agent, e.g. , camptothecin, by weight of the polymer, can vary, for example, the loading of the therapeutic agent, e.g. , camptothecin, can be at least about 3% by weight, e.g. , at least about 5%, at least about 8%, at least about 10%, at least about 13%, at least about 15%, or at least about 20%.

In an embodiment, at least a portion of the L moieties of L-D is absent. In an embodiment, each L is independently an amino acid or derivative thereof (e.g. , glycine).

In an embodiment, the coupling of the polymer with the plurality of L-D moieties results in the formation of a plurality of amide bonds.

In certain instances, the CDPs are random copolymers, in which the different subunits and/or other monomeric units are distributed randomly throughout the polymer chain. Thus, where the formula X_m-Y_n-Z₀ appears, wherein X, Y and Z are polymer subunits, these subunits may be randomly interspersed throughout the polymer backbone. In part, the term "random" is intended to refer to the situation in which the particular distribution or incorporation of monomeric units in a polymer that has more than one type of monomeric units is not directed or controlled directly by the synthetic protocol, but instead results from features inherent to the polymer system, such as the reactivity, amounts of subunits and other characteristics of the synthetic reaction or other methods of manufacture, processing, or treatment. Pharmaceutical Compositions

In another aspect, the disclosure provides a composition, e.g. , a pharmaceutical composition, comprising a CDP-therapeutic agent antibody conjugate and a

pharmaceutically acceptable carrier or adjuvant.

In an embodiment, a pharmaceutical composition may include a pharmaceutically acceptable salt of a CDP-therapeutic agent antibody conjugate, e.g. , a CDP-therapeutic agent antibody conjugate described herein. Pharmaceutically acceptable salts of the compounds described herein include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate and undecanoate. Salts derived from appropriate bases include alkali metal (e.g. , sodium), alkaline earth metal (e.g. , magnesium), ammonium and N-(alkyl)₄ ⁺ salts. This disclosure also envisions the quaternization of any basic nitrogen-containing groups of the compounds described herein. Water or oil-soluble or dispersible products may be obtained by such

quaternization.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gailate, aipha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

A composition may include a liquid used for suspending a CDP-therapeutic agent antibody conjugate, which may be any liquid solution compatible with the CDP- therapeutic agent antibody conjugate, which is also suitable to be used in pharmaceutical compositions, such as a pharmaceutically acceptable nontoxic liquid. Suitable suspending liquids including but are not limited to suspending liquids selected from the group consisting of water, aqueous sucrose syrups, corn syrups, sorbitol, polyethylene glycol, propylene glycol, and mixtures thereof.

A composition described herein may also include another component, such as an antioxidant, antibacterial, buffer, bulking agent, chelating agent, an inert gas, a tonicity agent and/or a viscosity agent.

In an embodiment, the CDP-therapeutic agent antibody conjugate is provided in lyophilized form and is reconstituted prior to administration to a subject. The lyophilized CDP-therapeutic agent antibody conjugate can be reconstituted by a diluent solution, such as a salt or saline solution, e.g. , a sodium chloride solution having a pH between 6 and 9, lactated Ringer' s injection solution, or a commercially available diluent, such as PLASMA-LYTE A Injection pH 7.4® (Baxter, Deerfield, IL).

In an embodiment, a lyophilized formulation includes a lyoprotectant or stabilizer to maintain physical and chemical stability by protecting the CDP-therapeutic agent antibody conjugate from damage from crystal formation and the fusion process during freeze-drying. The lyoprotectant or stabilizer can be one or more of polyethylene glycol (PEG), a PEG lipid conjugate (e.g. , PEG-ceramide or D-alpha-tocopheryl polyethylene glycol 1000 succinate), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), polyoxyethylene esters, poloxomers, Tweens, lecithins, saccharides, oligosaccharides, polysaccharides and polyols (e.g. , trehalose, mannitol, sorbitol, lactose, sucrose, glucose and dextran), salts and crown ethers.

In an embodiment, the lyophilized CDP-therapeutic agent antibody conjugate is reconstituted with a mixture of equal parts by volume of Dehydrated Alcohol, USP and a nonionic surfactant, such as a polyoxyethylated castor oil surfactant available from GAF Corporation, Mount Olive, N.J., under the trademark, Cremophor EL. The lyophilized product and vehicle for reconstitution can be packaged separately in appropriately light- protected vials. To minimize the amount of surfactant in the reconstituted solution, only a sufficient amount of the vehicle may be provided to form a solution having a concentration of about 2 mg/mL to about 4 mg/mL of the CDP-therapeutic agent antibody conjugate. Once dissolution of the drug is achieved, the resulting solution is further diluted prior to injection with a suitable parenteral diluent. Such diluents are well known to those of ordinary skill in the art. These diluents are generally available in clinical facilities. It is, however, within the scope of the disclosure to package the subject CDP-therapeutic agent antibody conjugate with a third vial containing sufficient parenteral diluent to prepare the final concentration for administration. A typical diluent is Lactated Ringer's Injection.

The final dilution of the reconstituted CDP-therapeutic agent antibody conjugate may be carried out with other preparations having similar utility, for example, 5%

Dextrose Injection, Lactated Ringer's and Dextrose Injection, Sterile Water for Injection, and the like. However, because of its narrow pH range, pH 6.0 to 7.5, Lactated Ringer's Injection is most typical. Per 100 mL, Lactated Ringer's Injection contains Sodium Chloride USP 0.6 g, Sodium Lactate 0.31 g, Potassium chloride USP 0.03 g and Calcium Chloride2H20 USP 0.02 g. The osmolality is 275 mOsmol/L, which is very close to isotonicity.

The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

Routes of Administration

The pharmaceutical compositions described herein may be administered orally, parenterally (e.g. , via intravenous, subcutaneous, intracutaneous, intramuscular, intraarticular, intraarterial, 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.

Pharmaceutical compositions suitable for parenteral administration comprise one or more CDP-therapeutic agent antibody conjugate (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 CDP-therapeutic agent antibody conjugate 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 CDP-therapeutic agent antibody conjugate 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 non-aqueous 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 CDP-therapeutic agent antibody conjugate, 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 CDP-therapeutic agent antibody conjugate 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 CDP-therapeutic agent antibody conjugate 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 CDP-therapeutic agent antibody conjugate. Such materials include, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate. Compositions of the disclosure, 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 disclosure. Dosages and Dosage Regimens

The CDP-therapeutic agent antibody conjugate 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 disclosure 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 an embodiment, the CDP-therapeutic agent antibody conjugate is administered to a subject at a dosage of, e.g. , about 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.13 mg/kg, 0.15 mg/kg, 0.18 mg/kg, 0.20 mg/kg, 0.23 mg/kg, 0.25 mg/kg, 0.28 mg/kg, 0.30 mg/kg, 0.33 mg/kg, 0.35 mg/kg, 0.38 mg/kg, 0.40 mg/kg, 0.43 mg/kg, 0.45 mg/kg, 0.48 mg/kg, 0.50 mg/kg of the therapeutic agent. Administration can be at regular intervals, such as every 1, 2, 3, 4, or 5 days, or weekly, or every 2, 3, 4, 5, 6, or 7 or 8 weeks.

In an embodiment, the CDP-therapeutic agent antibody conjugate is administered to a subject at a dosage of, e.g. , about 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg of the antibody, e.g. , trastuzumab

(Herceptin). Administration can be at regular intervals, such as every 1, 2, 3, 4, or 5 days, or weekly, or every 2, 3, 4, 5, 6, or 7 or 8 weeks, e.g. , every weekly or every three weeks. The administration, e.g. , intravenous 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. In an embodiment, the CDP-therapeutic agent antibody conjugate is administered as a bolus infusion or intravenous push, e.g. , over a period of 15 minutes, 10 minutes, 5 minutes or less. In an embodiment, the CDP-therapeutic agent antibody conjugate is administered in an amount such the desired dose of the agent is administered. Preferably the dose of the CDP-therapeutic agent antibody conjugate is a dose described herein.

The administration, e.g. , subcutaneous administration, can be administered via injection under the skin. In an embodiment, the CDP-therapeutic agent antibody conjugate is administered in an amount such the desired dose of the agent is

administered. Preferably the dose of the CDP-therapeutic agent antibody conjugate is a dose described herein.

In an 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 receive an infusion once every 1, 2, 3 or 4 weeks until the disorder or a symptom of the disorder are cured, healed, alleviated, relieved, altered, remedied, ameliorated, palliated, improved or affected. Preferably, the dosing schedule is a dosing schedule described herein.

The CDP-therapeutic agent antibody conjugate can be administered as a first line therapy, e.g. , alone or in combination with an additional agent or agents. In other embodiments, a CDP-therapeutic agent antibody conjugate is administered after a subject has developed resistance to, has filed to respond to or has relapsed after a first line therapy. The CDP-therapeutic agent antibody conjugate can be administered in combination with a second agent. Preferably, the CDP-therapeutic agent antibody conjugate is administered in combination with a second agent described herein. Kits

A CDP-therapeutic agent antibody conjugate described herein may be provided in a kit. The kit includes a CDP-therapeutic agent antibody conjugate 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 CDP- therapeutic agent antibody conjugate for the methods described herein.

The informational material of the kits is not limited in its form. In an

embodiment, the informational material can include information about production of the CDP-therapeutic agent antibody conjugate, physical properties of the CDP-therapeutic agent antibody conjugate, concentration, date of expiration, batch or production site information, and so forth. In an embodiment, the informational material relates to methods for administering the CDP-therapeutic agent antibody conjugate.

In an embodiment, the informational material can include instructions to administer a CDP-therapeutic agent antibody conjugate 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 a CDP-therapeutic agent antibody conjugate 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 CDP-therapeutic agent antibody conjugate described herein into a pharmaceutically acceptable composition.

In an embodiment, the kit includes instructions to use the CDP-therapeutic agent antibody conjugate, such as for treatment of a subject. The instructions can include methods for reconstituting or diluting the CDP-therapeutic agent antibody conjugate for use with a particular subject or in combination with a particular agent. The instructions can also include methods for reconstituting or diluting the CDP-therapeutic agent antibody conjugate for use with a particular means of administration, such as by intravenous infusion or subcutaneous administration. Methods of Use

The CDP-therapeutic agent antibody conjugate, e.g. , a CDP-therapeutic agent monoclonal antibody conjugate, e.g. , a CDP-camptothecin trastuzumab (Herceptin) conjugate, e.g. , a CDP-camptothecin trastuzumab conjugate described herein, can be used in a method of treating a disorder, e.g. , a cancer, in a subject, e.g. , a human, the method comprising: administering a composition that comprises a CDP-therapeutic agent antibody conjugate, e.g. , a CDP-therapeutic agent monoclonal antibody conjugate, e.g. , a CDP-camptothecin trastuzumab (Herceptin) conjugate, e.g. , a CDP-camptothecin trastuzumab conjugate described herein, to a subject in an amount effective to treat the disorder, e.g. , cancer, in the subject, to thereby treat the disorder, e.g. , cancer.

In an embodiment, the cancer is HER2 overexpressing breast cancer, e.g. , metastatic HER2 overexpressing breast cancer.

In an embodiment, the cancer is HER2-overexpressing metastaic gastric or gastroesophageal junction adenocarcinoma.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES

The cyclodextrin containing polymer used in the following examples was of the following structure:

wherein the group

Example 1. Synthesis of thiol modified NDC conjugated to maleimide modified herceptin

Step 1: Synthesis of orthopyridyl disulfide protected amine-polyfethylene glycol)- thiol, MW 5,000 Da

Amine-poly(ethylene glycol)-thiol (762 mg, 0.15 mmol) and 2,2'-dipyridyl disulfide (168 mg, 0.76 mmol) were dissolved in 10 mL of methanol containing 2% acetic acid. The reaction mixture was stirred at room temperature overnight. Complete conversion of starting material was confirmed by HPLC and the reaction mixture was precipitated into 150 mL of a 1: 1 mixture of isopropyl alcohol and diethyl ether. The suspension was centrifuged and the solvents were decanted. The residual polymer was washed with three 15 mL portions of diethyl ether. The product was dissolved in dichloromethane, transferred to a sample vial and dried to give a white solid, 750 mg. In the following Steps 2a, 2b and 2c, cyclodextrin PEG copolymer was reacted with different ratios of camptothecin-glycinate^»TFA (X) and orthopyridyl disulfide protected amine-poly(ethylene glycol)-thiol (product from Step 1) (Y). In Step 2a, 80% of camptothecin-glycinate'TFA (X) and 20% orthopyridyl disulfide protected amine- poly(ethylene glycol)-thiol (product from Step 1) (Y) were employed. In Step 2b, 98% of camptothecin-glycinate'TFA (X) and 2% orthopyridyl disulfide protected amine- poly(ethylene glycol)-thiol (product from Step 1) (Y) were employed. In Step 2c, 99.6% of camptothecin-glycinate'TFA (X) and 0.4% orthopyridyl disulfide protected amine- poly(ethylene glycol)-thiol (product from Step 1) (Y) were employed. Step 2a: Synthesis of QPSS-PEG-co-CPT-Gly NDC (X=80%, v= 20%)

Camptothecin-glycinate'TFA (50 mg, 0.10 mmol), orthopyridyl disulfide protected amine-poly(ethylene glycol)-thiol (128 mg, 0.03 mmol), and cyclodextrin PEG copolymer (275 mg, 0.06 mmol monomer equivalent) were dissolved in dry DMF (2.75 mL). Hunig's base (44 mg, 0.34 mmol) followed by HATU (43 mg, 0.11 mmol) were added at room temperature and the reaction stirred for 40 minutes. The reaction mixture was precipitated into IPA (100 mL) and stirred for 10 min. The solvent was decanted and acetone (50 mL) was charged and stirred for 15 min. The acetone was decanted and a second portion was added for an additional 15 min. The solvent was decanted again and the remaining polymer dissolved in water (100 mL) at pH 3. The NDC was purified by tangential flow filtration (30k MWCO) with 750 mL of water at pH 3. The retentate was concentrated to a final volume of 17 mL.

Step 2b: Synthesis of OPSS-PEG-co-CPT-Gly NDC (X=98%, v= 2%)

Camptothecin-glycinate'TFA (61.5 mg, 0.12 mmol), orthopyridyl disulfide protected amine-poly(ethylene glycol)-thiol (11.4 mg, 0.002 mmol), and cyclodextrin PEG copolymer (275 mg, 0.06 mmol monomer equivalent) were dissolved in dry DMF (2.75 mL). Hunig's base (44 mg, 0.34 mmol) followed by HATU (43 mg, 0.11 mmol) were added at room temperature and the reaction stirred for 40 minutes. The reaction mixture was precipitated into IPA (100 mL) and stirred for 10 min. The solvent was decanted and acetone (50 mL) was charged and stirred for 15 min. The acetone was decanted and a second portion was added for an additional 15 min. The solvent was decanted again and the remaining polymer dissolved in water (100 mL) at pH 3. The NDC was purified by tangential flow filtration (30k MWCO) with 750 mL of water at pH 3. The retentate was concentrated to a final volume of 15 mL. Step 2c: Synthesis of OPSS-PEG-co-CPT-Gly NDC (X=99.6%, v= 0.4%)

Camptothecin-glycinate'TFA (62.7 mg, 0.12 mmol), orthopyridyl disulfide protected amine-poly(ethylene glycol)-thiol (2.6 mg, 0.0005 mmol), and cyclodextrin PEG copolymer (275 mg, 0.06 mmol monomer equivalent) were dissolved in dry DMF (2.75 mL). Hunig's base (44 mg, 0.34 mmol) followed by HATU (43 mg, 0.11 mmol) were added at room temperature and the reaction stirred for 40 minutes. The reaction mixture was precipitated into IPA (100 mL) and stirred for 10 min. The solvent was decanted and acetone (50 mL) was charged and stirred for 15 min. The acetone was decanted and a second portion was added for an additional 15 min. The solvent was decanted again and the remaining polymer dissolved in water (100 mL) at pH 3. The NDC was purified by tangential flow filtration (30k MWCO) with 750 mL of water at pH 3. The retentate was concentrated to a final volume of 12 mL.

Table 1. PEG Thiol Modified NDCs Reaction Conditions and Nanoparticle Properties

Lot 1, high PEG ! Lot 2, medium PEG ! Lot 3, low PEG

CPT:PEG feed 80:20 98:2 99.6:0.4

TS 15.6 mg/ml 12.75 mg/ml 15.13 ! mg/ml

CPT 0.99 mg/ml 1.3015 mg/ml I 1.52928 ! mg/ml

Pyr 0.077733 mg/ml ! 0.010571 mg/ml ! 0.005093 ! mg/ml

PEG-Pyr 3.02 mg/ml ! 0.411004 mg/ml ! 0.198004! mg/ml

CPX1175 11.5 mg/ml 11.0 mg/ml 13.4 ! mg/ml

CPT /1175 8.6 wt% 11.8 wt% 11.4 ! wt%

CPT /1175 theoretical 12.4 wt% 12.4 wt% 12.4 !wt%

CPT loading 69.0 % 95.1 % 92.0! %

PEG-Pyr/1175 26.2 wt% 3.7 wt% 1.5 ! wt%

PEG-Pyr/1176 theoretical 68 wt% 68 wt% 68! wt%

PEG-Pyr/1175 loading 38.5 % 5.5 % 2.2! %

Total loading 107.5 % 100.6 % 94.2! % size pH 3 41.57 nm 29.99 nm 23.67 ! nm

PDI 0.249 0.27 0.229! size pH 7.4 40.41 nm 30.78 nm 21.8 ! nm

PDI 0.247 0.274 0.224!

• Lot 1 refers to the nanoparticles of Step 2a

· Lot 2 refers to the nanoparticles of Step 2b

• Lot 3 refers to the nanoparticles of Step 2c

In all cases, the PEG thiol modified NDCs formed well loaded nanoparticles with low PDI. A potentially useful nuance to this invention is that increasing amounts of PEG conjugated to the cyclodextrin-PEG polymer backbone increases the NDC particle size. Step 3: Preparation of Maleimide Modified Herceptin

Herceptin antibody drug product was purchased from Myoderm USA. The antibody was dialyzed against IX PBS buffer to remove excipients. The desalted Herceptin was brought to a concentration of 2 mg/mL in PBS and sulfosuccinimidyl 4- [N-maleimidomethyl]cyclohexane-l-carboxylate (sulfo-SMCC) was added in a solution of PBS (4.8 mg/mL). The reaction mixture was stirred at room temperature for 30 minutes and was purified using a 7 kDa MWCO Zeba™ spin column. The reaction mixture was carried out in five molar excess ratios between sulfo-SMCC and Herceptin. A molar excess of 2.6, 6.6, 66, 130, and 330 gave respective maleimide per antibody ratios of 0.9, 1.9, 2.7, 3.5 and 6.4. Maleimide to antibody ratios were calculated by measuring the bulk antibody concentration using the bicinchoninic acid assay (BCA) and the bulk maleimide concentration using a fluorometric maleimide quantification assay kit (Abeam®). In a second variation of maleimide modified Herceptin, a maleimide- poly(ethylene glycol)skDa-NHS ester (Laysan Bio, Inc.) was used rather than sulfo- SMCC. This variation had the potential benefit of extending the maleimide group further from the antibody to allow more NDCs to conjugate without impeding the antibody's ability to bind its target. A maleimide-PEG-Herceptin was made using the same procedure described for using sulfo-SMCC. In this case, a 66 fold molar excess of maleimide-poly(ethylene glycol)5kDa-NHS ester gave an antibody with a maleimide per antibody ratio of 3.7.

Step 5: Disulfide deprotection and conjugation of resulting thiol modified NDC to maleimide modified herceptin A sample of OPSS-PEG-co-CPT-Gly NDC was treated with 1% by volume of

0.5M TCEP solution to liberate free sulfhydryls at the PEG chain end. This solution was purified from excess TCEP using a 7 kDa MWCO Zeba™ spin column. The NDC solution was diluted to an appropriate concentration and added to a stirring solution of maleimide modified antibody at 1 mg/mL. A respresentative schemative of the reaction of a CDP-camptothecin conjugate modified with a PEG thiol (sulfhydryl) linker and a maleimide modified trastuzumab (Herceptin) antibody to prepare nanoparticles comprising CDP-camptothecin trastuzumab (Herceptin) conjugates is depicted in FIG. 3. The reaction was stirred at room temperature for lh and then quenched with a 1M solution of cysteine^»HCl. The drug to antibody ratio (DAR) can be modulated by changing the stoichiometry between the sulfhydryl modified NDC and the maleimide modified Herceptin.

Step 5a: Conjugation Reaction #1 The NDC product from Step 2a (2.5 mL) was reduced with 25 uL of 0.5M TCEP at room temperature for 30 minutes. A portion of the reaction mixture (2 mL) was loaded onto a 7 kDa MWCO Zeba™ spin column and spun for 2 minutes at 1000 RPM. The reduced NDC solution was used immediately in conjugation reactions with maleimide modified Herceptin. Maleimide Herceptin (2.7 mal/antibody) was divided into 3

Eppendorf tubes in 1 mL aliquots. Reduced antibody was added to the tubes in a 1: 1, 2: 1, and 4: 1 ratio. The ratio of NDC to antibody was estimated using the concentration of camptothecin in the NDC solution, the percent loading of camptothecin per polymer strand, and the approximate number of polymer strands per NDC.

Exemplary Calculations:

Reactions were stirred at room temperature for 1 hour and then quenched with 10 uL of a 1M solution of cysteine^»HCl to quench the unreacted maleimide groups. The size of the thiol modified NDC starting material was 40.4 nm. After conjugation to the maleimide modified Herceptin, the particle sizes increased to 52.7, 63.4 and 71.9 nm for RXN 1 (1: 1 ratio NDC to antibody), RXN 2 (2: 1 NDC to antibody), and RXN 3 (4: 1 NDC to antibody), respectively. ANDCs (NDC-Antibody conjugate) release of camptothecin under physiological conditions was consistent with other glycinate ester linked drug delivery systems (FIG. 4).

Step 5b: Conjugation Reaction #2

The NDC product from Step 2b (6 mL) was reduced and conjugated to 3 different batches of maleimide-Herceptin (1.36 Mal/Ab, 1.45 Mal/Ab, 1.57 Mal/Ab) and one batch of maleimide-PEG-Herceptin (3.67 Mal/Ab). Conjugations were carried out in 0.2: 1, 1: 1, 2: 1, and 4: 1 NDC to Ab ratios. Samples were synthesized according to Conjugation Reaction #1. Samples are prepared and used in an ELISA binding assay to determine if maleimide modified Herceptin and Herceptin ANDCs are still effective at binding Her2. The reaction matrix and corresponding analytical data are summarized in Table 2 and Table 3.

Table 2. NDC-Herceptin Conjugates (ANDCs)

Table 3. Analytical Characterization of NDC-Herceptin Conjugates (ANDCs)

These data support that the ANDC construct was formed. In the cases of la-4a, a sub- stoichiometric amount of thiol modified NDC was added to the maleimide modified Herceptin. This resulted in excess unreacted maleimide modified Herceptin to be present. As more thiol modified NDC was added, the unreacted Herceptin peak decreased until it disappeared completely.

Example 2. ELISA binding assay of NDC-Herceptin Conjugates (ANDCs) Samples are prepared and used in an ELISA binding assay to determine if maleimide modified Herceptin and Herceptin ANDCs are still effective at binding Her2. Samples of CDP-camptothecin Herceptin conjugate particles are diluted (60μΙ_^ of sample to 360μί with water and PBS concentrate so the final solution is IX PBS). The samples are dispensed (60μί to 5 individual centrifuge tubes and hold the remainder in a 6th tube as stock solution (SS)). Sealed tubes are held at 37deg in a covered water bath for T=0, 16, 24, 48 and 72 hours.

Example 3. Synthesis of Cleavable OPSS-PEG-co-CPT-Gly NDC (X=94%, y= 6 % ) (Compound 4)

Step 1: Synthesis of hydroxyl terminated poly(ethylene glycol) orthopyridyl disulfide, MW 5,000 Da (Compound 1) Orthopyridyl disulfide-poly(ethylene glycol)- succinimidyl valerate, average MW

5,000 Da (500 mg, 0.1 mmol, Laysan Bio Inc) and 2-(2-aminoethoxy)ethanol (105 mg, 1.0 mmol) were dissolved in dichloromethane (2 mL). The solution stirred at room temperature overnight and was then precipitated into rapidly stirring diethyl ether (100 mL). The precipitate was isolated by centrifugation as a white solid (477 mg, 93% yield). Step 2: Synthesis of Boc-glvcine ester terminated polv(ethylene glycol) orthopyridyl disulfide, MW 5,000 Da (Compound 2)

Compound 1 (300 mg, 0.06 mmol), Boc-Glycine (52 mg, 0.3 mmol), and dimethylamino pyridine (7 mg, 0.06 mmol) where dissolved in a dichloromethane (1.5 mL) and dimethylformamide (0.2 mL). l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (57 mg, 0.3 mmol) was added and the solution stirred at room temperature overnight. The solution was precipitated into rapidly stirring diethyl ether (100 mL). The precipitate was isolated by centrifugation as a white solid (250 mg, 80% yield).

Step 3: Synthesis of Glycine ester terminated polv(ethylene glycol) orthopyridyl disulfide, MW 5,000 Da (Compound 3) Compound 2 (60 mg, 0.01 mmol) was dissolved in a 1: 1 vol/vol solution of dichloromethane and trifluoroacetic acid (0.5 mL). The solution stirred at room temperature overnight and was then precipitated into rapidly stirring diethyl ether (100 mL). The precipitate was isolated by centrifugation as a white solid (50 mg, 83% yield).

Step 5: Synthesis of cleavable OPSS-PEG-co-CPT-Gly NDC (X=94%, v= 6%) (Compound 4)

Camptothecin-glycinate'TFA (61 mg, 0.12 mmol), compound 3 (40 mg, 0.008 mmol), and cyclodextrin PEG copolymer (275 mg, 0.06 mmol monomer equivalent) were dissolved in dry DMF (2.75 mL). Hunig's base (44 mg, 0.34 mmol) followed by HATU (43 mg, 0.11 mmol) were added at room temperature and the reaction stirred for 40 minutes. The reaction mixture was precipitated into IPA (100 mL) and stirred for 10 min. The solvent was decanted and acetone (50 mL) was charged and stirred for 15 min. The acetone was decanted and a second portion was added for an additional 15 min. The solvent was decanted again and the remaining polymer dissolved in water (100 mL) at pH 3. The NDC was purified by tangential flow filtration (30k MWCO) with 750 mL of water at pH 3. The retentate was concentrated to a final volume of 17 mL.

Table 4. NDC Characterization Data

Example 4. Second Synthetic Approach to Cleavable NDCs

N. ¾ 0 Compound 5

Χ ,ΝΗΒοο

Compound 6

Step 1: Synthesis of Boc-glycine ester terminated poly(ethylene glycol) orthopyridyl disulfide, MW 5,000 Da (Compound 5)

Orthopyridyl disulfide protected amine-poly(ethylene glycol)-thiol (200 mg, 0.04 mmol) and 2-(((tert-butoxycarbonyl)glycyl)oxy)ethyl IH-imidazole-l-carboxylate (63 mg, 0.2 mmol) were dissolved in dichloromethane (1 mL) and diisproylethyl amine (30 μί, 0.2 mmol). The solution stirred at room temperature overnight and was then precipitated into rapidly stirring diethyl ether (100 mL). The precipitate was isolated by centrifugation as a white solid (170 mg, 85% yield).

Step 2: Synthesis of Glycine ester terminated poly(ethylene glycol) orthopyridyl disulfide, MW 5,000 Da (Compound 6) Compound 6 (137 mg, 0.027 mmol) was dissolved in a 1: 1 vol/vol solution of dichloromethane and trifluoroacetic acid (1 mL). The solution stirred at room temperature overnight and was then precipitated into rapidly stirring diethyl ether (100 mL). The precipitate was isolated by centrifugation as a white solid (130 mg, 95% yield). Step 3: Synthesis of cleavable OPSS-PEG-co-CPT-Gly NDC (X=80%, v= 20%)

(Compound 7)

Camptothecin-glycinate'TFA (50 mg, 0.10 mmol), compound 6 (128 mg, 0.026 mmol), and cyclodextrin PEG copolymer (275 mg, 0.06 mmol monomer equivalent) were dissolved in dry DMF (2.75 mL). Hunig's base (44 mg, 0.34 mmol) followed by HATU (43 mg, 0.11 mmol) were added at room temperature and the reaction stirred for 40 minutes. The reaction mixture was precipitated into IPA (100 mL) and stirred for 10 min. The solvent was decanted and acetone (50 mL) was charged and stirred for 15 min. The acetone was decanted and a second portion was added for an additional 15 min. The solvent was decanted again and the remaining polymer dissolved in water (100 mL) at pH 3. The NDC was purified by tangential flow filtration (30k MWCO) with 750 mL of water at pH 3. The retentate was concentrated to a final volume of 17 mL.

Table 5. NDC Characterization Data

21.04 mg/ml

1.0314 mg/ml

! Pyr I 0.127368 mg/ml

! PEG-Pyr I 4.95193 mg/ml

I CPX1175 I 15.05667 mg/ml

i CPT /1175 I 6.85012 wt%

! CPT /1175 theoretica l 12.4 wt%

! CPT loading I 55.2429 %

PEG-Pyr/1175 ! 32.88861 wt%

! PEG-Pyr/1175 theoretica l 68 wt%

PEG-Pyr/1175 loading ! 48.36561 %

Total loading I 103.6085 %

i size pH 3 41.46 nm

0.254

i size pH 7.4 39.66 nm

0.237

Step 4: Preparation of Maleimide Modified Herceptin

A solution of sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l- carboxylate (200 μί, 4.8 mg/mL) was added to a solution of Herceptin in PBS (2.5 mL at 2 mg/mL). The reaction stirred at room temperature for 30 minutes and was purified using a 7 kDa MWCO Zeba™ spin column.

Step 5: Disulfide reduction of cleavable OPSS-PEG-co-CPT-Gly NDC

Compound 4 (2 mL) was treated tris(2-carboxyethyl)phosphine (25 uL) and stirred at room temperature for 30 minutes. The reaction was purified using a 7 kDa MWCO Zeba™ spin column.

Step 6: Cleavable ANDC conjugation reaction

Maleimide modified antibody (250 μί) was added to a vial containing freshly reduced cleavable HS-PEG-co-CPT-Gly NDC (250 μί). The reaction was allowed to stir at room temperature for 15 minutes. A second batch was prepared using 500 μΐ_^ of each component. The two batches were pooled to give 1.5 mL of crude ANDC solution.

The diameter of the purified ANDC constructs measured by DLS was 39.28 nm, PDI: 0.271. The drug to antibody molar ratio (DAR) was determined to be 287. The ANDC binding to its target, Her2, was determined to be 83% compared to native Herceptin (see FIGs. 5A-5E and 6) using the HPLC conditions shown in Table 7.

Example 5. Analytical Methods for ANDCs

Method 1. ANDC particle size determination:

1) Dilute ANDC suspension with PBS to between l-5mg/mL total solids.

2) Aliquot to an appropriate cuvette and measure using manufacturer's automatic settings for DLS instrument.

3) Report size and PDI.

DLS settings (Malvern Zetasizer Nano):

Seek Optimum Position

Automatic

Method 2. ANDC total solids method for the NDC key intermediary

A gravimetric method was used to determine the total solids for the total drug-polymer conjugate which was linked with antibody to form an ANDC.

1) Weigh an appropriate weighing container that has a weight less than 100 times the expected dried mass of the CDP drug substance (Wl).

2) Transfer an adequate volume Drug Substance Solution to the weighing vessel such that the expected remaining solids is greater than 1% of the vessel weight.

3) Transfer the vessel to a drying oven set at 100°C and dry the sample until no

liquid remains (usually overnight).

4) After drying, allow container to equilibrate to ambient temperature.

5) Accurately weigh the container and contents (W2).

6) Calaculate Total solids (mg) = weight after drying in mg (W2) - tare weight in mg (Wl).

Method 3. Camptothecin (CPT) assay for CPT concentration in NDCs prepared for ANDC formulations with CPT.

Camptothecin (CPT) concentration in the NDC formulations with camptothecin API, following base digestion, was determined using HPLC with UV detection and external camptothecin reference material in 50:50 DMSO/water and 2% TFA. This method was used for analyzing released CPT from CPT-NDC with PEG-PyrSH in varying amounts covalently attached to NDC. This method separated the digested CPT from the NDC complex using a C18 5μιη 300 A column with a water/ ACN (0.1%TFA) solvent system and monitoring at X^360nm. ΙΟμΙ_^ of sample was diluted with 30uL water and lOuL 2NM NaOH. The digestion was allowed to sit at room temperature for 5 minutes, and then diluted to 200μί with 50ul water and lOOul 2% TFA in DMSO. The sample concentration was calculated using standard response factor.

HPLC conditions:

Method 4. Determination of PEG-PyrSH concentration in Cerulean NDC key

intermediary for ANDC formulations.

PEG-PyrSH concentration in NDC formulations with camptothecin API, following tris(2- carboxyethyl)phosphine (TCEP) reduction, was determined using HPLC with UV detection and 2,2' -dipyridyldisulfide (DPS) reference material. This method was used for the analysis of released pyridylthiol from PEG-PyrSH covalently attached to NDC using reductive release. This method was used to separate the free PyrSH from the NDC complex using a C18 column with a water/ ACN (0.1%TFA) solvent system and UV monitoring at

The standard was prepared by dissolving 0.02mg/mL 2,2'- dipyridyldisulfide in 5% TCEP/water and allowing the mixture to sit at room temperature for at least 5 minutes (Note: 2,2' -dipyridyldisulfide reduces to 2(PyrSH) in

5%TCEP/water). ΙΟμί of sample was diluted to 200μί with 5% TCEP/water for a dilution factor (DF) of 20, and the mixture was allowed to sit at room temperature for 5 minutes. AUC of the PyrSH peak at X^270nm was measured, and the concentration was determined using reference material response factor. Area Released Sample*DF(20)/RF (PyrSH) / mg/mL= Released PyrSH mg/mL. The concentration of the PEG-PyrSH, the average molecular weight (Mw), and the polydispersity (PDI) of the PEG must be known to calculate the average molecular number (PDI=Mn/Mw). Therefore, the concentration of PEG-PyrSH is {Cone PyrSH_(mg_/mL)*{PEG(Mn)+PyrSH₍Mw)}/PyrSH₍Mw) = Cone PEG- PyrSH mg/mL}. Report cone PEG-PyrSH mg/mL

HPLC conditions

Method 5. Determination of camptothecin (CPT) and PEG-PyrSH loading in the NDC formulations with camptothecin API and PEG-PyrSH functional groups as key intermediary for ANDC formulations.

This method was used to determine CPT and PEG-PyrSH loading vs. theoretical values for the bi-functional PEG-cyclodextrin polymer conjugate containing those two entities using concentration data from HPLC analyses and total solids value from gravimetric analysis.

1) Determine the concentration of residual polymer (RP) minus the functional groups [(total solids mg/mL) - (PEG-PyrSH mg/mL) - (CPT mg/mL)= RP mg/mL] . Determine wt% of CPT vs RP [(CPT mg/mL)/(RP mg/mL)* 100 = CPT wt% vs RP].

2) Determine %Loading of CPT wt% vs theoretical wt% [(CPT wt% vs RP)/(CPT theoretical wt% vs RP)* 100 = CPT %loading].

3) Determine wt% of PEG-PyrSH vs RP [(PEG-PyrSH mg/mL)/(RP mg/mL)* 100 = PEG-PyrSH wt% vs RP].

4) Determine %Loading of PEG-PyrSH wt% vs theoretical wt% [(PEG-PyrSH wt% vs RP)/(PEG-PyrSH theoretical wt% vs RP)* 100 = PEG-PyrSH %loading].

5) Determine total loading efficiency% [(CPT %loading) + (PEG-PyrSH %loading) = total loading efficiency] .

6) Report CPT %loading, PEG-PyrSH loading, and total loading efficiency.

Representative determination

Residual polymer concentration 11.0 i mg/ml i

CPT / RP 11.8 j wt%

j CPT / RP theoretical % loading 12.4 j wt%

CPT % loading 95.1 j % j

PEG-PyrSH wt% 3.7 j wt%

PEG-PyrSH / RP theoretical 68 I wt%

% loading

PEG-PyrSH % loading 5.5 1 ^% i

Total loading efficiency 100.6 1 ^% i

Method 6. ANDC in-vitro release method for camptothecin release in PBS at 37C: In- vitro determination of Antibody-Nanoparticle-Drug-Conjugates concentration of released vs total camptothecin in the ANDC formulations held in PBS buffer at 37C using HPLC and UV detection with camptothecin external standard

This method was used for analysis of in-vitro released camptothecin vs total

camptothecin (released and conjugated to ANDC). This method was used to separate the free camptothecin from the conjugated camptothecin using a 300A C4 column with a water/ ACN (0.1%TFA) solvent system following protein precipitation.

1) Dissolve camptothecin 50% DMSO at 0.025mg/mL for external standard.

2) Dilute 60μί of sample to 360μί with water and PBS concentrate so the final solution is IX PBS.

3) Dispense

to 5 individual centrifuge tubes and hold the remainder in a 6^th tube as stock solution (SS).

4) Hold sealed tubes at 37deg in a covered water bath for T=0, 16, 24, 48 and 72 hours.

5) For each time point analysis inject a sample for released API.

6) Subtract the concentration of free API from the total API to determine the amount of conjugated API.

7) Divide the released API concentration from the total API concentration to

determine the % release for each time point. 8) Stock solution for total API analysis was digested by diluting 40 uL SS with 20μί 2N NaOH, vortexed and allowed to digest at RT for 15 minutes. Post digestion the samples were quenched and precipitated with ΙΟΟμί of 2% TFA/ACN, centrifuged and the supernatant analyzed with a total dilution factor of 24.

9) Samples for analysis were diluted and precipitated with 180μί 2% TFA/ACN on ice, centrifuged and the supernatant analyzed with a total dilution factor of 24.

10) Report % released CPT of total CPT.

HPLC conditions

C4, 5μιη, 300A, 4.6 X 50mm

A: 0.1% TFA in water

B: 0.1% TFA in ACN

1.0 mL/min

40°C

10 iL

UV/Vis, 360 nm

Method 7. ANDC assay for unconjugated trastuzumab concentration in ANDC formulations: Determination of unconiugated antibody (AB) concentration in the ANDC formulations with camptothecin API, using HPLC with UV detection and external reference material

This method was used for analysis of unconjugated AB from ANDC with in varying amounts of AB covalently attached to NDC. This method was used to separate the reacted AB from the ANDC complex using an Sepax PS-DVB Proteomix RP-1000 column with a water/ ACN (0.1%TFA) solvent system and monitoring

1) Prepare antibody standard at lmg.mL in PBS, determine response factor. Note:

Unconjugated AB has been modified and has a slightly longer retention time than does the unmodified reference material. Specificity determination to be made by spectral identification max = 280nm whereas ANDC has max = 360nm.

2) Calculate concentration of free antibody (AB) [Area Released Sample*DF(20)/RF (AB) / mg/mL= Unconjugated trastuzumab Cone mg/mL].

3) Report concentration unconjugated trastuzumab mg/mL.

HPLC conditions

15 90 1.0

16 35 1.0

19 35 1.0

Method 8. Purification of cleavable and non-cleavable ANDCs

Purification of the ANDC included removal of unconjugated CPT-NDC, which necessitated analysis of the Antibody component of the ANDC. The camptothecin (CPT) absorption obscured the absorption of antibody for HPLC traces at 280 nm, therefore, the CPT signal was subtracted. Camptothecin has equal absorption at 280nm and 304nm subtracting the signal of 304nm from that of 280nm the contribution of camptothecin absorption at 280nm is eliminated. Antibody absorption at 304nm is minor therefore the resultant area for ANDC peaks at 280nm-(304nm) was equivalent to the antibody contribution. This response was used to determine the proper sample collection times.

Instrumental Conditions:

Method 9. Determination of Antibody Drug Ratios (PAR) for ANDCs: This method is for the analysis of antibody and camptothecin molar ratio in ANDC.

Standard preparation: preparation of camptothecin NDC was at a camptothecin concentration similar to that expected for the ANDC in PBS, and preparation of

Herceptin Ab was at a concentration similar to that expected for the ANDC in PBS. Antibody concentration analysis for ANDC where the camptothecin absorption obscures the absorption of antibody for HPLC traces at 280nm. However, because camptothecin has equal absorption at 280nm and 304nm subtracting the signal of 304nm from that of 280nm the contribution of camptothecin absorption at 280 nm was eliminated. Antibody absorption at 304nm was negligible so the resultant area for ANDC peaks at 280nm- (304nm) was equivalent to the antibody contribution. This response was used to calculate the moles of antibody in the ANDC compared to the Ab response factor from Herceptin standard material. Camptothecin concentration analysis for the ANDC was performed using the Antibody peak overlaid on the trace for 360nm. The tail was manually integrated for the 360nm (CPT) peak dropping a perpendicular so the integrated area matched the peak base from the antibody trace. This response was used to calculate the moles of CPT in the ANDC using the camptothecin response factor from the CPT- NDC reference material. Free, unconjugated NDC was determined by subtracting the area manually integrated for CPT from the total area for CPT at 360nm. That difference was CPT concentration in unconjugated NDC.

Instrumental Conditions:

Method 10. Binding determination of antibody, modified antibody, and ANDCs to HER2 antigen in solution

This method was for analysis of antibody, modified antibody, and ANDC binding to antigen in lieu of ELISA due to ANDC steric issues.

1) Stock Solution (SS): dissolve HER2 (90kDa label claim) @ 0.34mg/mL (3.3nM).

2) Dilute sample to 0.5mg/mL antibody concentration equimolar to the HER2

(3.3nM) in PBS. If the antibody concentration is less than 0.5mg/mL dilute standard to maintain equimolar ratio.

3) Perform analysis doing the following: Dilute an aliquot of standard SS 50% with

PBS (std 1), Dilute an aliquot of sample 50% with PBS (control sample), Mix equal parts of standard SS and sample (binding sample) at room temp for 5min in HPLC vial. 4) The mixed sample should develop a new peak that is either resolved from the dilute sample (antibody analysis) or from standard 1 (ANDC analysis). For ANDC the camptothecin absorption will obscure the absorption of antibody for HPLC traces at 280nm. However, because camptothecin has equal absorption at 280nm and 304nm subtracting the signal of 304nm from that of 280nm, the contribution of camptothecin absorption at 280nm is eliminated.

5) Antibody absorption at 304nm was negligible so the resultant area for ANDC peaks at 280nm-(304nm) was equivalent to the antibody contribution only.

6) Concurrently, HER2 was dye labeled with dylite 647 and binding evaluated by area % for the newly formed peak as compared to the total area at 650nm.

Instrumental Conditions:

Sepax Zenix SEC-300 4.6X300mm

PN: 213300-4630

A: PBS

0.35 mL/min

40°C

ΙΟ μΙ,

UV/Vis, 280 nm 360nm 304nm

Flow

Time (min) %A

(mL/min)

0 100 0.35

15 100 0.35

Example 6. ANDC Cell Penetration Study Cell Culture:

SK-BR-3 (HER2+) and HCC1806 (HER2-) cells were purchased from ATCC. Cells were routinely passaged in suggested media from supplier and maintained at 37°C in 5% C0₂ and split at 90% confluence. Cell Viability Assay:

Cell proliferation and cytotoxicity assays were performed using CellTiter96 Aqueous one solution (Promega). The viability assay was performed according to the manufactures' instructions. Briefly, SK-BR-3 and HCT116 cells were seeded into 96- well cell culture plates. Individual wells, in triplicate, were treated with CRLX101 or ANDC at concentrations ranging from 10 μΜ to 0.0015 μΜ. At 72 hours, 20 μΐ of CellTiter96 was applied to each well and allowed to incubate for 2 hours at 37°C.

Following the incubation, the absorbance of each well was captured using a SpectraMax platereader. Cell viability was calculated by subtracting the background signal obtained from blank wells containing medium only (FIG. 7). Percent of cell viability was calculated using the equation mean of treatment / mean of control * 100. The IC₅₀ value was calculated using GraphPad Prism.

Visualization of ANDC:

SK-BR-3 and HCC1806 cells were grown on glass coverslips in 24-well plates at a sub-confluent density. Cells were exposed to rhodamine-labelled ANDCs for 2-6 hours at 37°. To terminate uptake, cells were transferred to ice and washed with phosphate- buffered saline (PBS) prior to fixation by 4% (wt/vol) paraformaldehyde in PBS.

Following fixation, cells were washed with PBS and nuclei visualization was completed by labeling with DRAQ5 for 5 minutes. Coverslips were subsequently mounted using Aqua-Poly/Mount (Polysciences). Samples were imaged using a Leica SP5 inverted confocal scanning microscope using a Zeiss 63x Plan Apo oil immersion objective (FIG. 8). The excitation of fluorescence was generated with a white light laser and

corresponding emission wavelengths were collected by sequential scanning and capturing of individual emission wavelengths. Micrographs were collected using the associated Leica application software and analyzed using ImageJ.

Synthesis of Rhodamine-Labelled ANDCs:

Example 7. ANDC Pharmacokinetic (PK) Studies Study 1. PK #1

A PK study was performed in female Ncr nude mice with ANDC and CRLXlOl, both a single treatment IV at 8 mg/kg, the dose volume of 10 mL/kg (FIG. 9). Blood was collected into EDTA tubes and centrifuged to separate the plasma. Samples were collected at 1 hr, 24 hrs and 72 hrs and frozen on dry ice for storage at -80°C until analyses, 3 mice per time point.

Study 2. PK #2

A PK study was performed comparing CRLX101 (8 mg/kg) (FIG. 10A) and

ANDC (8 mg/kg CPT+ 6.5 mg/kg Herceptin combined) (FIG. 10B) as single IV treatments in HCT-116 tumor-bearing female Ncr nude mice. HCT-116 tumors were implanted as 2.5xl0⁶ cells suspended in DMEM medium and injected in a 100 volume above the mammary fat pad. The treatments started on Day 11 post-tumor implantation, when the mean tumor volume was 166 mm .

Blood and tumor samples were collected at 0.5 hr, 2 hrs, 7 hrs, 24 hrs, 48 hrs, 72 hrs, 120 hrs and 168 hrs. Blood was collected into EDTA tubes and centrifuged to separate the plasma. Plasma and tumor samples were frozen on dry ice for storage at - 80°C until analyses. Three mice per time point.

At the same dose of 8 mg/kg, the ANDC was better tolerated than the CRLX101

(FIG. 11). The ANDC group lost a maximum of 4% mean body weight (Day 2, i.e., 1 day after the treatment), whereas the CRLX101 group lost a maximum of 17% mean body weight loss (Day 6).

Study 3. PK #3

A PK study was performed to compare CRLX101 (8 mg/kg) (FIG. 12A) and

ANDC (8 mg/kg CPT + 6.5 mg/kg Herceptin combined) (FIG. 12B) as single IV treatments in SK-BR-3 tumor-bearing female Ncr nude mice. SK-BR-3 tumors were implanted as 4xl0⁶ cells suspended in DMEM medium and injected in a 100 volume above the mammary fat pad. The treatments started on Day 9 post-tumor implantation, when the mean tumor volume was 258 mm .

Blood and tumor samples were collected at 0.5 hr, 2 hrs, 7 hrs, 24 hrs, 48 hrs, 72 hrs, 120 hrs and 168 hrs, with an additional 336 hr time point for ANDC. Blood was collected into EDTA tubes and centrifuged to separate the plasma. Plasma and tumor samples were frozen on dry ice for storage at -80 °C until analyses. Three mice per time point.

At the dose of 8 mg/kg, the ANDC group lost a maximum of 10% mean body weight (on Day 6, i.e., 5 days after the treatment), whereas the CRLX101 group lost a maximum of 14% mean body weight loss (on Day 4) (FIG. 13).

Example 8. Bioanalytical Methods for ANDCs

Method 1. Total Camptothecin (CPT) assay in mouse plasma for PK samples from ANDC formulation dosing

This method was used for quantitation of total CPT in mouse plasma PK samples generated from PK Studies conducted using the ANDC formulations of CPT-NDC with PEG-PyrSH in varying amounts covalently attached to NDC. To 60 ul mouse plasma, 10 ul of the internal standard solution (2 ug/ml 7-Ethyl Camptothecin in Acetonitrile) and 10 ul 2N NaOH were added and incubated for 15 minutes at room temperature resulting in the release all bound CPT from the ANDC formulation. After 15 minutes, 10 ul 50% formic acid in water, and 200 ul acetonitrile were added. Samples were vortexed, centrifuged and the clear supernatant analyzed by LCMS. External standard quantitation using the same CPT/ANDC formulation dosed in the PK study in the concentration range of 50-10,000 ng/ml is performed by spiking the formulation used into mouse plasma based on the theoretical amount of CPT in the original CPT/ANDC formulation.

Absolute sensitivity is instrument dependent using acceptance criteria of <20% accuracy and linearity >0.99.

LCMS conditions:

LCMS Conditions: Agilent 641 OB

Method 2. Released Camptothecin (CPT) assay in mouse plasma for PK samples from ANDC formulation dosing

This method is for quantitation of released CPT in mouse plasma PK samples generated from the ANDC formulations of CPT-NDC with PEG-PyrSH in varying amounts covalently attached to NDC. Sample Preparation: to 60 ul mouse plasma, 10 ul of the internal standard solution (2 ug/ml 7-Ethyl Camptothecin in Acetonitrile), 10 ul 50% formic acid in water, and 200 ul acetonitrile were added. Samples were vortexed, centrifuged and the clear supernatant analyzed by LCMS. External standard quantitation using curve concentration range of 1-2500 ng/ml is performed. Absolute sensitivity is instrument dependent with acceptance criteria of <20% accuracy and linearity >0.99.

LCMS conditions:

LCMS Conditions: Agilent 641 OB

Method 3. Total Camptothecin (CPT) assay in mouse tumors for PK samples from ANDC formulation dosing

This method was used for quantitation of total CPT in mouse tumor PK samples generated from PK Studies conducted using the ANDC formulations of CPT-NDC with PEG-PyrSH in varying amounts covalently attached to NDC. Tumors were collected, weighed and transferred into Lysing Matrix D (1.4 mm spheres) bead mill tubes. Tumor processing was performed by adding 0.4 ml 1XPBS containing protease inhibitor (e.g. Roche: complete,cat 116974900) and processed under cool conditions using a MPBio Fast Prep-24 5G bead mill until fully homogenized. After homogenization, 100 ul acetonitrile was added, the tubes were vortexed and the processed bead mill tubes were centrifuged. The clear tumor homogenate supernatant was further processed for LCMS analysis. To 60 ul clear tumor homogenate, 10 ul of the internal standard solution (2 ug/ml 7-Ethyl Camptothecin in Acetonitrile) and 10 ul 2N NaOH were added and mixture was incubated for 15 minutes at room temperature. The NaOH step released all bound CPT from the ANDC formulation. After 15 minutes, 10 ul 50% formic acid in water, and 200 ul acetonitrile were added. Samples were vortexed, centrifuged and the clear supernatant analyzed by LCMS. External standard quantitation using the same CPT/ ANDC formulation dosed in the PK study in the concentration range of 50-10,000 ng/ml based is performed by spiking the formulation used into control tumor homogenate based on the theoretical amount of CPT in the original CPT/ ANDC formulation.

Absolute sensitivity is instrument dependent using acceptance criteria of <20% accuracy and linearity >0.99. Conversion to ng/mg tumor was performed by dividing ng/ml tumor homogenate by the tumor weight/500 ul processing dilution step.

LCMS conditions

LCMS Conditions: Agilent 641 OB

Method 4. Released Camptothecin (CPT) assay in mouse tumors for PK samples from ANDC formulation dosing

This method was used for quantitation of released CPT in mouse tumor PK samples generated from PK Studies conducted using the ANDC formulations of CPT-NDC with PEG-PyrSH in varying amounts covalently attached to NDC. Tumors were collected, weighed and transferred into Lysing Matrix D (1.4 mm spheres) bead mill tubes. Tumor processing was performed by adding 0.4 ml 1XPBS containing protease inhibitor (e.g. Roche: complete, cat 116974900) and processed under cool conditions using a MPBio Fast Prep-24 5G bead mill until fully homogenized. After homogenization, 100 ul acetonitrile was added, tubes were vortexed and the processed bead mill tubes were centrifuged. The clear tumor homogenate supernatant was further processed for LCMS analysis. To 60 ul clear tumor homogenate add 10 ul of the internal standard solution (2 ug/ml 7-Ethyl CPT in Acetonitrile), 10 ul 50% formic acid in water, and 200 ul acetonitrile. Samples were vortexed, centrifuged and the clear supernatant analyzed by LCMS. External standard quantitation using a CPT in the concentration range of 5-2500 ng/ml based is performed by spiking CPT standard into control tumor homogenate. Absolute sensitivity is instrument dependent using acceptance criteria of <20% accuracy and linearity >0.99. Conversion to ng/mg tumor is performed by dividing ng/ml tumor homogenate by the [tumor weight/500 ul] processing dilution. Conversion to ng/mg tumor was performed by dividing ng/ml tumor homogenate by the tumor weight/500 ul processing dilution step.

LCMS conditions

LCMS Conditions: Agilent 641 OB

Example 9. ANDC In Vivo Tolerability and Efficacy Studies Study 1. Tolerability #1

ANDC was administered to non-tumor-bearing female Ncr nude mice IV once. The doses were 4, 6 and 8 mg/kg, the dose volumes were 10 mL/kg, 3 mice per treatment (FIG. 14). CRLX101 was administered at 6 mg/kg and Herceptin at 10 mg/kg and 20 mg/kg. Three days later, body weights and liver, spleen and kidney weights were measured. Clinical observations were carried out daily. The tolerability endpoints monitored included changes in body weight and clinical observations (lethargy, low body temperature, piloerection, etc.). A mouse reached an endpoint for health reasons with a >20% body weight loss or becoming moribund (combination of lethargy, low body temperature, etc.), at which time the mouse was removed from the study.

The highest dose, 8 mg/kg, caused a 5% body weight loss. CRLX101 and

Herceptin at 20 mg/kg caused only a 1% body weight loss in 3 days. There were no changes in tissue weights by any of the treatments. The amount of weight loss due to the ANDC at 8 mg/kg did not suggest poor tolerability in the 3 day time frame. Study 2. Tolerability #2

ANDC was administered to non-tumor-bearing female Ncr nude mice IV once. The doses were 10, 12, 14, 18 and 22 mg/kg, the dose volume was 10 ml/kg, 3 mice per treatment. The highest dose was the maximum possible considering the concentration (1 mg/mL) and the highest volume that could be dosed (22 ml/kg).

Body weights were measured 3 days and 7 days after the treatments (FIG. 15). All doses were well tolerated, with a maximum of 1% weight loss in the 18 mg/kg group 3 days after the treatment, and where 22 mg/kg caused no body weight loss at the time points measured (FIG. 16). Study 3. Efficacy

An efficacy study was performed comparing ANDC, CRLX01 and Herceptin in the SK-BR-3 tumor model in female Ncr nude mice (FIG. 17). The doses of ANDC were 2 and 25 mg/kg. The doses of CRLXlOl were 2 and 6 mg/kg. The dose of Herceptin was 48.4 mg/kg, corresponding to the highest dose of ANDC. A mixture of CRLXlOl + Herceptin was administered, 2 + 4.84 mg/kg, respectively, corresponding to the low dose of ANDC. All treatments were given only one time, and the route was IV with a dose volume of 10 mL/kg for all the treatments except the ANDC at 22 mg/kg. The ANDC treatment of 22 mg/kg had a dose volume of 22 mL/kg.

SK-BR-3 tumors were implanted as 3xl0⁶ cells suspended in DMEM medium and injected in a 100 uL volume above the mammary fat pad. The treatments started on Day 9 post-tumor implantation, when the mean tumor volume was 146 mm .

Tumor volumes were measured using the equation (width*width*length)/2, in mm . Tumor growth delay (TGD) was calculated using the equation (day treatment group mean reaches 1000 mm endpoint) - (day Vehicle control group mean reaches 1000 mm endpoint), in days.

Body weights were recorded when tumor volumes were measured (FIG. 18). Clinical observations were carried out daily. The tolerability endpoints monitored included changes in body weight and clinical observations (lethargy, low body temperature, piloerection, etc.). A mouse reached an endpoint for health reasons with a >20% body weight loss or becoming moribund (combination of lethargy, low body temperature, etc.), at which time the mouse was removed from the study. CRLXIOI at 6 mg/kg caused the greatest body weight loss, a mean of 9% three days later. Herceptin at the high dose of 48.4 mg/kg caused no body weight loss. The high dose of ANDC caused a maximum mean body weight loss of 9%, but this was at 4 times the dose of CRLXIOI. The efficacy of CRLXIOI 6 mg/kg and ANDC 25 mg/kg were similar, with a tumor growth delay of 21 days and 24 days, respectively.

Other embodiments are in the claims.

Claims

CLAIMS We claim:

1. A cyclodextrin-containing polymer (CDP)-therapeutic agent antibody conjugate.

2. The CDP-therapeutic agent antibody conjugate of claim 1, wherein the conjugate is a CDP-therapeutic agent monoclonal antibody conjugate.

3. The CDP-therapeutic agent antibody conjugate of claim 1, wherein the conjugate is a CDP-camptothecin trastuzumab conjugate.

4. The CDP-therapeutic agent antibody conjugate of claim 3, wherein camptothecin and trastuzumab (Herceptin) are attached or conjugated to the CDP via a covalent linkage or via a linker.

5. The CDP-therapeutic agent antibody conjugate of claim 1, wherein the conjugate forms a nanoparticle.

6. The CDP-therapeutic agent antibody conjugate of claim 1, wherein the therapeutic agent is attached to the CDP through a hydroxyl group of the therapeutic agent.

7. The CDP-therapeutic agent antibody conjugate of claim 1, wherein the antibody is attached to the CDP through the nitrogen of the the antibody.

8. The CDP-therapeutic agent antibody conjugate of claim 1, wherein the conjugate comprises a therapeutic agent coupled via a linker to a CDP, and an antibody coupled via a linker, to the same CDP.

9. The CDP-therapeutic agent antibody conjugate of claim 1, wherein the conjugate is the conjugate depicted in FIG. 1.

10. A method of treating a disorder in a subject, the method comprising: administering a composition that comprises a CDP-therapeutic agent antibody conjugate, to a subject in an amount effective to treat the disorder in the subject, to thereby treat the disorder.

11. The method of claim 10, wherein the disorder is a cancer.

12. The method of claim 10, wherein the cancer is HER2 overexpressing breast cancer.

13. The method of claim 12, wherein the cancer is metastatic HER2 overexpressing breast cancer.

14. The method ofclaim 11, wherein the cancer is HER2-overexpressing metastaic gastric or gastroesophageal junction adenocarcinoma.

15. The method of claim 11, wherein the cancer is selected from ovarian cancer, stomach cancer, uterine cancer, uterine serous carcinoma, and non-small cell lung cancer.

16. A method of making a CDP-therapeutic agent antibody conjugate, the method comprising conjugating a plurality of therapeutic agents and a pluraility of antibodies to a CDP.

17. The method of claim 16, comprising conjugating a plurality of antibodies to a CDP-therapeutic agent conjugate modified with a thiol linker.

18. A method of making a nanoparticle comprising a CDP-therapeutic agent antibody conjugate, wherein the CDP-therapeutic agent antibody conjugate is contacted with an antisolvent, thereby producing a nanoparticle comprising a CDP-therapeutic agent antibody conjugate.

19. The method of claim 18, wherein the antisolvent is a solvent in which the

CDP-therapeutic agent antibody conjugate is not soluble.

20. The method of claim 18, wherein the method further comprises filtering the nanoparticle.

21. A method of formulating a CDP-therapeutic agent antibody conjugate or a nanoparticle comprising a CDP-therapeutic agent antibody conjugate into a

pharmaceutical composition, the method comprising combining a CDP-therapeutic agent antibody conjugate or a nanoparticle comprising a CDP-therapeutic agent antibody conjugate with a pharmaceutically acceptable excipient.