HIGH MOLECULAR WEIGHT ZWITTERION-CONTAINING

POLYMERS

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 61/548,146, filed October 17, 201 1 , which is incorporated in its entirety herein for all purposes.

BACKGROUND OF THE INVENTION

[0002] An arms race of sorts is happening right now amongst the big phanna companies who are all trying to deliver 'medically differentiated products'. Biopharmaceuticals are seen as a key vehicle. The belief is that differentiation will come not necessarily through target novelty but through novel drug formats. These formats will be flexible such that resulting drugs can be biology centric rather than format centric. This next wave of

biopharmaceuticals will be modular, multifunctional, and targeted. These drugs wi ll be designed with a view towards understanding the broader disease biology being targeted and applying that knowledge in a multifaceted drug. Antibodies are fantastic drugs, but despite a significant amount of antibody protein engineering they are and will continue to be a rigid and inflexible format.

[0003] The pharma protein engineers are looking to smaller protein formats. There was a wave of progress in the 2006 timeframe with the likes of adnectins (developed by Adnexus and acquired by BMS), avimers (developed by Avidia and acquired by Amgen), diabodies (developed by Domantis and acquired by GSK), Haptogen (acquired by Wyeth), BiTES (developed by icromet), camelids (developed by Ablynx), peptides (developed by the likes of Gryphon Therapeutics and Compugen and many others). But the conversion of these platform technologies into multiple products in the pharma pipeline has been slow to materialize. Over the past two decades, the problems besetting these non-whole antibody formats related to suboptimal affinity, poor stability, low manufacturing yield, as well as tools development. To a large degree, these problems have been or are being solved. But the Achilles heel of these formats remains their inadequate in vivo residence time, an issue which is holding back a wave of important product opportunities. [0004] Whole antibodies have an elimination half life in vivo upwards of 250 hours, corresponding to more than one month of physical residency in the body. This makes them an excellent product format from a dosing point of view. Often they can achieve monthly or less frequent injection. The trajectory is also towards subcutaneous injection in smaller volumes (l mL, 0.8mL, 0.4mL), more stable liquid formulations (versus lyophilized formulations requiring physician reconstitution), storage at higher concentrations (50mg/mL, l OOmg/mL, 200mg/mL) and at higher temperatures (-80 degrees, -20 degrees, 2 - 8 degrees, room temperature).

[0005] Antibodies are a tough act to follow, especially with all of the activity in the broad antibody discovery and development ecosystem. But antibodies do leave much to be desired. They are ungainly, inflexible, large, single-target limited, manufactured in mammalian systems, overall poorly characterized and are central to many different in vivo biologies of which target binding, epithelial FcRn receptor recycling, antibody-dependent cell-mediated cytotoxicity (ADCC), complement dependent cytoxicity (CDC), avidity, higher order architectures, to name just a few.

[0006] The smaller, modular formats can make a major contribution towards the development of safer, targeted, multifunctional, higher efficacy, wel l-characterized and cheaper therapeutics. In addition, there is a similar need to improve the serum residence time and associated physical properties of other types of drug agents such as recombinant proteins and peptides (either native or mutein) and oligonucleotides. The challenge is to devise a technical solution that dramatically increases in vivo residence time for these soluble biopharmaceuticals (the performance issue), does so without forcing compromises in other key parameters such as drug solubility, stability, viscosity, characterizability (the related physical properties issues), and employs an approach that allows predictability across target classes and across the drug development path from early animal studies through to manufacturing scale-up and late-stage human clinical trials (the portfolio planning issue).

[0007] The first attempted class of solutions is biology-based and depends on fusing the protein agents to transferrin, albumin, immunoglobulin gamma (IgG), IgG constant region (IgG-Fc) and/or other serum proteins. But fusing a biology-based serum extension moiety to a functional biologic moiety increases the number and complexity of concurrent biological interactions. These non-target-mediated interactions rarely promote the desired therapeutic action of the drug, but rather more often detract from the desired therapeutic action of the drug in complex and poorly understood ways. The net impact is to undermine predictability, performance, and safety. [0008] The second attempted class of solutions is based broadly on a set of approaches that make use of polymers of different types which are attached to the drug. These polymers function largely on the basis of their ability to bind and structure water. The bound water decreases clearance by the myriad in vivo clearance mechanisms, both passive and active, while also improving physical properties of the polymer-drug conjugate such as solubility, stability, viscosity. This second class of solutions is subcategorized further in two ways: (1 ) by the water binding entity within the polymer, and (2) how the polymer is attached to the drug agent. Relating to (1 ), there are a number of different polymeric water binding moieties in use, such as sugars (carbohydrates), amino acids (hydrophilic protein domains), polyethylene oxide, polyoxazoline, polyvinyl alcohol, polyvinyl pyrrolidone, etc. Relating to (2), the distinction is largely whether the polymer is added to the drug agent by the cellular machinery or whether it is added in a semi-synthetic conjugation step.

[0009] Relating to polymers added to the drug agent by cellular machinery (i.e. not through a semi-synthetic step), one example is the addition of hydrophilic carbohydrate polymers to the surface of a translated protein through a cell-mediated glycosylation process by adding or modifying a glycosylation site at the level of the coding nucleotide sequence (e.g. Aranesp). Another example is the addition of a string of hydrophilic amino acids during protein translation by adding a series of repeating nucleotide units at the level of the open reading frame codons (i.e. Amunix's XTEN platform).

[0010] Relating to the semi-synthetics: The most experience exists with PEGylation in which polymers of polyethylene oxide are functionalized and then conjugated to the drug agent. Also, Fresenius employs a HESylation approach in which long-chain maize starches are functionalized and then conjugated to the drug agent. Also, Serina Therapeutics' employs a hydrophilic polyoxazoline backbone (as opposed to the polyethylene backbone of PEG). Another method termed po!yPEG as described by Haddleton et al employs a polymer backbone capable of radical polymerization and a water binding entity that is either a short string of PEG or a sugar.

[0011] How well do these different technology approaches work in practice? In general, despite significant time and money spent by biopharma and pharma, the general conclusion is that these technologies are not delivering the level of performance benefit needed (especially in vivo residence time) and furthermore are at the flat of the curve in terms of their ability to deliver further progress through additional engineering. The level of improvement required depends on the drug and its biology and the required product profile , but in many cases is as high as three to fourfold. Many companies are working to achieve this level of improvement but in practice the technologies employed are falling short and delivering incremental improvements that are overall niche in their applicability.

[0012] For example:

[0013] PEGylation of an antibody fragment scFv (approximately 22 kDa in size) inhibitor of GM-CSF (Micromet data) with a 40 kDa branched PEG resulted in a murine elimination half life after intravenous injection of 59 hours which is inadequate. To be useful, the murine half-life should be over 150 hours (a 3x improvement) and preferably over 250 hours (a 4x improvement).

[0014] PEGylation of a recombinant interferon alfa of approximately 19.5 kDa with a 40 kDa branched PEG (Pegasys data) results in a murine elimination half life after subcutaneous injection of approximately 50 hours and a human half life in the range of 80 hours. Pegasys is dosed weekly in humans.

[0015] PEGylation of a Fab' antibody fragment of approximately 50 kDa against IL-8 (Genentech data, Leong et al, 2001 ) with a series of PEG polymers of increasing size and architecture. Half lives in rabbits after intravenous injection ranged from 44 hours with a

PEG 20 kDa linear to 105 hours with a PEG 40 kDa branched. This can be correlated against the half-life of the approved product Cimzia which has a Fab' against TNFa conjugated with a 40 kDa branched polymer. Human half life after subcutaneous injection is 3 1 1 hours and is sufficient (as approved by the FDA for rheumatoid arthritis) for monthly subcutaneous dosing. But the properties driven by the PEG moiety (solubility, stability, viscosity) are not sufficient to enable the full dose amount (400mg) to be formulated in a single vial for subcutaneous injection (limit l mL, preferably 0.8mL or less). Rather, Cimzia is formulated preferably as a solid and in two vials for two separate injections each delivering 200mg of product. Furthermore, the PEG reagent is very expensive and constitutes up to twenty percent of the average wholesale price of the drug. Therefore, the Cimzia product is not very competitive in the marketplace versus Humira (anti-TNFa antibody, in a liquid formulation, in a single use syringe, administered by single subcutaneous injection, twice monthly) and even less so versus Simponi (anti-TNFa antibody, in a liquid formulation, in a single use syringe, administered by single subcutaneous injection, once monthly).

[0016] PEGylation of a peptide mimetic (approximately 4kDa) of erythropoietin receptor (Hematide data) with a 40 kDa branched PEG polymer after subcutaneous injection showed between 23 and 3 1 hour half-life in rats (dose dependent). In monkeys the half-life ranged between 15 hours and 60 hours (Fan et al Experimental Hematology, 34, 2006). The projected dose frequency for the molecule is monthly. In this case, the ability to dose monthly with this molecule is enabled by a pharmacodynamic effect whose duration far exceeds the physical half-life and residence time of the drug itself. This property holds for certain potent agonistic drugs but generally does not hold for inhibitors that need to maintain a minimal inhibitory concentration nor does it hold for enzymes nor for high dose agonistic proteins.

[0017] Interferon beta (approximately 20 kDa) was PEGylated with a 40 kDa linear PEG polymer. Avonex. an unPEGylated form, demonstrates a mean terminal half life in monkeys after intravenous injection of 5.5 hours and a half-life of 10 hours after intramuscular injection. Conjugation of a 40 kDa linear PEG polymer can demonstrate a half life of approximately fifteen hours after intravenous administration and thirty hours after subcutaneous administration. Conjugation of a 40 kDa branched PEG polymer can demonstrate a half life of thirty hours after intravenous administration and sixty hours after subcutaneous administration. The projected dose frequency is twice monthly, so the ability to dose twice monthly with this molecule is enabled by a biological or pharmacodynamic effect whose duration exceeds the physical half-life and residence time of the drug itself. For an attractive target product profile to challenge the existing interferon beta products, a once a month dose frequency is required. Alternatively, a polymer conjugate that was dosed twice monthly but with very flat, potentially zero order, kinetics could be ideal. This is obtainable with a highly biocompatible conjugate and dosed at a lower overall dose. Furthermore, interferon beta is an unstable and overall 'difficult' protein to work with and further improvement in solubility and stability is desired.

[0018] PEGylation of recombinant human Factor VIII (upwards of 300 kDa) with a 60 kDa branched PEG polymer has been performed. UnPEGylated FVIII demonstrates a twelve to fourteen hour circulating half-life in humans. It is used acutely in response to a bleeding crisis. It is also being used for prophylaxis via three times weekly intravenous infusions. The murine mean terminal half-life is six hours in the unPEGylated form and eleven hours with a site-directed PEGylated form . In rabbits, with a full-length FVIII protein, an unPEGylated form showed a mean terminal half life of 6.7 hours. With a form PEGylated with a 60kDa branched PEG, the half life increased to twelve hours. The magnitude of increase in half-life of PEG-FVIII correlates to the increase in PEG mass. A key goal, however, is to enable prophylaxis with a once weekly intravenous infusion. The benefit delivered even by the very large (and expensive 60kDa PEG reagent) is not thought to, nor is it likely to, enable the once weekly dose frequency. It needs an additional >2x preferably a 4x versus PEG to be a game changer. Another in vivo performance metric to improve would be to substantially decrease the incidence of neutralizing antibodies generated against the administered FVIII drug. This goal is inadequately met via FVTII-PEG conjugates. Another in vitro performance metric to improve would be to achieve a stable, high concentration formulation sufficient to enable subcutaneous dosing rather than intravenous dosing - this would also require improvement of the in vivo immunogenicity properties as the subcutaneous areas are high in immune- stimulating antigen presenting cells. Recently, a Biogen-generated fusion of FVIII to immunoglobulin Fc fragment was tested and demonstrated to have similar level of in vivo half-life as the PEGylated FVIII but interestingly very poor bioavailability presumably due to FcRn-mediated endothelial cell clearance of the drug. These data have led FVIII drug developers to conclude the existing technologies have "hit a wall".

[0019] The Amunix XTEN technology fuses approximately 850 hydrophilic amino acids (approximately 80kDa in size) to the GLP-1 peptide. This boosts the half-life to sixty hours in a cynomolgus monkey which is slightly inferior to a GLP-1 equivalent conjugated to a 40kDa branched PEG polymer. So a polymer of 2x increased size delivers essentially the same performance benefit. A similar level of benefit was seen with XTEN attached to human growth hormone. In terms of trying to extend further the level of half life benefit, there are a number of challenges. First and foremost, the hydrophilic amino acids used to bind and structure the water are non-optimal in terms of their water binding characteristics. Second, the requisite use of the ribosomal translation machinery to add the polymer limits the architecture to single arm, linear structures which have been shown in many PEGylation examples to be inferior to branched architectures when holding molecular weight constant and increasing the level of branching. Third, a peptide bond used as a polymer backbone is sufficiently unstable such that it will demonstrate a polydispersity, which heterogeneity becomes limiting in practical terms such that the length of the hydrophilic polymer cannot be easily increased to achieve half lives superior to the 40kDa branched PEG (this on top of other complexity related to the use of multiple long repeating units in the encoding plasmid vector which itself becomes limiting). This technology then becomes niche in its application, for example, to allow a peptide formerly made synthetically via chemical synthesis to be made in a cell-based system which has some perceived advantages (as well as new disadvantages) but overall with similar in vivo performance as possible with other technologies, especially in vivo elimination half life.

[0020] rfiEPO is a 30.4 kDa protein with 165 amino acids and 3 N-linked plus 1 O-linked glycosylation site. 40% of the mass is carbohydrate. The carbohydrates are not necessary for activity in vitro, but absolutely necessary for activity in vivo. Aranesp is a form of human erythropoietin modified at the genetic level to contain 5 N-linked oligosaccharide chains versus the native form which contains 3 chains. The additional carbohydrates increase the approximate molecular weight of the glycoprotein from 30kDa to 37kDa. In humans, the change increases mean terminal half life after intravenous injection from 7 hours to 21 hours and after subcutaneous injection from 16 hours to 46 hours, which is an approximate threefold improvement in both cases. Mircera which is a PEGylated form of recombinant human erythropietin demonstrated in vivo half life after subcutaneous injection of approximately 140 hours but in chronic renal disease patients, where patients because of renal filtration of the drug show a more than 2x increase in half life as well as a decreased receptor affinity which decreases mechanistic clearance, meaning the actual physical half life is less than 70 hours and in line with Affymax's Hematide peptidomimetic (PEGylated with a 40kDa branched PEG).

[0021] The HESylation technology employs a semi-synthetic conjugation of a maize derived starch polymer to a drug. Data shows that a 1 OOkDa HESylation polymer is equivalent to a 30kDa linear PEG polymer on erythropoietin in mice (Mircera product equivalent). It is possible to use a bigger polymer, but the approach is fundamentally limited by the nature of the starch water binding. Also, equivalence of a 1 OOkDa polymer to a 30kDa linear PEG (which is itself inferior to a 40kDa branched PEG) shows that there is a long way to go in terms of performance before this can equal a 40kDa branched PEG much less provide a requisite 4x benefit.

[0022] These examples are illustrative of several of the approaches being tried and the overall performance they achieve. In short, these approaches and technologies fall short. For non-antibody scaffolds, they converge and hit the wall at elimination half lives of around 60 to 80 hours in monkey. Although the line varies, it is generally desired to achieve at least 100 hour mean terminal half life in monkeys in order to enable once weekly dosing in humans. And when dose frequency is longer than the half life, this places additional demands on the formulation's solubility, stability, and viscosity. For other types of proteins, such as Factor VIII, the absolute value of the starting half life and thus the requisite target value is lower, but the performance multiple required to get to an attractive target product profile is similar and on the order of 3x to 4x. The question, then, is how to get here?

[0023] First, some more background. Efforts to formulate biologically active agents for delivery must deal with a variety of variables including the route of administration, the biological stability of the active agent and the solubility of the active agents in physiologically compatible media. Choices made in formulating biologically active agents and the selected routes of administration can affect the bioavailability of the active agents. For example, the choice of parenteral administration into the systemic circulation for biologically active proteins and polypeptides avoids the proteolytic environment found in the gastrointestinal tract. However, even where direct administration, such as by injection, of biologically active agents is possible, formulations may be unsatisfactory for a variety of reasons including the generation of an immune response to the administered agent and responses to any excipients including burning and stinging. Even if the active agent is not immunogenic and satisfactory excipients can be employed, biologically active agents can have a limited solubility and short biological half life that can require repeated administration or continuous infusion, which can be painful and/or inconvenient.

[0024] For some biologically active agents, a degree of success has been achieved in developing suitable formulations of functional agents by conjugating the agents to water soluble polymers. The conjugation of biologically active agents to water soluble polymers is generally viewed as providing a variety of benefits for the delivery of biologically active agents, and in particular, proteins and peptides. Among the water soluble polymers employed, polyethylene glycol (PEG) has been most widely conjugated to a variety of biologically active agents including biologically active peptides. A reduction in

immunogenicity or antigenicity, increased half-life, increased solubility, decreased clearance by the kidney and decreased enzymatic degradation have been attributed to conjugates of a variety of water soluble polymers and functional agents, including PEG conjugates. As a result of these attributes, the polymer conjugates of biologically active agents require less frequent dosing and may permit the use of less of the active agent to achieve a therapeutic endpoint. Less frequent dosing reduces the overall number of injections, which can be painful and which require inconvenient visits to healthcare professionals.

[0025] Although some success has been achieved with PEG conjugation, "PEGylation" of biologically active agents remains a challenge. As drug developers progress beyond very potent agonistic proteins such as erythropoietin and the various interferons, the benefits of the PEG hydrophilic polymer are insufficient to drive (i) in vitro the increases in solubility, stability and the decreases in viscosity, and (ii) in vivo the increases in bioavailability, serum and/or tissue half-life and the decreases in immunogenicity that are necessary for a commercially successful product.

[0026] Branched forms of PEG for use in conjugate preparation have been introduced to alleviate some of the difficulties and limitations encountered with the use of long straight PEG polymer chains. Experience to date demonstrates that branched forms of PEG deliver a "curve-shift" in performance benefit versus linear straight PEG polymers chains of same total molecular weight. While branched polymers may overcome some of the limitations associated with conjugates formed with long linear PEG polymers, neither branched nor linear PEG polymer conjugates adequately resolve the issues associated with the use of conjugated functional agents, in particular, inhibitory agents. PEGylation does, though, represent the state of the art in conjugation of hydrophilic polymers to target agents.

PEGylated compound products, among them peginterferon alfa-2a (PEGASYS), pegfilgrastim (Neulasta), pegaptanib (Macugen), and certolizumab pegol (Cimzia), had over $6 billion in annual sales in 2009. Functional ized PEG (suitable for conjugation) is manufactured through a laborious process that involves polymerization of short linear polymers which are then multiply functionalized then attached as two conjugation reactions to a lysine residue which becomes a two-arm PEG reagent. Due to the number of synthetic steps and the need for high quality, multiple chromatography steps are required. Low polydispersity (<1.2) linear PEG polymers have a size restriction of approximately 20kDa, 30kDa or 40kDa with 20kDa being the economically feasible limit. When formed into a branched reagent, then, the final reagent size is 40 kDa (2 x 20 kDa), 60 kDa (2 x 30 kDa), 80 kDa (2 x 40 kDa). The larger the size, the more expensive to manufacture with low polydispersity. Also, the larger the size, the less optimal the solubility, stability, and viscosity of the polymer and the associated polymer-drug conjugate.

[0027] In summary, PEG polymers work well with low-dose, high-potency agonistic molecules such as erythropoietin and interferon. However, despite its commercial success, PEGylated products have inadequate stability and solubility, the PEG reagent is expensive to manufacture and, most important, PEGylated products have limited further upside in terms of improving in vivo and in vitro performance.

[0028] In view of the recognized advantages of conjugating functional agents to water soluble polymers, and the limitations of water soluble polymers such as PEG in forming conjugates suitable for therapeutic purposes, additional water soluble polymers for forming conjugates with functional agents are desirable. Water soluble polymers, particularly those which have many of the advantages of PEG for use in conjugate formation, and which do not suffer from the disadvantages observed with PEG as a conjugating agent would be desirable for use in forming therapeutic and diagnostic agents.

[0029] PEGylation does nonetheless point the way to a solution to the entire

biocompatibility issue. PEG works because of the polymer's hydrophilic characteristics which shield the conjugated biological agent from the myriad non-specific in vivo clearance mechanisms in the body. The importance of water is generally recognized, but the special insight in this technology is to dig deeper to appreciate that it is how the water is bound and the associated water structure that is critical to the performance enhancement. PEG works because of its hydrophilic nature, but the water is not tightly bound to the polymer and thus the conjugated agent. Water molecules are in free exchange between the PEGylated compound and the surrounding bulk water, enabling clearance systems to recognize the protein. The answer is to find a way to "glue" water so tightly to the polymer and thus conjugated moiety such as to tightly mask the complex entirely from non-specific interactions. To accomplish, it is necessary for the polymer to maintain both positive and negative charges, thus being net neutral, an essential zwitterion. Certain zwitterionic polymers hold and will not release water molecules bound to their structures.

[0030] To make further progress, then, it is necessary to take a closer look at: (i) other examples of hydrophilic moieties that bind water to a greater extent and with more favorable physical properties and therefore with improved fundamental biocompatibility in vivo and in vitro, and (ii) examples of much bigger, extended form polymers (size and architecture) which is the related key driver of the in vivo and in vitro performance.

[0031] What is important for these polymers is the extent to which they bind water molecules and the physical properties of those water binding interactions. This combination of properties drives the fundamental biocompatibility of the polymer and the extent to which such a polymer can impart biocompatibility to a functional agent to which it is conjugated. The ideal technology would use a water binding moiety which very tightly if not irreversibly binds a large amount of water, would format these water binding moieties into a polymer backbone of sufficient length and flexibility to shield a range of desired drugs and formats, may have an extended form (i.e. multi-armed) architecture, would be functionalized for high efficiency conj ugation to the drug moiety, would be manufactured inexpensively with a minimal number of production steps, and would demonstrate very high quality as judged analytically and very high performance judged in functional in vivo (terminal half-life, immunogenicity, bioactivity) and in vitro (solubility, stability, viscosity, bioactivity) systems. A technology that allowed for the maximization of these elements would take the field to new levels of in vivo and in vitro performance.

[0032] One such technology uses as the water binding moiety the phosphorylcholine derived 2-methacryloyloxyethyl phosphorylcholine (HEMA-PC) or a related zwitterion, on a polymer of total size greater than 50 kDa peak molecular weight (Mp) as measured by multi- angle light scattering, with the possibility for highly branched architectures or pseudo architectures, functionalized for site-specific conjugation to a biopharmaceutical(s) of interest, manufactured with techniques enabling a well characterized therapeutic with high quality and low polydispersity, and when conjugated to a biopharmaceutical imparts a dramatic increase in mean terminal half-life versus an equivalent biopharmaceutical as modified with another half-life extension technology (for example, as conjugated with a PEG polymer) and which imparts solubility, stability, viscosity, and characterizability parameters to the conjugate that are a multiple of that seen with PEG or other technologies.

[0033] Of critical importance is the size of the polymer. When used for therapeutic purposes in the context of soluble polymer-drug conjugates, the prior art teaches that there is a well-defined and described trade-off between the size of the polymer and its quality. The polydispersity index (a key proxy for quality) is particularly important as it speaks to the heterogeneity of the underlying statistical polymer which when conjugated to a

pharmaceutical of interest imparts such heterogeneity to the drug itself which significantly complicates the reliable synthesis of the therapeutic protein required for consistent effectiveness. and which is undesirable from a manufacturing, regulatory, clinical, and patient point of view.

[0034] The present invention describes very large polymers with very high quality and very low polydispersity index which are functionalized for chemical conjugation for example to a soluble drug. Importantly, the polymers are not inert, nor are they destined for attachment to a surface or gelled as hydrogel. This is wholly new, surprising, very useful and has not been described previously. For their therapeutic intent, a well-defined drug substance is essential.

This manifests itself at the level of the polymer, the pharmaceutical, and the conjugate.

Notably, there is a body of work on polymers having been made using a variety of approaches and components with unfunctionalized polymers. That body of work is not directly relevant here where a required step is a specific conjugation.

[0035] The current state of the art as it relates to functionalized polymers, constructed from hydrophilic monomers by conventional, pseudo or controlled radical polymerization, is that only low molecular weight polymers (typically <50 kDa) have been described. In addition, as this molecular weight is approached, control of molecular weight, as evidenced by the polydispersity index (PDI), is lost.

[0036] For instance, Ishihara et al (2004, Biomaterials 25, 71-76) utilized controlled radical polymerization to construct linear polymers of 2-methacryloyloxyethyl phosphorylcholine (HEMA-PC) up to a molecular weight of 37 kDa. The PD1 was 1.35, which is too high to be pharmaceutically relevant. In addition, these authors clearly stated, "In this method, it is hard to control the molecular weight distribution and increase the molecular weight." Lewis et al (US Patent 2004/0063881 ) also describe homopolymerization of this monomer using controlled radical polymerization, and reported molecular weights up to 1 1 kDa with a PD1 of 1.45. In a later publication, Lewis et al (2008, Bioconjugate Chem. 19, 2144-2155) again synthesized functionalized homopolymers of HEMA-PC this time to molecular weights up to 37 kDa. The PDI was 2.01. They stated that they achieved good control only at very limited (insufficient) molecular weights, with polydispersity increasing dramatically. They report loss of control at their high end molecular weight range (37 kDa) which they attribute to fast conversion at higher monomer concentrations which leads to the conclusion that it is not possible to create high molecular weight polymers of this type with tight control of polydispersity.

[0037] For instance, Hadd!eton et al (2004, JACS 126, 13220- 13221 ) utilized controlled radical polymerization to construct small linear polymers of poly(methoxyPEG)methacrylates for use in conjugation with proteins and in a size range of 1 1 ,000 to 34,000 Daltons. In an attempt to build the larger of these polymers, the authors increased the reaction temperature and sought out catalysts that could drive a faster polymerization. In a later publication, Haddleton et al (2005, JACS 127, 2966-2973) again synthesized functionalized

homopolymers of poly(methoxyPEG) methacrylates via controlled radical polymerization for protein conjugation in the size range of 4.1 to 35.4 kDa with PDI's ranging upwards of 1.25 even at this small and insufficient molecular weight distribution. In a subsequent publication, Haddleton et al (2007, JACS 129, 15156- 15163) again synthesized functionalized polymers via controlled radical polymerization for protein conjugation in the low size range of 8 to 30 kDA with PDI range of 1.20 - 1.28. Haddleton et al's mindset and approach teach away from the methods that need to be used to make high molecular weight, low polydispersity polymers relevant to this invention. Further, the focus on low molecular weight polymers for protein conjugation reflects a lack of understanding as to the size, architecture, and quality of polymers needed to carry the biopharmaceutical field to the next level.

[0038] The present invention describes high molecular weight zwitterion-containing polymers (>50 kDa peak molecular weight measured using multi-angle light scattering) with concomitantly low PDIs. This is surprising in light of the foregoing summary of the current state of the art. - BRIEF SUMMARY OF THE INVENTION

[0039] In some embodiments, the present invention provides a polymer having at least two polymer arms each having a plurality of monomers each independently selected from acrylate, methacrylate, acrylamide, methacrylamide, styrene, vinyl-pyridine,

vinyl-pyrrolidone or vinyl-ester, wherein each monomer includes a hydrophilic group. The polymer also includes an initiator fragment linked to a proximal end of the polymer arm, wherein the initator moiety is suitable for radical polymerization. The polymer also includes an end group linked to a distal end of the polymer arm. At least one of the initiator fragment and the end group of the polymer includes a functional agent or a linking group.

[0040] In other embodiments, the present invention provides a conjugate including at least one polymer having at least two polymer arms each having a plurality of monomers each independently selected from the group consisting of acrylate, methacrylate, acrylamide, methacrylamide, styrene, vinyl-pyridine, vinyl-pyrrolidone or vinyl-ester, wherein each monomer includes a hydrophilic group, an initiator fragment linked to a proximal end of the polymer arm, wherein the initator moiety is suitable for radical polymerization, and an end group linked to a distal end of the polymer arm. The conjugates of the present invention also include at least one functional agent having a bioactive agent or a diagnostic agent, linked to the initiator fragment or the end group.

[0041] In some other embodiments, the present invention provides a polymer of the formula:

wherein R can be H, L -A , LG or L -LG . Each M and M ^~ can be independently selected from acrylate, methacrylate, acrylamide, methacrylamide, styrene, vinyl-pyridine, vinyl-pyrrolidone or vinyl-ester. Each of G ¹ and G ² is each independently a hydrophilic group. Each group I is an initiator fragment and Γ a radical scavenger such that the combination of l-Γ is an initiator, I ¹ , for the polymerization of the polymer via radical polymerization. Alternatively, each F can be independently selected from H, halogen or Ci_6 alkyl. Each L ¹ , L ² and L ³ can be a linker. Each A ¹ can be a functional agent. Each LG ¹ can be a linking group. Subscripts x and y' can each independently be an integer of from 1 to 1000. Each subscript z can be independently an integer of from 1 to 10. Subscript s can be an integer of from 2 to 1 00.

[0042] In some embodiments, the present invention provides an initiator of the formula:

wherein each Γ can independently be halogen, -SCN, or -NCS; L ⁴ and L ⁵ can each independently be a bond or a linker, such that one of L ⁴ and L ⁵ is a linker; C is a bond or a core group; LG ² is a linking group; and subscript p is an integer from 2 to 1 00.

BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 shows a scheme for the preparation of the random copolymers of the present invention. The initiator Ι-Γ is cleaved into initiator fragment I and radical scavenger Γ. The initiator fragment I then reacts with comonomers M ¹ and M ² to initiate the polymerization process and generate species A. The radical scavenger Γ can then reversibly react with species A to form species B. Alternatively, species A can react with additional monomers to continue propagation of the polymer (species C). Concomitantly, the growing polymer chain of species C reversibly reacts with radical scavenger F to form the random copolymer, species D.

DETAILED DESCRIPTION OF THE INVENTION

I. General

[0044] The present invention provides high MW polymers having hydrophilic groups or zwitterions, such as phosphorylcholine, and at least one functional agent (as defined herein). Phosphorylcholine as a highly biocompatible molecule drives fundamental biocompatibility. It also has chaperone type functions, in terms of protecting proteins under temperature or other stress. It also can allow other functions such as reversible cellular uptake. The functional agent can be a bioactive agent such as a drug, therapeutic protein or targeting agent, as well as a detection agent, imaging agent, labeling agent or diagnostic agent. The high MW polymers are useful for the treatment of a variety of conditions and disease states by selecting one or more appropriate functional agents. More than one bioactive agent can be linked to the high MW polymer, thus enabling treatment of notjust a single disease symptom or mechanism, but rather the whole disease. In addition, the high MW polymers are useful for diagnostic and imaging purposes by attachment of suitable targeting agents and imaging agents. The high MW polymers can include both therapeutic and diagnostic agents in a single polymer, providing theranostic agents that treat the disease as well as detect and diagnose. The polymers can be linked to the bioactive agent(s) via stable or unstable linkages.

[0045] The polymers can be prepared via a conventional free-radical polymerization or controlled/living radical polymerization, such as atom transfer radical polymerization (ATRP), using monomers that contain zwitterions, such as phosphorylcholine. The initiators used for preparation of the high MW polymers can have multiple initiating sites such that multi-arm polymers, such as stars, can be prepared. The initiator can also contain either the bioactive agent, or linking groups that are able to link to the bioactive agent.

[0046] The invention also describes new ways to achieve branched polymer architectures on a bioactive surface. The concept is one of "branching points" or "proximal attachment points" on the target molecule such as to recreate an effective >2 arm polymer with >1 arm polymers attached to a localized site(s) on a target molecule. In the prior art, indiscriminate PEGylation of a protein with a non site-specific reagent (for example an NHS functionalized PEG reagent) would result in multiple PEG polymers conjugated to multiple amine groups scattered through the protein. Here, what is described is preferably a one step approach in which the target agent is modified to locate two unique conjugation sites (for example, cysteine amino acids) such that once the tertiary structure of the protein or peptide or agent is formed, the two sites will be in proximity one to the other. Then, this modified target agent is used in a conjugation reaction with a polymer containing the corresponding conjugation chemistry (for example, thiol reactive). The result is a single target agent which is conjugated with two polymers in close proximity to one another, thereby creating a branching point or "pseudo" branch. In another embodiment, the target agent would contain a single unique site, for example a free cysteine, and a tri(hetero)functional linking agent would be employed to attach >2 linear polymers to this single site, again creating a "pseudo" branch.

[0047] The invention also describes new ways to achieve very high efficiency and site specific conjugation to peptides and proteins by way of inteins.

II. Definitions [0048] "Polymer" refers to a series of monomer groups linked together. The high MW polymers are prepared from monomers that include, but are not limited to, acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinyl-pyridine, vinyl-pyrrolidone and vinyl esters such as vinyl acetate. Additional monomers are useful in the high MW polymers of the present invention. When two different monomers are used, the two monomers are called "comonomers," meaning that the different monomers are copolymerized to form a single polymer. The polymer can be linear or branched. When the polymer is branched, each polymer chain is referred to as a "polymer arm." The end of the polymer arm linked to the initiator moiety is the proximal end, and the growing-chain end of the polymer arm is the distal end. On the growing chain-end of the polymer arm, the polymer arm end group can be the radical scavenger, or another group.

[0049] "Hydrophilic group" refers to a compound or polymer that attracts water, and is typically water soluble. Examples of hydrophilic groups include hydrophilic polymers and zwitterionic moieties. Other hydrophilic groups include, but are not limited to, hydroxy, amine, carboxylic acid, amide, sulfonate and phosphonate. Hydrophilic polymers include, but are not limited to, polyethylene oxide, polyoxazoline, cellulose, starch and other polysaccharides. Zwitterionic moiety refers to a compound having both a positive and a negative charge. Zwitterionic moieties useful in the high MW polymers can include a quaternary nitrogen and a negatively charged phosphate, such as phosphorylcholine:

RO-P(=0)(0>0-CH ₂ CH2-N ⁺ ( e) ₃ . Other zwitterionic moieties are useful in the high MW polymers of the present invention, and Patents WO 1994/016748 and WO 1994/016749 are incorporated in their entirety herein.

[0050] "Initiator" refers to a compound capable of initiating a polymerization using the comonomers of the present invention. The polymerization can be a conventional free radical polymerization or a controlled/living radical polymerization, such as Atom Transfer Radical Polymerization (ATRP), Reversible Addition-Fragmentation-Termi nation (RAFT) polymerization or nitroxide mediated polymerization (NMP). The polymerization can be a "pseudo" controlled polymerization, such as degenerative transfer. When the initiator is suitable for ATRP, it contains a labile bond which can homolytically cleave to form an initiator fragment, I, being a radical capable of initiating a radical polymerization, and a radical scavenger, Γ, which reacts with the radical of the growing polymer chain to reversibly terminate the polymerization. The radical scavenger F is typically a halogen, but can also be an organic moiety, such as a nitrile.

[0051] "Linker" refers to a chemical moiety that l inks two groups together. The linker can be cleavable or non-cleavable. Cleavable linkers can be hydrolyzable, enzymatically cleavable, pH sensitive, photolabile, or disulfide linkers, among others. Other linkers include homobifunctional and heterobifunctional linkers. A "linking group" is a functional group capable of forming a covalent linkage consisting of one or more bonds to a bioactive agent. Nonlimiting examples include those illustrated in Table 1 .

[0052] "Hydrolyzable linker" refers to a chemical linkage or bond, such as a covalent bond, that undergoes hydrolysis under physiological conditions. The tendency of a bond to hydrolyze may depend not only on the general type of linkage connecting two central atoms between which the bond is severed, but also on the substituents attached to these central atoms. Non-limiting examples of hydrolytically susceptible linkages include esters of carboxylic acids, phosphate esters, acetals, ketals. acyloxyalkyl ether, imines, orthoesters, and some amide linkages.

[0053] "Enzymatically cleavable linker" refers to a linkage that is subject to degradation by one or more enzymes. Some hydrolytically susceptible linkages may also be enzymatically degradable. For example esterases may act on esters of carboxylic acid or phosphate esters, and proteases may act on peptide bonds and some amide linkages.

[0054] "pH sensitive linker" refers to a linkage that is stable at one pH and subject to degradation at another pH. For example, the pH sensitive linker can be stable at neutral or basic conditions, but labile at mildly acidic conditions.

[0055] "Photolabile linker" refers to a linkage, such as a covalent bond, that cleaves upon exposure to light. The photolabile linker includes an aromatic moiety in order to absorb the incoming light, which then triggers a rearrangement of the bonds in order to cleave the two groups linked by the photolabile linker.

[0056] "Self-immolative or double prodrug linker" refers to a linkage in which the main function of the linker is to release a functional agent only after selective trigger activation (for example, a drop in pH or the presence of a tissue-specific enzyme) followed by spontaneous chemical breakdown to release the functional agent.

[0057] "Functional agent" is defined to include a bioactive agent or a diagnostic agent. A "bioactive agent" is defined to include any agent, drug, compound, or mixture thereof that targets a specific biological location (targeting agent) and/or provides some local or systemic physiological or pharmacologic effect that can be demonstrated in vivo or in vitro.

Non-limiting examples include drugs, vaccines, antibodies, antibody fragments, scFvs, diabodies, avimers, vitamins and cofactors, polysaccharides, carbohydrates, steroids, lipids, fats, proteins, peptides, polypeptides, nucleotides, oligonucleotides, polynucleotides, and nucleic acids (e.g., mR A, tRNA, snRNA, RNAi, D A, cDNA, antisense constructs, ribozymes, etc). A "diagnostic agent" is defined to include any agent that enables the detection or imaging of a tissue or disease. Examples of diagnostic agents include, but are not limited to, radiolabels, fluorophores and dyes.

[0058] "Therapeutic protein" refers to peptides or proteins that include an amino acid sequence which in whole or in part makes up a drug and can be used in human or animal pharmaceutical applications. Numerous therapeutic proteins are known to practitioners of skill in the art including, without limitation, those disclosed herein.

[0059] "Phosphorylcholine," also denoted as "PC," refers to the following:

where * denotes the point of attachment. The phosphorylcholine is a zwitterionic group and includes salts (such as inner salts), and protonated and deprotonated forms thereof.

[0060] "Phosphorylchol ine containing polymer" is a polymer that contains

phosphorylcholine. It is specifically contemplated that in each instance where a

phosphorylcholine containing polymer is specified in this application for a particular use, a single phosphorylcholine can also be employed in such use. "Zwitterion containing polymer" refers to a polymer that contains a zwitterion.

[0061] "Poly(acryIoyloxyethyl phosphorylcholine) containing polymer" refers to a polymer of acrylic acid containing at least one acryloyloxyethyl phosphorylcholine monomer such as 2-methacryIoyloxyethyl phosphorylcholine (i. e. , 2-inethacryloyl-2'-trimethylammonium ethyl phosphate).

[0062] "Contacting" refers to the process of bringing into contact at least two distinct species such that they can react. It should be appreciated, however, that the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture.

[0063] "Water-soluble polymer" refers to a polymer that is soluble in water. A solution of a water-soluble polymer may transmit at least about 75%, more preferably at least about 95% of light, transmitted by the same solution after filtering. On a weight basis, a water-soluble polymer or segment thereof may be at least about 35%, at least about 50%, about 70%, about 85%), about 95% or 100% (by weight of dry polymer) soluble in water. [0064] "Molecular weight" in the context of the polymer can be expressed as either a number average molecular weight, or a weight average molecular weight or a peak molecular weight. Unless otherwise indicated, all references to molecular weight herein refer to the peak molecular weight. These molecular weight determinations, number average, weight average and peak, can be measured using gel permeation chromatography or other liquid chromatography techniques. Other methods for measuring molecular weight values can also be used, such as the use of end-group analysis or the measurement of colligative properties (e.g. , freezing-point depression, boiling-point elevation, or osmotic pressure) to determine number average molecular weight, or the use of light scattering techniques,

ultracentrifugation or viscometry to determine weight average molecular weight. The polymeric reagents of the invention are typically polydisperse (i.e. , number average molecular weight and weight average molecular weight of the polymers are not equal), possessing low polydispersity values of preferably less than about 1 .5, as judged by gel permeation chromatography. In other embodiments the polydispersities may be in the range of about 1.4 to about 1 .2, more preferably less than about 1.15, still more preferably less than about 1.10, yet still more preferably less than about 1.05, and most preferably less than about 1.03.

[0065] The phrase "a" or "an" entity as used herein refers to one or more of that entity ; for example, a compound refers to one or more compounds or at least one compound. As such, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein.

[0066] "About" as used herein means variation one might see in measurements taken among different instruments, samples, and sample preparations.

[0067] "Protected,", "protected form", "protecting group" and "protective group" refer to the presence of a group (i. e., the protecting group) that prevents or blocks reaction of a particular chemically reactive functional group in a molecule under certain reaction conditions. Protecting group will vary depending upon the type of chemically reactive group being protected as well as the reaction conditions to be employed and the presence of additional reactive or protecting groups in the molecule, if any. The skilled artisan will recognize protecting groups known in the art, such as those found in the treatise by Greene et al., "Protective Groups In Organic Synthesis," 3 ^rd Edition, John Wiley and Sons, Inc., New York, 1999.

[0068] "Spacer," and "spacer group" are used interchangeably herein to refer to an atom or a collection of atoms optionally used to link interconnecting moieties such as a terminus of a water-soluble polymer and a reactive group of a functional agent and a reactive group. A spacer may be lrydrolytically stable or may include a hydrolytically susceptible or enzymatically degradable linkage.

[0069] "Alkyl" refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. For example, Cj-Ce alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Other alkyl groups include, but are not limited to heptyl, octyl, nonyl, decyl, etc. Alkyl can include any number of carbons, such as 1 -2, 1 -3, 1 -4, 1 -5, 1 -6, 1 -7, 1 -8, 1 -9, 1 - 10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6 and 5-6. The alkyl group is typically monovalent, but can be divalent, such as when the alkyl group links two moieties together.

[0070] The term "lower" referred to above and hereinafter in connection with organic radicals or compounds respectively defines a compound or radical which can be branched or unbranched with up to and including 7, preferably up to and including 4 and (as unbranched) one or two carbon atoms.

[0071] "Alkylene" refers to an alkyl group, as defined above, linking at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkylene can be linked to the same atom or different atoms of the alkylene. For instance, a straight chain alkylene can be the bivalent radical of -(CH2) _n, where n is I , 2, 3, 4, 5 or 6. Alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene and hexylene.

[0072] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be a variety of groups selected from : -OR', =0, =NR',

=N-OR', -NR'R", -SR', -halogen, -SiR'R"R"', -OC(0)R\ -C(0)R\ -C0 ₂ R', -CONR'R", -O C(0)NR'R", -NR"C(0)R', -NR'-C(0)NR"R'", -NR"C(0) ₂ R', -NH-C(NH ₂ )=NH, -NR'C(N H ₂ )=NH, -NH-C(NH ₂ )=NR\ -S(0)R', -S(0) ₂ R', -S(0) ₂ NR'R", -CN and -N0 ₂ in a number ranging from zero to (2m'+l ), where m' is the total number of carbon atoms in such radical. R', R" and R"' each independently refer to hydrogen, unsubstituted (Cj-Cg)alkyl and heteroalkyl, unsubstituted aryl, aryl substituted with 1 -3 halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(Ci -C ₄ )alkyl groups. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include 1 -pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is meant to include groups such as haloalkyl (e.g., -CF ₃ and -CH ₂ CF ₃ ) and acyl

(e.g., -C(0)CH ₃ , -C(0)CF ₃ , -C(0)CH ₂ OCH ₃ , and the like). Preferably, the substituted alkyl and heteroalkyl groups have from 1 to 4 substituents, more preferably 1 , 2 or 3 substituents. Exceptions are those perhalo alkyl groups (e.g., pentafluoroethyl and the like) which are also preferred and contemplated by the present invention.

[0073] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to: -OR', =0, =NR\

=N-OR', -NR'R", -SR', -halogen, -SiR'R"R"', -OC(0)R', -C(0)R', -C0 ₂ R', -CONR'R", -O C(0)NR'R", -NR"C(0)R', -NR'-C(0)NR"R'", -NR"C(0) ₂ R', -NR-C(NR'R"R'")=NR"", -N R-C(NR'R")=NR"', -S(0)R', -S(0) ₂ R', -S(0) ₂ NR'R", -NRS0 ₂ R', -CN and -N0 ₂ in a number ranging from zero to (2m'+l ), where m ' is the total number of carbon atoms in such radical. R', R", R"' and R"" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., aryl substituted with 1 -3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R', R", R"' and R"" groups when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include, but not be limited to, 1 -pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF ₃ and -CH ₂ CF ₃ ) and acyl

(e.g., -C(0)CH ₃ , -C(0)CF ₃ , -C(0)CH ₂ OCH ₃ , and the like).

[0074] "Alkoxy" refers to alkyl group having an oxygen atom that either connects the alkoxy group to the point of attachment or is linked to two carbons of the alkoxy group. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc. The alkoxy groups can be further substituted with a variety of substituents described within. For example, the alkoxy groups can be substituted with halogens to form a "halo-alkoxy" group.

[0075] "Carboxyalkyl" means an alkyl group (as defined herein) substituted with a carboxy group. The term "carboxy cycloalkyl" means an cycloalkyl group (as defined herein) substituted with a carboxy group. The term alkoxyalkyl means an alkyl group (as defined herein) substituted with an alkoxy group. The term "carboxy" employed herein refers to carboxylic acids and their esters.

[0076] "Haloalkyl" refers to alkyl as defined above where some or all of the hydrogen atoms are substituted with halogen atoms. Halogen (halo) preferably represents chloro or fluoro, but may also be bromo or iodo. For example, haloalkyl includes trifluoromethyl, fluoromethyl, 1 ,2,3,4,5-pentafluoro-phenyl, etc. The term "perfluoro" defines a compound or radical which has all available hydrogens that are replaced with fluorine. For example, perfluorophenyl refers to 1 ,2,3,4,5-pentafluorophen l, perfluoromethyl refers to

1 , 1 , 1 -trifluoromethyl, and perfluoromethoxy refers to 1 , 1 , 1 -trifluoromethoxy .

[0077] "Fluoro-substituted alkyl" refers to an alkyl group where one, some, or al l hydrogen atoms have been replaced by fluorine.

[0078] "Cytokine" in the context of this invention is a member of a group of protein signaling molecules that may participate in cell-cell communication in immune and inflammatory responses. Cytokines are typically small, water-soluble glycoproteins that have a mass of about 8-35 kDa.

[0079] "Cycloalkyl" refers to a cyclic hydrocarbon group that contains from about 3 to 12, from 3 to 10, or from 3 to 7 endocyclic carbon atoms. Cycloalkyl groups include fused, bridged and spiro ring structures.

[0080] "Endocyclic" refers to an atom or group of atoms which comprise part of a cyclic ring structure.

[0081] "Exocyclic" refers to an atom or group of atoms which are attached but do not define the cyclic ring structure.

[0082] "Cyclic alkyl ether" refers to a 4 or 5 member cyclic alkyl group having 3 or 4 endocyclic carbon atoms and 1 endocyclic oxygen or sulfur atom (e.g. , oxetane, thietane, tetrahydrofuran, tetrahydrothiophene); or a 6 to 7 member cyclic alkyl group having 1 or 2 endocyclic oxygen or sulfur atoms (e.g. , tetrahydropyran, 1 ,3-dioxane, 1 ,4-dioxane, tetrahydrothiopyran, 1 ,3-dithiane, 1 ,4-dithiane, 1 ,4-oxathiane).

[0083] "Alkenyl" refers to either a straight chain or branched hydrocarbon of 2 to 6 carbon atoms, having at least one double bond. Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, isopropenyl, 1 -butenyl, 2-butenyl, isobutenyl, butadienyl, 1 -pentenyl, 2-pentenyl, isopentenyl, 1 ,3-pentadienyl, 1 ,4-pentadienyl, 1 -hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1 ,4-hexadienyl, 1 ,5-hexadienyl, 2,4-hexadienyl, or

1 ,3,5-hexatrienyl. Alkenyl groups can also have from 2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6 and 5 to 6 carbons. The alkenyl group is typically monovalent, but can be divalent, such as when the alkenyl group links two moieties together.

[0084] "Alkenylene" refers to an alkenyl group, as defined above, linking at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkenylene can be linked to the same atom or different atoms of the alkenylene. Alkenylene groups include, but are not limited to, ethenylene, propenylene, isopropenylene, butenylene, isobutenylene, sec-butenylene, pentenylene and hexenylene.

[0085] "Alkynyl" refers to either a straight chain or branched hydrocarbon of 2 to 6 carbon atoms, having at least one triple bond. Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1 -butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiynyl, 1 -pentynyl, 2-pentynyl, isopentynyl, 1 ,3-pentadiynyl, 1 ,4-pentadiynyl, 1 -hexynyl, 2-hexynyl, 3-hexynyl, 1 ,3-hexadiynyl, 1 ,4-hexadiynyl, 1 ,5-hexadiynyl, 2,4-hexadiynyl, or

1 ,3,5-hexatriynyl. Alkynyl groups can also have from 2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6 and 5 to 6 carbons. The alkynyl group is typically monovalent, but can be divalent, such as when the alkynyl group links two moieties together.

[0086] "Alkynylene" refers to an alkynyl group, as defined above, linking at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkynylene can be linked to the same atom or different atoms of the alkynylene. Alkynylene groups include, but are not limited to, ethynylene, propynylene, butynylene, sec-butynylene, pentynylene and hexynylene.

[0087] "Cycloalkyl" refers to a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly containing from 3 to 12 ring atoms, or the number of atoms indicated. Monocyclic rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Bicyclic and polycyclic rings include, for example, norbornane, decahydronaphthalene and adamantane. For example, C;,.8cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and norbornane.

[0088] "Cycloalkylene" refers to a cycloalkyl group, as defined above, linking at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the cycloalkylene can be linked to the same atom or different atoms of the cycloalkylene.

Cycloalkylene groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cyclooctylene. [0089] "Heterocycloalkyl" refers to a ring system having from 3 ring members to about 20 ring members and from 1 to about 5 heteroatoms such as N, O and S. Additional heteroatoms can also be useful, including, but not limited to, B, Al, Si and P. The heteroatoms can also be oxidized, such as, but not limited to, -S(O)- and -S(0)2-. For example, heterocycle includes, but is not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, morpholino, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, piperidinyl, indolinyl, quinuclidinyl and l ,4-dioxa-8-aza-spiro[4.5]dec-8-yl.

[0090] "Heterocycloalkylene" refers to a heterocyclalkyl group, as defined above, linking at least two other groups. The two moieties linked to the heterocycloalkylene can be linked to the same atom or different atoms of the heterocycloalkylene.

[0091] "Aryl" refers to a monocyclic or fused bicyclic, tricyclic or greater, aromatic ring assembly containing 6 to 16 ring carbon atoms. For example, aryl may be phenyl, benzyl or naphthyi, preferably phenyl. "Arylene" means a divalent radical derived from an aryl group. Aryl groups can be mono-, di- or tri-substituted by one, two or three radicals selected from alkyl, alkoxy, aryl, hydroxy, halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy and oxy-C2-C3-alkylene; all of which are optionally further substituted, for instance as hereinbefore defined; or 1 - or 2-naphthyl; or 1 - or 2-phenanthrenyl.

Alkylenedioxy is a divalent substitute attached to two adjacent carbon atoms of phenyl, e.g. methylenedioxy or ethylenedioxy. Oxy-C2-C3-alkylene is also a divalent substituent attached to two adjacent carbon atoms of phenyl, e.g. oxyethylene or oxypropylene. An example for oxy- Ci-Cs-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yI.

[0092] Preferred as aryl is naphthyi, phenyl or phenyl mono- or disubstituted by alkoxy, phenyl, halogen, alkyl or trifluoromethyl, especially phenyl or phenyl-mono- or disubstituted by alkoxy, halogen or trifluoromethyl, and in particular phenyl.

[0093] Examples of substituted phenyl groups as R are, e.g. 4-chlorophen- 1 -yl,

3,4-dichlorophen- l -yl, 4-mefhoxyphen-l -yl, 4-methylphen- l -yl, 4-aminomethylphen- l-yl, 4-methoxyethylaminomethylphen- l -yl, 4-hydroxyethylaminomethylphen- l -yl,

4-hydroxyethyl-(methyl)-aminomethylphen- l -yl, 3-aminomethylphen- l -yl,

4-N-acetylaminomethylphen-l -yl, 4-aminophen-l -yl, 3-aminophen- l -yl, 2-aminophen- l -yl, 4-phenyl-phen- l -yl, 4-(imidazol- l -yl)-phen- l, 4-(imidazol- l -ylmethyl)-phen- l -yl, 4-(morpholin- 1 -y l)-phen- 1 -yl, 4-(morpholin- 1 -y lmethyl)-phen- 1 -yl,

4-(2-methoxyethylaminomethyl)-phen-l -yl and 4-(pyrrolidin- l -ylmethyl)-phen-l -yl, 4-(thiophenyl)-phen- l -yl, 4-(3-thiophenyl)-phen-l -yl, 4-(4-methylpiperazin-l-yl)-phen- l -yl, and 4-(piperidinyl)-phenyl and 4-(pyridinyl)-phenyl optionally substituted in the heterocyclic ring.

[0094] "Arylene" refers to an aryl group, as defined above, linking at least two other groups. The two moieties linked to the arylene are linked to different atoms of the arylene. Arylene groups include, but are not limited to, phenylene.

[0095] "Arylene-oxy" refers to an arylene group, as defined above, where one of the moieties linked to the arylene is linked through an oxygen atom. Arylene-oxy groups include, but are not limited to, phenylene-oxy.

[0096] Similarly, substituents for the aryl and heteroaryl groups are varied and are selected from: -halogen, -OR', -OC(0)R', -NR'R", -SR', -R\ -CN, -N0 ₂ , -C0 ₂ R\ -CONR'R", -C(O) R\ -OC(0)NR'R", -NR"C(0)R', -NR"C(0) ₂ R\

,-NR'-C(0)NR"R"\ -NH-C( H ₂ )=NH, -NR'C(NH ₂ )=NH, -NH-C(NH ₂ )=NR', -S(0)R', -S(0 ) ₂ R', -S(0) ₂ NR'R", -N ₃ , -CH(Ph) ₂ , peril uoro(Ci-C )alkoxy, and peril uoro(C _r C ₄ )alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R', R" and R'" are independently selected from hydrogen, (Ci -Cs)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C i-C4)alkyl, and (unsubstituted aryl)oxy-(C i -Chalky 1.

[0097] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(0)-(CH ₂ ) _q -U-, wherein T and U are independently -NH-, -0-, -CH ₂ - or a single bond, and q is an integer of from 0 to 2. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH ₂ ) _r -B-, wherein A and B are independently -CH ₂ -, -0-, -NH-, -S-, -S(0 , -S(0) ₂ -, -S(0) ₂ NR'- or a single bond, and r is an integer of from 1 to 3. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the

formula -(CH ₂ ) _s -X-(CH ₂ ) _r , where s and t are independently integers of from 0 to 3, and X is -0-, -NR'-, -S-, -S(O)-, -S(0) ₂ ~, or -S(0) ₂ NR'-. The substituent R'

in -NR'- and -S(0) ₂ NR'- is selected from hydrogen or unsubstituted (Ci-C6)alkyl.

[0098] "Heteroaryl" refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, where from 1 to 4 of the ring atoms are a heteroatom each N, O or S. For example, heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any other radicals substituted, especially mono- or di-substituted, by e.g. alkyl, nitro or halogen. Pyridyl represents 2-, 3- or 4-pyridyl, advantageously 2- or 3-pyridyl. Thienyl represents 2- or 3-thienyl. Quinolinyl represents preferably 2-, 3- or 4-quinolinyl.

Isoquinolinyl represents preferably 1 -, 3- or 4-isoquinolinyl. Benzopyranyl,

benzothiopyranyl represents preferably 3-benzopyranyl or 3-benzothiopyranyl, respectively. Thiazolyl represents preferably 2- or 4-thiazolyl, and most preferred, 4-thiazolyl. Triazolyl is preferably 1-, 2- or 5-(l ,2,4-triazolyl). Tetrazolyl is preferably 5-tetrazolyl.

[0099] Preferably, heteroaryl is pyridyl, indolyl, quinolinyl, pyrrolyl, thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl, benzothienyl, oxazolyl, indazolyl, or any of the radicals substituted, especially mono- or di-substituted.

[0100] As used herein, the term "heteroalkyl" refers to an alkyl group having from 1 to 3 heteroatoms such as N, O and S. Additional heteroatoms can also be useful, including, but not limited to, B, Al, Si and P. The heteroatoms can also be oxidized, such as, but not limited to, -S(O)- and -S(0)2-. For example, heteroalkyl can include ethers, thioethers, alkyl-amines and alkyl-thiols.

[0101] As used herein, the term "heteroalkylene" refers to a heteroalkyl group, as defined above, linking at least two other groups. The two moieties linked to the heteroalkylene can be linked to the same atom or different atoms of the heteroalkylene.

[0102] "Electrophile" refers to an ion or atom or collection of atoms, which may be ionic, having an electrophilic center, i. e., a center that is electron seeking, capable of reacting with a nucleophile. An electrophile (or electrophilic reagent) is a reagent that forms a bond to its reaction partner (the nucleophile) by accepting both bonding electrons from that reaction partner.

[0103] "Nucleophile" refers to an ion or atom or collection of atoms, which may be ionic, having a nucleophilic center, i.e., a center that is seeking an electrophilic center or capable of reacting with an electrophile. A nucleophile (or nucleophilic reagent) is a reagent that forms a bond to its reaction partner (the electrophile) by donating both bonding electrons. A "nucleophilic group" refers to a nucleophile after it has reacted with a reactive group. Non limiting examples include amino, hydroxyl, alkoxy, haloalkoxy and the like.

[0104] " aleimido" refers to a pyrrole-2,5-dione- l -yl group having the structure:

which upon reaction with a sulfliydryl {e.g., a thio alkyl) forms an -S-maleimido group having the structure

where "·" indicates the point of attachment for the maleimido group and "^''indicates the point of attachment of the sulfur atom the thiol to the remainder of the original sulfhydryl bearing group.

[0105] For the purpose of this disclosure, "naturally occurring amino acids" found in proteins and polypeptides are L-alanine, L-arginine, L-asparagine, L-aspartic acid,

L-cysteine, L-glutamine, L-glutamic acid, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and or L-valine. "Non-naturally occurring amino acids" found in proteins are any amino acid other than those recited as naturally occurring amino acids. Non-naturally occurring amino acids include, without limitation, the D isomers of the naturally occurring amino acids, and mixtures of D and L isomers of the naturally occurring amino acids. Other amino acids, such as 4-hydroxyprol ine, desmosine, isodesmosine, 5-hydroxylysine, epsilon-N-methyllysine, 3-methylhistidine, although found in naturally occurring proteins, are considered to be non-naturally occurring amino acids found in proteins for the purpose of this disclosure as they are generally introduced by means other than ribosomal translation of mRNA.

[0106] "Linear" in reference to the geometry, architecture or overall structure of a polymer, refers to polymer having a single polymer arm.

[0107] "Branched," in reference to the geometry, architecture or overall structure of a polymer, refers to polymer having 2 or more polymer "arms" extending from a core structure, such as an L group, that may be derived from an initiator employed in an atom transfer radical polymerization reaction. A branched polymer may possess 2 polymer arms, 3 polymer arms, 4 polymer arms, 5 polymer arms, 6 polymer arms, 7 polymer arms, 8 polymer arms, 9 polymer arms or more. For the purpose of this disclosure, compounds having three or more polymer arms extending from a single linear group are denoted as having a "comb" structure or "comb" architecture. Branched can also be achieved through "statistical" structures to create broader dendrimer-like architectures. The group linking the polymer arms can be a small molecule having multiple attachment points, such as glycerol, or more complex structures having 4 or more polymer attachment points, such as dendrimers and hyperbranched structures. The group can also be a nanoparticle appropriately functionalized to allow attachment of multiple polymer arms.

[0108] "Pharmaceutically acceptable" composition or "pharmaceutical composition" refers to a composition comprising a compound of the invention and a pharmaceutically acceptable excipient or pharmaceutically acceptable excipients.

[0109] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to an excipient that can be included in the compositions of the invention and that causes no significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer's, normal sucrose, normal glucose and the like.

[0110] "Patient" or "subject in need thereof refers to a living organism suffering from or prone to a condition that can be prevented or treated by administration of a pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals and other non-mammalian animals.

[0111] "Therapeutically effective amount" refers to an amount of a conjugated functional agent or of a pharmaceutical composition useful for treating, ameliorating, or preventing an identified disease or condition, or for exhibiting a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art.

[0112] The "biological half-life" of a substance is a pharmacokinetic parameter which specifies the time required for one half of the substance to be removed from an organism following introduction of the substance into the organism.

III. High Molecular Weight Polymers

[0113] The present invention provides a high molecular weight polymer having hydrophilic groups and a functional group or linking group. In some embodiments, the present invention provides a polymer having at least two polymer arms each having a plurality of monomers each independently selected from acrylate, methacrylate, acrylamidc, methacrylamide, styrene, vinyl-pyridine, vinyl-pyrrolidone or a vinyl ester such as vinyl acetate, wherein each monomer includes a hydrophilic group. The polymer also includes an initiator fragment linked to a proximal end of the polymer arm, wherein the initiator moiety is suitable for radical polymerization. The polymer also includes an end group linked to a distal end of the polymer arm. At least one of the initiator fragment and the end group of the polymer includes a functional agent or a linking group.

[0114] In other embodiments, the present invention provides a polymer having a polymer arm having a plurality of monomers each independently selected from acrylate, methacrylate, acrylamide, methacrylamide, styrene, vinyl-pyridine, vinyl-pyrrolidone or a vinyl ester such as vinyl acetate, wherein each monomer includes a hydrophilic group. The polymer also includes an initiator fragment linked to a proximal end of the polymer arm, wherein the initiator moiety is suitable for radical polymerization. The polymer also includes an end group linked to a distal end of the polymer arm. At least one of the initiator fragment and the end group of the polymer includes a functional agent or a linking group. In addition, the polymer has a peak molecular weight (Mp) of from about 50 kDa to about 1 ,500 kDa, as measured by multi-angle light scattering.

[0115] The polymers of the present invention can have any suitable molecular weight. Exemplary molecular weights for the high W polymers of the present invention can be from about 50 to about 1 ,500 kilo-Daltons (kDa). In some embodiments, the high MW polymers of the present invention can have a molecular weight of about 50 kDa, about 100 kDa, about 200 kDa, about 250 kDa, about 300 kDa, about 350 kDa, about 400 kDa, about 450 kDa, about 500 kDa, about 650 kDa, about 750 kDa, about 1 ,000 kDa or about 1 ,500 kDa.

[0116] In some other embodiments, the present invention provides a polymer of the formula:

wherein R can be H, L -A , LG or L -LG . Each M and M can be independently selected from acrylate, methacrylate, acrylamide, methacrylamide, styrene, vinyl-pyridine, vinyl-pyrrolidone or vinyl-ester. Each of G ¹ and G ² is each independently a hydrophilic group. Each group I is an initiator fragment and Γ a radical scavenger such that the combination of Ι-Γ is an initiator, I ¹ , for the polymerization of the polymer via radical polymerization. Alternatively, each Γ can be independently selected from H, halogen or C| _6 alkyl. Each L ¹ , L ² and L ³ can be a linker. Each A ¹ can be a functional agent. Each LG ¹ can be a linking group. Subscripts x and y' can each independently be an integer of from 1 to 1000. Each subscript z can be independently an integer of from 1 to 10. Subscript s can be an integer of from 2 to 100.

[0117] In other embodiments, the present invention provides a polymer of Formula I:

wherein R ¹ of formula I can be H, LAA ¹ , LG ¹ or L ³ -LG' . Each M ¹ and M ² of formula I can be independently selected from acrylate, methacrylate, acrylamide, methacrylamide, styrene, vinyl-pyridine, vinyl-pyrrolidone or vinyl-ester. Each of ZW and ZW ¹ of formula I can be independently a zwitterionic moiety. Each I is an initiator fragment and Γ a radical scavenger such that the combination of I-P is an initiator, I ¹ , for the polymerization of the polymer of formula I via radical polymerization. Alternatively, each F can be independently selected from H, halogen or Ci.6 alkyl. Each L ¹ , L ² and L ³ of formula 1 can be a linker. Each A ¹ of formula I can be a functional agent. Each LG ¹ of formula I can be a linking group. Subscripts x and y' of formula I can each independently be an integer of from 1 to 1000. Each subscript z of formula I can be independently an integer of from 1 to 10. Subscript s of formula I can be an integer of from 2 to 100. The sum of s, x, y ¹ and z can be such that the polymer of formula I has a peak molecular weight of from about 50kDa to about l ,500kDa, as measured by multi-angle light scattering.

[0118] In other embodiments, the polymer can have the formula:

In some other embodiments, the polymer can have the formula

wherein R ² can be selected from H or Ci_6 alkyl, and PC can be phosphorylcholine. [0119] The high MW polymers of the present invention can also have any suitable number of comonomers, M ² . For example, the number of comonomers, subscript z, can be from 1 to 10, such as 1,2, 3, 4, 5, 6, 7, 8, 9 or 10. The number of comonomers, subscript z, can also be from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, the high MW polymer of the present invention can have two different monomers where subscript z is 1, such as in formula la:

Additional comonomers M ^" can be present in the high MW polymers of the present invention, such as M ^2a , M ^2b , M ^c , M ^2d , M ^2e , M ^2f , M ² , M ^2h , etc., and are defined as above for M ² , where each comonomer is present in a same or different y' value, and each comonomer having a corresponding ZW ¹ group attached.

[0120] The different monomers of the high MW polymers can also be present in any suitable ratio. For example, the M ² monomers, collectively or individually, can be present relative to the M ¹ monomer in a ratio of 100: 1 , 50: 1 , 40: 1 , 30: 1 , 20: 1 , 10: 1 , 9: 1 , 8: 1 , 7: 1 , 6: 1 , 5:1, 4:1,3:1,2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:30, 1:40, 1:50 and 1 : 100. In addition, each M ² monomer can be present in any suitable ratio relative to the M ¹ or any other M ² monomer, such as 100:1, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1,3:1,2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:30, 1:40, 1:50 and 1:100.

[0121] The high MW polymers of the present invention can have any suitable architecture. For example, the high M W polymers can be linear or branched. When the high MW polymers are branched, they can have any suitable number of polymer arms, as defined by subscript s of formula I, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 and up to 100 arms. In some embodiments, subscript s can be from 2 to 32, 2 to 16, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In some embodiments, subscript s can be 2, 3, 4, 5,6, 8, 9 or 12. In other embodiments, subscript s can be 3, 6, or 9. In some embodiments, subscript s can be 3. In still other embodiments, subscript s can be 6. In some other embodiments, subscript s can be 9. The high MW polymers of the present invention can adopt any suitable architecture. For example, the high MW polymers can be linear, branched, stars, dendrimers, combs, etc. [0122] A functional agent of the high MW polymers can be linked to the initiator fragment I, or the radical scavenger Γ, or both. When multiple functional agents are present, L ¹ can be a branching linker such that two or more functional agents can be linked to the initiator fragment I. In some embodiments, the high MW polymer has formula lb:

In formula lb, functional agent A ¹ can be a drug, therapeutic protein or a targeting agent. Linker L ¹ can be a cleavable linker, such as when attached to a drug or therapeutic protein to facilitate release of the drug or therapeutic protein. Alternatively, linker L ¹ can be a non-cleavable linker.

[0123] When multiple comonomers M ² are present, each comonomer M ² can have a different zwitterionic group attached. For example, the high MW polymer can have formula Ic:

wherein each of ZW ^la and ZW ^lb are as defined above for ZW, and each of y ^{, a} and y ^{l b} are as defined above for y ¹ .

[0124] In some embodiments, the high MW polymers have linking groups LG linked to the initiator fragment I, such as shown in the structures below:

[0125] In some embodiments, the high MW polymers of the present invention can be modified via a subsequent polymerization with one or more additional monomers. For example, in formula Ic above, monomers M ¹ and M ^2a can be copolymerized in a first polymerization, and monomer M ^2b can be polymerized in a second polymerization. A block copolymer would be formed having two blocks, the first block being a high MW polymer of ' and M , and the second block a homopolymer of M . Alternatively, following polymerization of monomers M ¹ and M ^2a , monomer M ^2b can be copolymerized with monomer M ^2c , thus forming a block copolymer where the first block is a high MW polymer of M ¹ and M ^2a , and the second block is a high MW polymer of M ^2b and M ^2c . Additional polymer structures can be prepared by copolymerizing monomers M ¹ , M ^2a and M ^2b in a first polymerization, followed by copolymerization of monomers M , M , and others, in a second copolymerization. Additional blocks can be prepared by yet a third polymerization using additional monomers. Such polymers provide blocks of copolymers that can have different properties, drugs and functional agents.

[0126] In some embodiments, the polymer can be





[0129] In some embodiments, R ¹ is L ³ -A ', LG ¹ or L ^J -LG' ; A ¹ is a drug, an antibody, an antibody fragment, a single domain antibody, an avimer, an adnectin, diabodies. a vitamin, a cofactor, a polysaccharide, a carbohydrate, a steroid, a lipid, a fat, a protein, a peptide, a polypeptide, a nucleotide, an oligonucleotide, a polynucleotide, a nucleic acid, a radiolabel, a contrast agent, a fluorophore or a dye; L ^J is -(CH ₂ CH ₂ 0)i.io-; and LG ¹ is maleimide, acetal, vinyl, allyl, aldehyde, -C(0)0-C] .6 alkyl, hydroxy, diol, ketal, azide, alkyne, carboxylic acid, or succinimide. In other embodiments, each LG ¹ can be hydroxy, carboxy, vinyl, vinyloxy, allyl, allyloxy, aldehyde, azide, ethyne, propyne, propargyl, -C(0)0-C] .6 alkyl,

A. Initiators

[0130] The high W polymers of the present invention are polymerized using any suitable initiator. Initiators useful in the present invention can be described by the formula: I-(F)m, where subscript m is an integer from 1 to 100. The initiator fragment I can be any group that initiates the polymerization. The radical scavenger F can be any group that will reversibly terminate the growing polymer chain. The radical scavenger F can be a halogen such as bromine, allowing the end of the polymer to be functional ized after polymerization. In some embodiments, the radical scavenger F is referred to as an end group. In addition, the initiator fragment 1 can optionally be functionalized with an R ¹ group that can include a variety of functional groups to tune the functionality of the high MW polymer.

[0131] Initiators useful in the present invention can have a single radical scavenger F, or any suitable number of branches such that there are multiple radical scavengers F each capable of reversibly terminating a growing polymer chain. When the initiator fragment I is branched and is capable of initiating multiple polymer chains, subscript m is greater than one such that there are as many radical scavengers F as there are growing polymer chains.

[0132] The polymer of the present invention can have a plurality of polymer arms. For example, the polymer can have from 2 to about 100 polymer arms, or from about 2 to about 50 polymer arms, or from about 2 to about 20 polymer arms, or from 2 to about 10 polymer arms, or from about 2 to about 8 polymer arms, or from about 2 to about 4 polymer arms. The polymer can also have any sutiable polydispersity index (PDI), as measured by the weight average molecular weight (M _w ) divided by the number average molecular weight (M _n ), where a PDI of 1 .0 indicates a perfectly monodisperse polymer. For example, the PDI can be less than about 2.0, or less than about 1 .9, 1.8, 1.7, 1 .6, 1.5, 1 .4, 1 .3, 1 .2 or 1.1 .

[0133] In some embodiments, the initiator fragment is linked to the proximal end of from 2 to about 100 polymer arms. In some other embodiments, the polymer has a polydispersity index of less than about 2.0. In sti ll other embodiments, the initiator fragment is linked to the proximal end of 2 polymer arms. In yet other embodiments, the initiator fragment is linked to the proximal end of 4 polymer arms. In other embodiments, the initiator fragment can be linked to the proximal end of 2, 3, 4, 5, 6, 8, 9 or 12 polymer arms. In some embodiments, the initiator fragment can be linked to the proximal end of 9 polymer arms.

[0134] Pseudo-branched polymers can also be obtained by linking multiple linear, unbranched, polymers of the present invention to a single functional agent such that the polymers are in close proximity. The proximity can be obtained by linking the polymers to nearby points on the functional agent, cysteines on a protein, for example. Alternatively, the proximity can be afforded by the structure of the functional agent, a protein for example, such that polymers attached to disparate regions of the protein are brought into close proximity due to the folding and secondary and tertiary structure of the protein. The close proximity of the two polymers of the present invention on a single functional agent, regardless of how the proximity is achieved, can impart properties similar to that of a polymer of the present invention having a plurality of polymer arms.

[0135] The bond between initiator fragment I and radical scavenger F is labile, such that during the polymerization process monomers M ¹ and comonomers M ² are inserted between initiator fragment I and radical scavenger Γ. For example, during a free radical polymerization, such as ATRP, initiator fragment I and radical scavenger F dissociate, as shown in Figure 1 , to form radicals of I and F. The radical of initiator fragment I then reacts with the monomers in solution to grow the polymer and forms a propagating polymer radical (species A and species C of Figure 1 ). During the polymerization process, the radical of the radical scavenger F will reversibly react with the propagating polymer radical to temporarily stop polymer growth. The bond between the monomer and the radical savenger F is also labile, such that the bond can cleave and allow the propagating polymer radical to react with additional monomer to grow the polymer. The end result of the polymerization process is that initiator fragment 1 is at one end of the polymer chain and radical scavenger F is at the opposite end of the polymer chain.

[0136] The radical of initiator fragment I is typically on a secondary or tertiary carbon, and can be stabilized by an adjacent carbonyl carbon. The radical scavenger F is typically a halogen, such as bromine, chlorine or iodine. Together, initiator fragment I and radical scavenger F form the initiator I ' useful in the preparation of the high MW polymers of the present invention.

[0137] A broad variety of initiators can be used to prepare the high MW polymers of the invention, including a number of initiators set forth in US 6,852,816 (incorporated herein by reference). In some embodiments, the initiators employed for ATRP reactions to prepare high MW polymers of the invention are selected from alkanes, cycloalkanes, alkyl carboxylic acids or esters thereof, cycloalkylcarboxylic acids or esters thereof, ethers and cyclic alkyl ethers, alkyl aryl groups, alkyl amides, alkyl-aryl carboxylic acids and esters thereof, and also bearing one radical scavenger F where unbranched high MW polymers are prepared, and more than one radical scavenger F where branched molecules are prepared.

[0138] Radical scavengers F useful in the present invention include, but are not limited to, halogens, such as Br, CI and I, thiocyanate (-SCN) and isothiocyanate (-N=C=S). Other groups are useful for the radical scavenger F of the present invention. In some embodiments, the radical scavenger I ' is bromine.

[0139] Initiators employed for ATRP reactions can be hydroxylated. In some

embodiments, the initiators employed for ATRP reactions to prepare high MW polymers of the invention are selected from alkanes, cycloalkanes, alkyl carboxylic acids or esters thereof, cycloalkylcarboxylic acids or esters thereof, ethers, cycl ic alkyl ethers, alkyl aryl groups, alkyl amides, alkyl-aryl carboxylic acids and esters thereof, bearing a hydroxyl group, and also bearing one radical scavenger F where unbranched high MW polymers are to be prepared, or alternatively, more than one radical scavenger F where branched molecules are to be prepared.

[0140] Initiators employed for ATRP reactions can bear one or more amine groups. In some embodiments, the initiators employed for ATRP reactions to prepare high MW polymers of the invention are alkanes, cycloalkanes, alkyl carboxylic acids or esters thereof, cycloalkylcarboxylic acids or esters thereof, ethers, cyclic alkyl ethers alkyl aryl groups, alkyl amides, alkyl-aryl carboxylic acids and esters thereof, bearing an amine group and also bearing one radical scavenger F where unbranched high MW polymers are to be prepared, or alternatively, more than one radical scavenger F where branched molecules are to be prepared.

[0141] Alkylcarboxylic acids, including alkyl dicarboxylic acids, having at least one radical scavenger F, and substituted with amino or hydroxy groups can also be employed as initiators. In some embodiments of the invention where ATRP is employed to prepare high MW polymers of the present invention, the initiators can be alkylcarboxylic acids bearing one or more halogens selected from chlorine and bromine.

[0142] Alkanes substituted with two or more groups selected from -COOH, -OH and -NH , and at least one radical scavenger F, can also be employed as initiators for the preparation of high MW polymers where ATRP is employed to prepare high MW polymers of the present invention.

[0143] Initiators can also contain one or more groups including, but not limited to, -Oi l, amino, monoalkylamino, dialkylamino, -O-alkyl, -COOH, -COO-alkyl, or phosphate groups (or protected forms thereof).

[0144] A broad variety of initiators are commercially available, for example bromoacetic acid N-hydroxysuccinimide ester available from Sigma-Aldrich (St. Louis, MO). Suitably protected forms of those initiators can be prepared using standard methods in the art as necessary.

[0145] Other initiators include thermal, redox or photo initiators, including, for example, alkyl peroxide, substituted alkyl peroxides, aryl peroxides, substituted aryl peroxides, acyl peroxides, alkyl hydroperoxides, substituted aryl hydroperoxides, aryl hydroperoxides, substituted aryl hydroperoxides, heteroalkyl peroxides, substituted heteroalkyl peroxides, heteroalkyl hydroperoxides, substituted heteroalkyl hydroperoxides, heteroaryl peroxides, substituted heteroaryl peroxides, heteroaryl hydroperoxides, substituted heteroaryl hydroperoxides, alkyl peresters, substituted alkyl peresters, aryl peresters, substituted aryl peresters, azo compounds and halide compounds. Specific initiators include cumene hydroperoxide (CHP), tert-butyl hydroperoxide (TBHP), tert-butyl perbenzoate, (TBPB), sodium carbonateperoxide, benzoyl peroxide (BPO), lauroyl peroxide (LPO), methylethyl ketone 45%, potassium persulfate, ammonium persulfate,

2,2-azobis(2,4-dimethyl-valeronitrile), l , l -azobis(cyclo-hexanecarbonitrile),

2,2-azobis(N,N-dimethyleneisobut 'ramidine) dihydrochloride, and 2,2-azobis

(2-amido-propane) dihydrochloride. Redox pairs such as persulfate/sulfite and Fe (2+) peroxide or ammonium persulfate and Ν,Ν,Ν'Ν'-tetramethyIethylenediamine (TEMED).

[0146] Still other initiators useful for preparing the high MW polymers of the present invention, are branched. Suitable initiators having a single branch point include the following: where radical R can be any of the following:

[0147] In some embodiments, the initiator can

which is a protected maleimide that can be deprotected after polymerization to form the maleimide for reaction with additional functional groups.

[0148] Additional branched initiators include, but are not limited to, the following, where radical R is as defined above:

[0149] In some embodiments, the branched initiators include, but are not limited to, the following:

[0150] Other branched initiators useful for preparing the high MW polymers of the present invention include the following:

where radical R is as defined above, and radical X can be CHO, SO2CI, S02CH=CH2, NHCOCH ₂ I, N=C=0 and N=C=S, among others. Additional X groups can include the following:

Still other initiators include, but are not limited to, the following:

[0151] In other embodiments, the initiator can have several branch points to afford a plurality of polymer arms, such as:

where radical R is as defined above. In some other embodiments, the initiator can have the following structure:

[0152] In some other embodiments, the initiator can have the following structures:





As described above, the initiator can be added to the polymerization mixture separately, or can be incorporated into another molecule, such as a monomer (hyperbranched structure) or a polymer fragment (such as graft copolymers). Initiation of the polymerization can be accomplished by heat, UV light, or other methods known to one of skill in the art.

[0153] In some embodiments, the initiator I-] ' of the present invention has the formula:

(F) _r -Sp'-C-Sp ² -r

where the initiator fragment I corresponds to F-Sp'-C-Sp ² . Each radical F is a functional group for reaction with a functional agent or linking group of the present invention. Radical r is from 1 to 10. Radicals Sp ¹ and Sp ² are spacers and can be any suitable group for forming a covalent bond, such as Ci_ ₆ alkyl, aryl or heteroaryl. Radical C can be any core providing one or a plurality of points for linking to one or more spacers, Sp ² (which can be the same or different), and one or more radical scavengers, Γ, and providing one or a plurality of points for linking to one or more spacers, Sp' (which can be the same or different), and one or more functional groups, F (which can be the same or different). Core C can be any suitable structure, such as a branched structure, a crosslinked structure including heteroatoms, such as silsesquiloxanes, and a linear, short polymer with multiple pendant functional groups. In addition, core C can be attached to the one or more Sp ¹ and Sp ² spacers by any suitable group for forming a covalent bond including, but not limited to, esters, amides, ethers, and ketones. Radical scavenger Γ is a radically transferable atom or group such as, but not limited to, a halogen, CI, Br, I, OR ¹⁰ , SR ^{1 1} , SeR ^{1 1} , OC(=0)R ⁿ , OP(=0)R ⁿ , 0P(=0)(0R" ) ₂ , 0-(R" )2, S-C(=S)N(R") ₂ , CN, NC, SCN, CNS, OCN, CNO, N ₃ , OH, O, C 1 -C6-alkoxy, (S0 ), P0 ₄ , HPO ₄ , H ₂ PO ₄ , triflate, hexafluorophosphate, methanesulfonate, arylsulfonate, carboxylic acid halide. R ¹⁰ is an alkyl of from 1 to 20 carbon atoms or an alkyl of from 1 to 20 carbon atoms in which each of the hydrogen atoms may be replaced by a halide, alkenyl of from 2 to 20 carbon atoms, alkynyl of from 2 to 10 carbon atoms, phenyl, phenyl substituted with from 1 to 5 halogen atoms or alkyl groups with from 1 to 4 carbon atoms, aralkyl, aryl, aryl substituted alkyl, in which the aryl group is phenyl or substituted phenyl and the alkyl group is from 1 to 6 carbon atoms, and R ^{1 1} is aryl or a straight or branched C1-C20 alkyl group or where an N(R ^] ')2 group is present, the two R ¹ ' groups may be joined to form a 5-, 6- or 7-member heterocyclic ring. Spacer Sp ¹ covalently links functional group F and core C while spacer Sp ² covalently links core C and radical scavenger P.

[0154] In other embodiments, the initiator of the present invention has the formula:

LG ² — L ⁵ -C- L ⁴ — I' wherein each P is independently selected from halogen, -SCN, or -NCS. L ⁴ and L ³ are each independently a bond or a linker, such that one of L ⁴ and L ³ is a l inker. C is a bond or a core group. LG ² is a linking group. And subscript p is from 1 to 100, wherein when subscript p is 1 , C is a bond, and when subscript p is from 2 to 100, C is a core group. In some embodiments, subscript p is from 2 to 100. In other embodiments, subscript p is from 3 to 20. Subscript p can also be 3, 6, 9, or 12. In some embodiments, subscript p is 9.

[0155] In some other embodiments, the initiator has the formula:

wherein each R ^J and R is independently selected H, CN or C | _ alkyl, and X is O or NH.

[0156] In some embodiments, the core group C has the formula:

wherein B, B' and B" are each independently a branching unit, L, L' and L" can each independently be a bond or a linker; subscript k is 0 or 1 ; and subscripts n, n' and n" are each independently an integer of 0, 2 or 3, wherein at least one of n, 11 ' and n" is other than 0, and subscript p is equal to the product of n, n' and n". [0157] In some embodiments, subscript k is 0, and subscripts n and n' are both 3. In some embodiments, subscript k is 0, subscript n is 3 and subscript n' is 2. In some embodiments, subscript k is 0, subscript n is 2 and subscript n ' is 3.

[0158] Branching units B, B' and B" can be any suitable branching unit, and can have 2, 3, 4 or more branches. In some embodiments, the branching units can be any of the following:

In some embodiments, the branching units can be any of the following

[0159] In still other embodiments, the initiator can have any of the following structures:

53

B. Monomers

[0160] Monomers useful for preparing the high MW polymers of the present invention include any monomer capable of radical polymerization. Typically, such monomers have a vinyl group. Suitable monomers include, but are not limited to, acrylate, methacrylate, acrylamide, methacrylamide, styrene, vinyl-pyridine, vinyl-pyrrolidone and vinyl esters such as vinyl acetate monomers. Monomers useful in the present invention include a hydrophilic group. The hydrophilic group of the present invention can be any suitable hydrophilic group. For example, the hydrophilic group can include z itterionic groups and hydrophilic polymers. In some embodiments, each hydrophilic group includes a zwitterionic group. Zwitterion groups of the present invention include any compound having both a negative charge and a positive charge. Groups having a negative charge and suitable for use in the zwitterions of the present invention include, but are not limited to, phosphate, sulfate, other oxoanions, etc. Groups having a positive charge and suitable for use in the zwitterions of the present invention include, but are not limited to, ammonium ions. In some embodiments, the zwitterion can be phosphorylcholine. Other zwitterions useful in the present invention include those described in WO1994016748 and WGT 994016749 (incorporated herein by reference). Hydrophilic polymers useful in the present invention include polyethyleneoxide, polyoxazoline, cellulose, dextran, and other polysaccharide polymers. One of skill in the art will appreciate that other hydrophilic polymers are useful in the present invention.

[0161] Other hydrophilic groups include, but are not limited to, hydroxy, amine, carboxylic acid, amide, sulfonate and phosphonate. Monomers useful in the present invention that include such hydrophilic groups include, but are not lim ited to, acrylamide, N- isopropylacrylamide (NiPAAM) and other substituted acrylamide, aciylic acid, and others.

[0162] Monomers, M ¹ , containing the zwitterionic moiety, ZW, include, but are not limited to, the following:

Other monomers are well-known to one of skill in the art, and include vinyl acetate and derivatives thereof.

[0163] In some embodiments, the hydrophilic group can be a zwitterionic group. In some embodiments, the monomer can be 2-(methacryloyloxyethyl)-2'-(trimethylammoniumethyl) phosphate (HEMA-PC). In some other embodiments, the monomer can be 2- (acryloyloxyethyl)-2'-(trimethylammoniumethyl) phosphate.

C. Linkers

[0164] The high MW polymers of the present invention can also incorporate any suitable linker L. The linkers L ³ provide for attachment of the functional agents to the initiator fragment I and the linkers L ¹ and L ² provide for attachment of the zwitterionic groups to the comonomers M ¹ and M ² . The linkers can be cleavable or non-cleavable, homobifunctional or heterobifunctional. Other linkers can be both heterobifunctional and cleavable, or homobifunctional and cleavable.

[0165] Cleavable linkers include those that are hydrolyzable linkers, enzymatically cleavable linkers, pH sensitive linkers, disulfide linkers and photolabile linkers, among others. Hydrolyzable linkers include those that have an ester, carbonate or carbamate functional group in the linker such that reaction with water cleaves the linker. Enzymatically cleavable linkers include those that are cleaved by enzymes and can include an ester, amide, or carbamate functional group in the linker. pH sensitive linkers include those that are stable at one pH but are labile at another pH. For pH sensitive linkers, the change in pH can be from acidic to basic conditions, from basic to acidic conditions, from mildly acidic to strongly acidic conditions, or from mildly basic to strongly basic conditions. Suitable pH sensitive linkers are known to one of ski ll in the art and include, but are not limited to, ketals, acetals, imines or imminiums, siloxanes, silazanes, silanes, maleamates-amide bonds, ortho esters, hydrazones, activated carboxylic acid derivatives and vinyl ethers. Disulfide linkers are characterized by having a disulfide bond in the linker and are cleaved under reducing conditions. Photolabile linkers include those that are cleaved upon exposure to light, such as visible, infrared, ultraviolet, or electromagnetic radiation at other wavelengths. [0166] Other linkers useful in the present invention include those described in U.S. Patent Application Nos. 2008/0241 102 (assigned to Ascendis/Complex Biosystems) and

2008/01 52661 (assigned to Minis), and International Patent Application Nos. WO

2004/010957 and 2009/1 1 753 1 (assigned to Seattle Genetics) and 01/24763, 2009/134977 and 2010/126552 (assigned to Immunogen) (incorporated in their entirety herein). Mirus linkers useful in the present invention include, but are not limited to, the following:

Other linkers include those described in Bioconjugate Techniques, Greg T. Hermanson, Academic Press, 2d ed., 2008 (incorporated in its entirety herein), and those described in Angew. Chem. Int. Ed. 2009, 48, 6974-6998 (Bertozzi, C.R. and Sletten, E.M) (incorporated in its entirety herein).

[0167] The linkers of the present invention can have a length of up to 30 atoms, each atom independently C, N, O, S, and P. In some embodiments, the linkers L ¹ , L ² , L ³ , L ⁴ , or L ⁵ , or L, L' and L"can be any of the following: -C i ₂ alkyl-, -C3-12 cycloalkyl-, -(C| .g alkyI)-(C3-| ₂ cycloalkylHCa.8 alkyl)-, -(CH ₂ ),_, ₂ 0-, (-(CH ₂ ),. ₆ -0-(CH ₂ ),. ₆ -),. ₁₂ -,

(-(CH2) I-4-N H-(CH ₂ )M) _M 2- _S (-(CH ₂ ),_4-0-(CH ₂ ),_ ₄ )i-i2-0-, -(CH ₂ ),-8-CONR-(CH ₂ CH ₂ 0) ₁ . , 2-, -(CH ₂ )i -8-CONR-(CH ₂ CH ₂ 0) ₁ .8-NH-(CH ₂ ) ₁ .6-, -(CH ₂ )i-8-CONR-CH ₂ CH ₂ -(OCH ₂ CH2)o-6- NHCO-(CH ₂ ) ,.3-, -C(0)-(CH ₂ ), .6-NHC(0)-(CH ₂ )i. ₆ -, -C(OHCH ₂ )i-6-OC(0)-(CH ₂ )i.6-, - C(0)-(CH ₂ ),. ₆ -, -OC(0)-(C ,. ₆ alkyl), -NHC(0)-(C ,. ₆ alkyl),

(-(CH ₂ )M-0-(CH ₂ )I -4-)I-I 20-(CH2)M 2- ₅ -(CH ₂ ),-, 2-(C=0)-0-, -(CH ₂ ),., ₂ -0-(C=0)-, -(phenyl (CH ₂ ) , .3-(C=0)-0-, -(phenyl)-(CH ₂ ),- ₃ -(C=0)-NH-, -(C, - ₆ alkyl)-(C=O)-O-(C ₀ - ₆

alkyl)-, -(CH ₂ )i_, ₂ -(C=0)-0-(CH ₂ ),., ₂ -, -C H(OH)-CH(OH)-(C=0)-0-, -CH(OH)-CH(OH)-( C=0)-NH-, -S-maleimido-(CH ₂ )i_6-, -S-maleimido-(Ci_ ₃

alkyl)-(C=0)-N H-, -S-maleimido-(Ci. ₃ alkyl)-(C ₅ - ₆ cycloalkyl)-(C ₀ - ₃ alkyl)-, -(C, _-3 alkyl)-(C ₅ -6 cycloalkyl)-(C ₀ - ₃ alkyl)-(C=0)-0-, -(C|. ₃ alkyl)-(C ₅ - ₆ cycloalkyl)-(C ₀ - ₃

alkyl)-(C=0)-NH-, -S-maleimido-(C ₀ . ₃ alkyl)-phenyl-(Co-3alkyl)-, -(C0-3

-(phenyl)-(CH ₂ ),

=0)-0-(CH ₂ ) ₂ -0-(C=0)-(CH ₂ ) ₂ -(C=0)-NH-, -(C,- ₆ alkyl)-(C=0)-N-(C _r6 alkyl)-, acetal, ketal, acyloxyalkyl ether, -N=CH-, -(d- ₆ alkyl)-S-S-(C ₀ - ₆ alkyl)- , -(C,- ₅ alkyl)-S-S-(C i- ₆ alkyl)-(C=0)-0-, -(C ,- ₆ alkyl)-S-S-(C,- ₆ alkyl)-(C=0)-NH-, -S-S-(CH ₂ )u-(C=0)-NH-(CH ₂ )i-4-NH-(C=0)- (CH ₂ ),. ₃ -, -S-S-(C ₀ - ₃ alkyl)-(phenyl)-, -S-S-(Ci _-3 -alkyl>(phenyl)-(O0)-NH-(CH ₂ )i.s-, -(C1.3

alkyl)-(phenyl)-(C=0)-NH-(CH ₂ )i- ₅ -(C=0)-NH-, -S-S-(Ci _-3 -alkyl)-, -(C,. ₃ -alkyl)-(phenyl)-( C=0)-NH-, -0-(C,-C ₆ alkyl)-S(0 ₂ )-(C,- ₆

alkyl)-0-(O0)-NH-, -S-S-(CH ₂ ) _U3 -(C=0)-, -(CH ₂ ),. ₃ -(C=0)-NH-N=C-S-S-(CH ₂ ),. ₃ -(C=0) -NH-(CH ₂ )|.5-, -(CH ₂ ),. ₃ -(C=0)-NH-(CH ₂ )i- ₅ -(C=0)-NH-, -(CH ₂ ) ₀ - ₃ -(heteroatyl)-(CH ₂ )o-3-, -(CH ₂ )o. ₃ -pheny]-(CH ₂ )o- ₃ -, -N=C(R)-, -(C,. ₆ alkyl)-C(R)= -(C,. ₆ alkyl)-, -(C, . ₆

alkyl)-(aryl)-C(R)=N-(C,_6 alkyl)-, -(C,„ ₆ alkyl)-C(R)=N-(aryl)-(C,_ ₆ alkyl)-, and -(d. ₅ alkyl)-O-P(O)(OH)-O-(C ₀ -6 alkyl)-, wherein R is H, C,_ ₆ alkyl, C ₃ . ₆ cycloalkyl, or an aryl group.

[0168] In some other embodiments, linkers L ¹ , L ² and L ³ can be any of the

following: -C,-C, ₂ alkyl-, -C ₃ -C, ₂ cycloalkyl-, (-(CH ₂ )i. ₆ -0-(CH ₂ )i-6-)i-! 2-,

(-(CH2)I-4-NH-(CH ₂ ) ) I-12-, -(CH ₂ ), ., ₂ 0-,

(-(CH ₂ ) I.4-0-(CH ₂ ) ₁ . ₄ ) _I -,2-0-, -(CH ₂ ) ₂ -(CO)-0-, -(CH ₂ ), . , ₂ -(CO)-N H-, -(CH ₂ ) _{M 2} -0-(CO)-, -(CH ₂ ),., ₂ -NH-(CO)-,

(-(CH ₂ ) ₁ . ₄ -0-(CH ₂ ), . ₄ ) ₁ . _{1 2} -0-(CH ₂ ),. ₁₂ -, -(CH ₂ ) _{M 2} -(CO)-0-(CH ₂ ),_ ₁₂ -, -(CH ₂ ) _{M 2} -(CO)-NH-( CH ₂ ) _M2 -, -(CH ₂ ), _| ₂ -0-(CO)-(CH ₂ ),. _l2 -, -(CH ₂ ),., 2-NH-(COHCH ₂ ) ₂ -, -(C ₃ -C _{l 2} cycloalkyl)-, -(C ,-C ₈ alkyl)-(C ₃ -Ci ₂ cycloalkyl)-, -(C ₃ -C, ₂

cycloalkyl)-(C,. ₈ alkyl)-, cycloalkyl)-(C , . ₈ alkyl)-,

and -(CH ₂ ) ₀ - ₃ -aryl-(CH ₂ ) ₀ - ₃ -.

[0169J In some embodiments, linkers L, L' and L" can be -C(0)-(CH ₂ ),. ₆ - HC(0)-(CH ₂ ),. 6-, -C(0)-(CH ₂ ),.6-OC(0)-(CH ₂ ),. ₆ -, or -C(0)-(CH ₂ ),. ₆ -.

[0170] In still other embodiments, each of linkers L ¹ , L ² and L ³ is a cleavable linker independently selected from hydrolyzable linkers, enzymatically cleavable linkers, pH sensitive linkers, disulfide linkers and photolabile linkers.

[0171] Other linkers useful in the present invention include self-immolative linkers. Useful self-immolative linkers are known to one of skill in the art, such as those useful for antibody drug conjugates. Exemplary self-immolative linkers are described in U.S. Patent No.

7,754,681 . D. Linking Groups LG

[0172] The linkers and functional agents of the present invention can react with a linking group on the initiator fragment I to form a bond. The linking groups LG of the present invention can be any suitable functional group capable of forming a bond to another functional group, thereby linking the two groups together. For example, linking groups LG useful in the present invention include those used in click chemistry, maleimide chemistry, and NHS-esters, among others. Linking groups involved in click chemistry include, but are not limited to, azides and alkynes that form a triazole ring via the Huisgen cycloaddition process (see U.S. Patent No. 7,375,234, incorporated herein in its entirety). The maleim ide chemistry involves reaction of the maleimide olefin with a nucleophile, such as -OH, -SH or -NH ₂ , to form a stable bond. Other linking groups include those described in Bioconjugate Techniques, Greg T. Hermanson, Academic Press, 2d ed., 2008 (incorporated in its entirety herein).

[0173] Some non-limiting examples of the reaction of the linking groups and some groups typically found or introduced into functional agents are set forth in Table 1.

Table I

Illustrative Groups Exemplary Reactive

that may react with Linking Groups Product Y-X a linking group (LG) (shown as appended to -X)

Y-NH ₂ R'OCH(OH)-X or Y-N=CH-X

hemiacetal or

Y-NH-CH-X following reduction

Y-NH ₂ R'(0=)C-X Y-N=CR'-X

ketone or

Y-NH-C(R')H-X following reduction

Y-NH ₂ (R'0) ₂ C(R')-X or Y-N=C(R')-X

or

Y-NH-C(R')H-X following reduction ketal

Y-NH ₂ R'OC(R')(OH)-X Y-N=C(R')-X

hemiketal or

Y-NH-C(R')H-X following reduction

Y-NH ₂ R'(S=)C-X Y-N=C(R')-X

ketone or

thione (thioketone) Y-NH-C(R')H-X following reduction

Y-NH ₂ (R'0)(R'S)C(R')-X or Y-N=C(R')-X

or

Y-NH-C(R')H-X following reduction monothioketal

Y-NH ₂ R'SC(R'XSH)-X or Y-N=C(R')-X

dithiohemiketal or

Y-NH-C(R')H-X following reduction

Y-NH ₂ (R'S) ₂ C(R')-X or Y-N=C(R')-X

or

Y-NH-C(R')H-X following r"" ^"S _\ '

reduction dithioketal

Y-SH Y-S-CH ₂ -C(OH)(R")-X-

^R " Y-OH epoxide (oxirane) Y-0-CH ₂ -C(OH)(R")-X-

Y-COOH (anion) Y-C(=0)0-CH ₂ -C(OH)(R")-X- Y-NHR" Y-NR"-CH ₂ -C(OH)(R' ' )-X-

R is Ci-6 alkyl, C ₃ - ₆ cycioalkyl, or an aryl group having 5-8 endocyclic atoms;

R is H, C| -6 alkyl, C ₃ -e cycioalkyl, or an aryl group having 5-8 endocyclic atoms;

R ^" is a carbonyl derivative *- (CO)-, * - (CO)-(CH ₂ )i. ₈ -S-S-,

*- (CO)-(CH ₂ )i .8-(CO)-0-, *- (CO)-(CH ₂ ),_8-0-(CO)-, * - (CO)-(CH ₂ ),. ₈ -(CO)-NH- , or

*- (C )-(CH ₂ )i. ₈ -NH-(C )-, or alternatively, R is carbonyl derivative of the form *- (C )-0-(CH ₂ ),_ ₈ -S-S-, *- (C0)-0-(CH ₂ ), _8-(CO)-0- ,

*- (C )-0-(CH ₂ )i_g-0-(CO)-, *- (C0)-0-(CH ₂ ) _K8 -(C )-NH- , or

*- (C )-0-(CH ₂ )i.s-NH-(C )-, where "*" indicates the point of attachment to succinimidyl or benzotriazolyl groups;

X and Y are each the active agent, linker, monomer or initiator fragment I. -C(0)NR ^la R ^lb , -NR ^la R ^{l b} ,

Cj.6 alkyl-NR ^la R ^Ib , -N(R ^la )C(0)R ^lb , -N(R ^,a )C(0)OR ^l , -N(R ^,a )C(0)NR ^la R ^{l b} , -OP(0)(OR ^u ) ₂ , -S(0) ₂ OR ^{l a} , -S(0) ₂ NR ^{, a} R ^lb , -CN, -N0 ₂ , cycloalkyl, heterocycloalkyl, aryl and heteroaryl

E. Functional agents [0174] Functional agents useful in the high MW polymers of the present invention include any biological agent or synthetic compound capable of targeting a particular ligand, receptor, complex, organelle, cell, tissue, epithelial sheet, or organ, or of treating a particular condition or disease state. In some embodiments, the bioactive agent is a drug, a therapeutic protein, a small molecule, a peptide, a peptoid, an oligonucleotide (aptamer, siRNA, microRNA), a nanoparticle, a carbohydrate, a lipid, a glycolipid, a phospholipid, or a targeting agent. Other functional agents useful in the high MW polymers of the present invention include, but are not limited to, radiolabels, contrast agents, fluorophores and dyes.

[0175] The functional agents can be linked to the initiator fragment I or the radical scavenger Γ, or both, of the high MW polymers. The functional agents can be linked to the initiator fragment I or the radical scavenger Γ either before or after polymerization via cleavable or non-cleavable linkers described above. The functional agent can also be physisorbed or ionically absorbed to the high MW polymer instead of covalently attached.

[0176] The preparation of the high MW polymers of the present invention linked to a functional agent can be conducted by first linking the functional agent to a linking group attached to an initiator fragment and subjecting the coupled functional agent to conditions suitable for synthesis of the inventive high MW polymers. In those cases, a suitable linking group can be an initiator (e.g., iodinated, brominated or chlorinated compound/group) for use in ATRP reactions. Such a reaction scheme is possible where the functional agent is compatible with the polymer polymerization reactions and any subsequent workup required. However, coupling of functional agents to preformed high MW polymers can be used where the functional agent is not compatible with conditions suitable for polymerization. In addition, where cost makes the loss of an agent to imperfect synthetic yields, oftentimes encountered particularly in multistep synthetic reactions, coupling of functional agent to preformed high MW polymers of the present invention can be employed.

[0177] Where a functional agent is not compatible with the conditions employed for polymerization reactions, it can be desirable to introduce the functional agent subsequent to the polymerization reaction. [0178] Bioactive agents, A, can be broadly selected. In some embodiments the bioactive agents can be selected from one or more drugs, vaccines, aptamers, avimer scaffolds based on human A domain scaffolds, diabodies, camelids, shark IgNAR antibodies, fibronectin type III scaffolds with modified specificities, antibodies, antibody fragments, vitamins and cofactors, polysaccharides, carbohydrates, steroids, lipids, fats, proteins, peptides, polypeptides, nucleotides, oligonucleotides, polynucleotides, and nucleic acids (e.g., mRNA, tRNA, snRNA, RNAi, microRNA, DNA, cDNA, antisense constructs, ribozymes, etc, and combinations thereof). In one embodiment, the bioactive agents can be selected from proteins, peptides, polypeptides, soluble or cell-bound, extracellular or intracellular, kinesins, molecular motors, enzymes, extracellular matrix materials and combinations thereof. In another embodiment, bioactive agents can be selected from nucleotides, oligonucleotides, polynucleotides, and nucleic acids (e.g. , mRNA, tRNA, snRNA, RNAi, DNA, cDNA, antisense constructs, ribozymes etc and combinations thereof). In another embodiment, bioactive agents can be selected from steroids, lipids, fats and combinations thereof. For example, the bioactive agent can bind to the extracellular matrix, such as when the extracellular matrix is hyaluronic acid or heparin sulfate proteoglycan and the bioactive agent is a positively charged moiety such as choline for non-specific, electrostatic, Velcro type binding interactions. In another embodiment, the bioactive agent can be a peptide sequence that binds non-specifically or specifically.

[0179] Bioactive agents can be designed and/or selected to have a full activity (such as a high level of agonism or antagonism). Alternatively, a multifunctional bioactive agent can be selected to modulate one target protein's activity while impacting fully another.

[0180] Just as mosaic proteins contain extracellular binding domains or sub-domains (example, VEGF and Heparin Binding Epidermal Growth Factor), sequences from these binding sites can be replicated as a bioactive agent for polymer attachment. More broadly, mosaic proteins represent strings of domains of many functions (target binding, extracellular matrix binding, spacers, avidity increases, enzymatic). The set of bioactives chosen for a particular application can be assembled in similar fashion to replicate a set of desired functional activities.

[0181] Other functional agents, A, include charged species such as choline, lysine, aspartic acid, glutamic acid, and hyaluronic acid, among others. The charged species are useful for facilitating ionic attachment, to vitreous for example. Therapeutic Proteins and Antibodies

[0182] In one particularly useful embodiment, the functional agent is a therapeutic protein. Numerous therapeutic proteins are disclosed throughout the application such as, and without limitation, erythropoietin, granulocyte colony stimulating factor (G-CSF), GM-CSF, interferon alpha, interferon beta, human growth hormone, imiglucerase, and RANK ligand.

[0183] In one embodiment, the functional agents can be selected from specifically identified polysaccharide, protein or peptide bioactive agents, including, but not limited to: Αβ, agalsidase, alefacept, alkaline phosphatase, aspariginase, amdoxovir (DAPD), antide, becaplermin, botulinum toxin including types A and B and lower molecular weight compounds with botulinum toxin activity, calcitonins, CDl d, cyanovirin, denileukin diftitox, erythropoietin (EPO), EPO agonists, dornase alpha, erythropoiesis stimulating protein (NESP), coagulation factors such as Factor V, Factor VII, Factor Vila, Factor VIII, B domain deleted Factor VIII, Factor IX, Factor X, Factor XII, Factor XIII, von Willebrand factor; ceredase, Fc gamma r2b, cerezyme, alpha-glucosidase, N-Acetylgalactosamine-6-sulfate sulfatase, collagen, cyclosporin, alpha defensins, beta defensins, desmopressin, exendin-4, cytokines, cytokine receptors, granulocyte colony stimulating factor (G-CSF),

thrombopoietin (TPO), alpha-1 proteinase inhibitor, elcatonin, granulocyte macrophage colony stimulating factor (GM-CSF), fibrinogen, filgrastim, growth hormones human growth hormone (hGH), somatropin, growth hormone releasing hormone (GHRH), GRO-beta, GRO-beta antibody, bone morphogenic proteins such as bone morphogenic protein-2, bone morphogenic protein-6, parathyroid hormone, parathyroid hormone related peptide, OP- 1 ; acidic fibroblast growth factor, basic fibroblast growth factor, Fibroblast Growth Factor 21 , CD40 ligand, 1COS, CD28, B7- 1 , B7-2, TLR and other innate immune receptors, heparin, human serum albumin, low molecular weight heparin (LMWH), interferon alpha, interferon beta, interferon gamma, interferon omega, interferon tau, consensus interferon; interleukins and interleukin receptors such as interleukin- 1 receptor, interleukin-2, interleukin-2 fusion proteins, interleukin- 1 receptor antagonist, interleukin-3, interleukin-4, interleukin-4 receptor, interleukin-6, interleukin-8, interleukin- 12, interleukin- 1 7, interleukin-21 , interleukin- 13 receptor, interleukin- 17 receptor; lactoferrin and lactoferrin fragments, luteinizing hormone releasing hormone (LHRH), insulin, pro-insulin, insulin analogues, amylin, C-peptide, somatostatin, somatostatin analogs including octreotide, vasopressin, follicle stimulating hormone (FSH), imiglucerase, influenza vaccine, insulin-like growth factor (IGF), insulintropin, macrophage colony stimulating factor (M-CSF), plasminogen activators such as alteplase, urokinase, reteplase, streptokinase, pamiteplase, lanoteplase, and teneteplase; nerve growth factor (NGF), trk A, trk B, osteoprotegerin, platelet-derived growth factor, tissue growth factors, transforming growth factor- 1 , vascular endothelial growth factor, leukemia inhibiting factor, keratinocyte growth factor ( GF), glial growth factor (GGF), T Cell receptors, CD molecules/antigens, tumor necrosis factor (TNF) (e.g., TNF-a and TNF-β), TNF receptors (e.g., TNF-a receptor and TNF-β receptor), CTLA4, CTLA4 receptor, monocyte chemoattractant protein- 1 , endothelial growth factors, parathyroid hormone (PTH), PTHrP, glucagon-like peptide, somatotropin, thymosin alpha 1 , rasburicase, thymosin alpha 1 Ilb/IIIa inhibitor, thymosin beta 1 0, thymosin beta 9, thymosin beta 4, alpha- 1 antitrypsin, phosphodiesterase (PDE) compounds, VLA-4 (very late antigen-4), VLA-4 inhibitors, bisphosponates, respiratory syncytial virus antibody, cystic fibrosis transmembrane regulator (CFTR) gene, deoxyribonuclease (Dnase), bactericidal/permeability increasing protein (BPI), and anti-CMV antibody. Exemplary monoclonal antibodies include etanercept (a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human 75 kD TNF receptor linked to the Fc portion of IgG l ), abciximab, adalimumab, afelimomab, alemtuzumab, antibody to B- lymphocyte, atlizumab, basiliximab, bevacizumab, biciromab, bertilimumab, CDP-484, CDP-571 , CDP-791 , CDP-860, CDP-870, cetuximab, clenoliximab, daclizumab, eculizumab, edrccolomab, efalizumab, epratuzumab, fontolizumab, gavilimomab, gemtuzumab ozogamicin, ibritumomab tiuxetan, infliximab, inolimomab, keliximab, labetuzumab, lerdelimumab, olizumab, radiolabeled lym- 1 , metelimumab, mepolizumab, mitumomab, muromonad-CD3, nebacumab, natalizumab, odulimomab, omalizumab, oregovomab, palivizumab, pemtumomab, pexelizumab, rhuMAb-VEGF, rituximab, satumomab pendetide, sevirumab, siplizumab, tositumomab, I ¹³¹ tositumomab, trastuzumab, tuvirumab, visilizumab, and fragments and mimetics thereof.

[0184] In one embodiment, the bioactive agent is a fusion protein. For example, and without limitation, the bioactive component can be an immunoglobulin or portion of an immunoglobulin fused to one or more certain useful peptide sequences. For example, the bioactive agent may contain an antibody Fc fragment. In one embodiment, the bioactive agent is a CTLA4 fusion protein. For example, the bioactive agent can be an Fc-CTLA4 fusion protein. In another embodiment, the bioactive agent is a Factor VIII fusion protein. For example, the bioactive agent can be an Fc-Factor VIII fusion protein.

[0185] In one particularly useful embodiment, the bioactive agent is a human protein or human polypeptide, for example, a hetcrologously produced human protein or human polypeptide. Numerous proteins and polypeptides are disclosed herein for which there is a corresponding human form (i.e., the protein or peptide is normally produced in human cells in the human body). Therefore, in one embodiment, the bioactive agent is the human form of each of the proteins and polypeptides disclosed herein for which there is a human form. Examples of such human proteins include, without limitation, human antibodies, human enzymes, human hormones and human cytokines such as granulocyte colony stimulation factor, granulocyte macrophage colony stimulation factor, interferons (e.g., alpha interferons and beta interferons), human growth hormone and erythropoietin.

[0186] Other examples of therapeutic proteins which (themselves or as the target of an antibody or antibody fragment or non-antibody protein) may serve as bioactive agents include, without limitation, factor VIII, b-domain deleted factor VIII, factor Vila, factor IX, factor X, anticoagulants; hirudin, alteplase, tpa, reteplase, tpa, tpa - 3 of 5 domains deleted, insulin, insulin lispro, insulin aspart, insulin glargine, long-acting insulin analogs, complement C5, hgh, glucagons, tsh, follitropin-beta, fsh, gm-csf, pdgh, ifn alpha2, ifn alpha2a, ifn alpha2b, inf-aphal , consensus ifn, ifn-beta, ifn-beta l b, ifn-beta l a, ifn-gamma (e.g., 1 and 2), ifn-lambda, ifn-delta, i 1-2, il- 1 1 , hbsag, ospa, murine mab directed against t- lymphocyte antigen, murine mab directed against tag-72, tumor-associated glycoprotein, fab fragments derived from chimeric mab directed against platelet surface receptor gpII(b)/III(a), murine mab fragment directed against tumor-associated antigen cal 25, lysyl oxidase, LOX2, murine mab fragment directed against human carcinoembryonic antigen, cea, murine mab fragment directed against human cardiac myosin, murine mab fragment directed against tumor surface antigen psma, murine mab fragments (fab/fab2 mix) directed against hmw-maa, murine mab fragment (fab) directed against carcinoma-associated antigen, mab fragments (fab) directed against nca 90, a surface granulocyte nonspecific cross reacting antigen, chimeric mab directed against cd20 antigen found on surface of b lymphocytes, humanized mab directed against the alpha chain of the il2 receptor, chimeric mab directed against the alpha chain of the il2 receptor, chimeric mab directed against tnf-alpha, humanized mab directed against an epitope on the surface of respirator synctial virus, humanized mab directed against her 2, human epidermal growth factor receptor 2, human mab directed against cytokeratin tumor-associated antigen anti-ctla4, chimeric mab directed against cd 20 surface antigen of b lymphocytes dornase-alpha dnase, beta glucocerebrosidase, tnf-alpha, il-2-diptheria toxin fusion protein, tnfr-lgg fragment fusion protein laronidase, dnaases, alefacept, darbepoetin alpha (colony stimulating factor), tositumomab, murine mab, alemtuzumab, rasburicase, agalsidase beta, teriparatide, parathyroid hormone derivatives, adalimumab (Iggl ), anakinra, biological modifier, nesiritide, human b-type natriuretic peptide (hbnp), colony stimulating factors, pegvisomant, human growth hormone receptor antagonist, recombinant activated protein c, omalizumab, immunoglobulin e (lge) blocker, lbritumomab tiuxetan, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigmentary hormones, somatomedin, erythropoietin, luteinizing hormone, chorionic gonadotropin, hypothalmic releasing factors, etanercept, antidiuretic hormones, prolactin and thyroid stimulating hormone. And any of these can be modified to have a site-specific conjugation point (a N-terminus, or C-terminus, or other location) using natural (for example, a serine to cysteine substitution) (for example, formylaldehyde per method of Redwood Biosciences) or non-natural amino acid. Non-natural amino acid residue(s) can be selected from the group consisting of: azidonorleucine, 3-(l-naphthyl)alanine, 3-(2-naphthyl)alanine, p-ethynyl-phenylalanine, p-propargly-oxy-phenylalanine, m-ethynyl-phenylalanine, 6- ethynyl-tryptophan, 5-ethynyl-tryptophan, (R)-2-amino-3-(4-ethynyl- l H-pyrol-3-yl)propanic acid, p-bromophenylalanine, p-iodophenylalanine, p-azidophenylalanine, p- acetylphenylalanine, 3-(6-chloroindolyl)alanine, 3-(6-bromoindolyl)alanine, 3-(5- bromoindolyl)alanine, azidohomoalanine, homopropargylglycine, p-chlorophenylalanine, - aminocaprylic acid, O-methyl-L-tyrosine, N-acetylgalactosamine-a-threonine, and N- acetylgalactosamine-a-serine.

[0187] Examples of therapeutic antibodies that may serve as bioactive agents (by themselves or fragments of such antibodies) include, but are not limited, to HERCEPTIN™ (Trastuzumab) (Genentech, CA) which is a humanized anti-HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer; REOPRO™ (abciximab) (Centocor) which is an anti-glycoprotein Ilb/IIIa receptor on the platelets for the prevention of clot formation; ZENAPAX™ (daclizumab) (Roche Pharmaceuticals, Switzerland) which is an immunosuppressive, humanized anti-CD25 monoclonal antibody for the prevention of acute renal allograft rejection; PANOREX™ which is a murine anti-17-IA cell surface antigen IgG2a antibody (Glaxo Wellcome/Centocor); BEC2 which is a murine anti-idiotype (GD3 epitope) IgG antibody (ImClone System); IMC-C225 which is a chimeric anti-EGFR IgG antibody (ImClone System); VITAXIN™ which is a humanized anti-aVp3 integrin antibody (Applied Molecular Evolution/Medlmmune); Campath; Campath 1 H/LDP-03 which is a humanized anti CD52 IgG 1 antibody (Leukosite); Smart M 195 which is a humanized anti-CD33 IgG antibody (Protein Design Lab/Kanebo); RITUXAN™ which is a chimeric anti-CD20 IgG 1 antibody (IDEC Pharm/Genentech, Roche/Zettyaku); LYMPHOCIDE™ which is a humanized anti-CD22 IgG antibody (Immunomedics); ICM3 is a humanized anti-ICAM3 antibody (ICOS Pharm); IDEC- 1 14 is a primate anti-CD80 antibody (IDEC Pharm/Mitsubishi); ZEVALIN™ is a radiolabelled murine anti-CD20 antibody (IDEC/Schering AG); IDEC- 131 is a humanized anti-CD40L antibody (IDEC/Eisai);

lDEC- 151 is a primatized anti-CD4 antibody (IDEC); IDEC- 152 is a primatized anti-CD23 antibody (IDEC/Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG (Protein Design Lab); 5G 1.1 is a humanized anti-complement factor 5 (CS) antibody (Alexion Pharm); D2E7 is a humanized anti-TNF-a antibody (CATIBASF); CDP870 is a humanized anti-TNF-a Fab fragment (Celltech); IDEC- 151 is a primatized anti-CD4 IgG 1 antibody (IDEC Pharm/SmithKline Beecham); MDX-CD4 is a human anti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571 is a humanized anti-TNF-α IgG4 antibody (Celltech); LDP-02 is a humanized anti-a4p7 antibody (LeukoSite/Genentech); OrthoClone OKT4A is a humanized anti-CD4 IgG antibody (Ortho Biotech); ANTOV A™ is a humanized anti-CD40L IgG antibody (Biogen); ANTEGREN™ is a humanized anti-VLA-4 IgG antibody (Elan); CAT-152, a human anti-TGF-Pa antibody (Cambridge Ab Tech); Cetuximab (BMS) is a monoclonal anti-EGF receptor (EGFr) antibody; Bevacizuma (Genentech) is an anti-VEGF human monoclonal antibody; Infliximab (Centocore, JJ) is a chimeric (mouse and human) monoclonal antibody used to treat autoimmune disorders; Gemtuzumab ozogamicin (Wyeth) is a monoclonal antibody used for chemotherapy; and Ranibizumab (Genentech) is a chimeric (mouse and human) monoclonal antibody used to treat macular degeneration.

[0188] Other antibodies, such as single domain antibodies are useful in the present invention. A single domain antibody (sdAb, called Nanobody by Ablynx) is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, the sdAb is able to bind selectively to a specific antigen. With a molecular weight of only 12-15 kDa, single domain antibodies are much smaller than common antibodies ( 150-160 kDa). A single domain antibody is a peptide chain of about 1 1 0 amino acids in length, comprising one variable domain (VH) of a heavy chain antibody, or of a common IgG.

[0189] Unlike whole antibodies, sdAbs do not show complement system triggered cytotoxicity because they lack an Fc region. Camelid and fish derived sdAbs are able to bind to hidden antigens that are not accessible to whole antibodies, for example to the active sites of enzymes.

[0190] A single domain antibody (sdAb) can be obtained by immunization of dromedaries, camels, llamas, alpacas or sharks with the desired antigen and subsequent isolation of the mRNA coding for heavy chain antibodies. Alternatively they can be made by screening synthetic libraries. Camelids are members of the biological family Camelidae, the only living family in the suborder Tylopoda. Camels, dromedaries, Bactrian Camels, llamas, alpacas, vicunas, and guanacos are in this group. Proteins, Peptides and Amino Acids

[0191] Proteins and peptides for use as bioactive agents as disclosed herein can be produced by any useful method including production by in vitro synthesis and by production in biological systems. Typical examples of in vitro synthesis methods which are well known in the art include solid-phase synthesis ("SPPS") and solid-phase fragment condensation

("SPFC"). Biological systems used for the production of proteins are also well known in the art. Bacteria (e.g., E coli and Bacillus sp. ) and yeast (e.g., Saccharomyces cerevisiae and Pichia pastoris) are widely used for the production of heterologous proteins. In addition, heterologous gene expression for the production of bioactive agents for use as disclosed herein can be accomplished using animal cell lines such as mammalian cell lines (e.g., CHO cells). In one particularly useful embodiment, the bioactive agents are produced in transgenic or cloned animals such as cows, sheep, goats and birds (e.g., chicken, quail, ducks and turkey), each as is understood in the art. See, for example, US Patent No. 6,781 ,030, issued August 24, 2004, the disclosure of which is incorporated in its entirety herein by reference.

[0192] Bioactive agents such as proteins produced in domesticated birds such as chickens can be referred to as "avian derived" bioactive agents (e.g., avian derived therapeutic proteins). Production of avian derived therapeutic proteins is known in the art and is described in, for example, US Patent No. 6,730,822, issued May 4, 2004, the disclosure of which is incorporated in its entirety herein by reference.

[0193] In embodiments where the bioactive agent is a protein or polypeptide, functional groups present in the amino acids of the protein polypeptide sequence can be used to link the agent to the high MW polymer. Linkages to protein or polypeptide bioactive agents can be made to naturally occurring amino acids in their sequence or to naturally occurring amino acids that have either been added to the sequence or inserted in place of another amino acid, for example the replacement of a serine by a cysteine.

[0194] Peptides useful in the present invention also include, but are not limited to, a macrocyclic peptide, a cyclotide, an aptamer, an LDL receptor A-domain, a protein scaffold (as discussed in US Patent Number 60/514,391 ), a soluble receptor, an enzyme, a peptide multimer, a domain multimer, an antibody fragment multimer, and a fusion protein.

[0195] Protein or polypeptide bioactive agents may also comprise non-naturally occurring amino acids in addition to the common naturally occurring amino acids found in proteins and polypeptides. In addition to being present for the purpose of altering the properties of a polypeptide or protein, non-naturally occurring amino acids can be introduced to provide a functional group that can be used to link the protein or polypeptide directly to high MW polymer. Furthermore, naturally occurring amino acids, e.g., cysteine, tyrosine, tryptophan can be used in this way.

[0196] Non-naturally occurring amino acids can be introduced into proteins and peptides by a variety of means. Some of the techniques for the introduction of non-natural amino acids are discussed in US Patent No. 5, 162,218 and US Patent No. 20080214439, the disclosure of which is incorporated in its entirety herein by reference. First, non-naturally occurring amino acids can be introduced by chemical modification of a polypeptide or protein on the amino acid side chain or at either the amino terminus or the carboxyl terminus. Non-limiting examples of chemical modification of a protein or peptide might be methylation by agents such as diazomethane, or the introduction of acetylation at an amino group present in lysine's side chain or at the amino terminus of a peptide or protein. Another example of the protein/polypeptide amino group modification to prepare a non-natural amino acid is the use of methyl 3-mercaptopropionimidate ester or 2-iminothiolane to introduce a thiol (sulfhydryl, -SH) bearing functionality linked to positions in a protein or polypeptide bearing a primary amine. Once introduced, such groups can be employed to form a covalent linkage to the protein or polypeptide.

[0197] Second, non-naturally occurring amino acids can be introduced into proteins and polypeptides during chemical synthesis. Synthetic methods are typically utilized for preparing polypeptides having fewer than about 200 amino acids, usually having fewer than about 150 amino acids, and more usually having 100 or fewer amino acids. Shorter proteins or polypeptides having less than about 75 or less than about 50 amino acids can be prepared by chemical synthesis.

[0198] The synthetic preparation methods that are particularly convenient for allowing the insertion of non-natural amino acids at a desired location are known in the art. Suitable synthetic polypeptide preparation methods can be based on Merrifield solid-phase synthesis methods where amino acids are sequentially added to a growing chain (Merrifield (1963) J. Am. Chem. Soc. 85 :2149-21 56). Automated systems for synthesizing polypeptides by such techniques are now commercially available from suppl iers such as Applied Biosystems, Inc., Foster City, Calif. 94404; New Brunswick Scientific, Edison, N.J. 08818; and Pharmacia, Inc., Biotechnology Group, Piscataway, N.J. 08854.

[0199] Examples of non-naturally occurring amino acids that can be introduced during chemical synthesis of polypeptides include, but are not limited to: D-amino acids and mixtures of D and L-forms of the 20 naturally occurring amino acids, N-formyl glycine, ornithine, norleucine, hydroxyproline, beta-alanine, hydroxyvaline, norvaline, phenylglycine, cyclohexylalanine, t-butylglycine (t-leucine, 2-amino-3,3-dimethylbutanoic acid), hydroxy-t-butylglycine, amino butyric acid, cycloleucine, 4-hydroxyproline, pyroglutamic acid (5-oxoproline), azetidine carboxylic acid, pipecolinic acid, indoline-2-carboxylic acid, tetrahydro-3-isoquinoline carboxylic acid, 2,4-diaminobutyricacid, 2,6-diaminopimelic acid, 2,4-diaminobutyricacid, 2,6-diaminopimelicacid, 2,3-diaminopropionicacid, 5-hydroxylysine, neuraminic acid, and 3,5-diiodotyrosine.

[0200] Third, non-naturally occurring amino acids can be introduced through biological synthesis in vivo or in vitro by insertion of a non-sense codon {e.g., an amber or ocher codon) in a DNA sequence (e.g., the gene) encoding the polypeptide at the codon corresponding to the position where the non-natural amino acid is to be inserted. Such techniques are discussed for example in US Patents No.: 5, 162,218 and 6,964,859, the disclosures of which are incorporated in their entirety herein by reference. A variety of methods can be used to insert the mutant codon including oligonucleotide-directed mutagenesis. The altered sequence is subsequently transcribed and translated, in vivo or in vitro in a system which provides a suppressor tRNA, directed against the nonsense codon that has been chemically or enzymatically acylated with the desired non-naturally occurring amino acid. The synthetic amino acid will be inserted at the location corresponding to the nonsense codon. For the preparation of larger and/or glycosylated polypeptides, recombinant preparation techniques of this type are usually preferred. Among the amino acids that can be introduced in this fashion are: formyl glycine, fluoroalanine, 2-Amino-3-mercapto-3-methylbutanoic acid, homocysteine, homoarginine and the like. Other similar approaches to obtain non-natural amino acids in a protein include methionine substitution methods.

[0201] Where non-naturally occurring amino acids have a functionality that is susceptible to selective modification, they are particularly useful for forming a covalent linkage to the protein or polypeptide. Circumstances where a functionality is susceptible to selective modification include those where the functionality is unique or where other functionalities that might react under the conditions of interest are hindered either stereochemically or otherwise.

[0202] Other antibodies, such as single domain antibodies are useful in the present invention. A single domain antibody (sdAb, called Nanobody by Ablynx) is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, the sdAb is able to bind selectively to a specific antigen. With a molecular weight of only 12-15 kDa, single domain antibodies are much smaller than common whole antibodies (150- 160 kDa). A single domain antibody is a peptide chain of about 1 10 amino acids in length, comprising one variable domain (VH) of a heavy chain antibody, or of a common IgG.

[0203] Unlike whole antibodies, sdAbs do not show complement system triggered cytotoxicity because they lack an Fc region. Camelid and fish derived sdAbs are able to bind to hidden antigens that are not accessible to whole antibodies, for example to the active sites of enzymes.

[0204] A single domain antibody (sdAb) can be obtained by immunization of dromedaries, camels, llamas, alpacas or sharks with the desired antigen and subsequent isolation of the mRNA coding for heavy chain antibodies. Alternatively they can be made by screening synthetic libraries. Camelids are members of the biological family Camelidae, the only living family in the suborder Tylopoda. Camels, dromedaries, Bactrian Camels, llamas, alpacas, vicunas, and guanacos are in this group.

[0205] Peptides useful in the present invention also include, but are not limited to, a macrocyclic peptide, a cyclotide, an LDL receptor A-domain, a protein scaffold (as discussed in US Patent Number 60/514,391 , incorporated in its entirety herein), a soluble receptor, an enzyme, a peptide multimer, a domain multimer, an antibody fragment multimer, and a fusion protein.

[0206] The invention also describes new ways to achieve branched polymer architectures on a bioactive surface. The concept is one of "branching points" or "proximal attachment points" on the target molecule such as to recreate an effective >2 arm polymer with >1 arm polymers attached to a localized site(s) on a target molecule. In the prior art, indiscriminate PEGylation of a protein with a non site-specific reagent (for example an NHS functional ized PEG reagent) would result in multiple PEG polymers conjugated to multiple amine groups scattered through the protein. Here, what is described is preferably a one step approach in which the target agent is modified to locate two unique conjugation sites (for example, cysteine amino acids) such that once the tertiary structure of the protein or peptide or agent is formed, the two sites will be in proximity one to the other. Then, this modified target agent is used in a conjugation reaction with a polymer containing the corresponding conjugation chemistry (for example, thiol reactive). The result is a single target agent which is conjugated with two polymers in close proximity to one another, thereby creating a branching point or "pseudo" branch. In another embodiment, the target agent would contain a single unique site, for example a free cysteine, and a tri(hetero)functional linking agent would be employed to attach >2 linear polymers to this single site, again creating a "pseudo" branch.

Drugs

[0207] In another embodiment, the bioactive agents can also be selected from specifically identified drug or therapeutic agents, including but not limited to: tacrine, memantine, rivastigmine, galantamine, donepezil, levetiracetam, repaglinide, atorvastatin, alefacept, tadalafil, vardenafil, sildenafil, fosamprenavir, oseltamivir, valacyclovir and valganciclovir, abarelix, adefovir, alfuzosin, alosetron, amifostine, amiodarone, aminocaproic acid, aminohippurate sodium, aminoglutethimide, aminolevulinic acid, aminosalicylic acid, amlodipine, amsacrine, anagrelide, anastrozole, aprepitant, aripiprazole, asparaginase, atazanavir, atomoxetine, anthracyclines, bexarotene, bicalutamide, bleomycin, bortezomib, buserelin, busulfan, cabergoline, capecitabine, carboplatin, carmustine, chlorambucin, cilastatin sodium, cisplatin, cladribine, clodronate, cyclophosphamide, cyproterone, cytarabine, camptothecins, 13-cis retinoic acid, all trans retinoic acid; dacarbazine, dactinomycin, daptomycin, daunorubicin, deferoxamine, dexamethasone, diclofenac, diethylstilbestrol, docetaxel, doxorubicin, dutasteride, eletriptan, emtricitabine, enfuvirtide, eplerenone, epirubicin, estramustine, ethinyl estradiol, etoposide, exemestane, ezetimibe, fentanyl, fexofenadine, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutarnide, fluticazone, fondaparinux, fulvestrant, gamma-hydroxybutyrate, gefitinib, gemcitabine, epinephrine, L-Dopa, hydroxyurea, icodextrin, idarubicin, ifosfam ide, imatinib, irinotecan, itraconazole, goserelin, laronidase, lansoprazole, letrozole, leucovorin, levamisole, lisinopril, lovothyroxine sodium, lomustine, mechlore hamine, medroxyprogesterone, megestrol, melphalan, memantine, mercaptopurine, mequinol, metaraminol bitartrate, methotrexate, metoclopramide, mexiletine, miglustat, mitomycin, mitotane, mitoxantrone, modafinil, naloxone, naproxen, nevirapine, nicotine, nilutamide, nitazoxanide, nitisinone, norethindrone, octreotide, oxaliplatin, palonosetron, pamidronate, pemetrexed, pergolide, pentostatin, pilcamycin, porfimer, prednisone, procarbazine, prochlorperazine, ondansetron, palonosetron, oxaliplatin, raltitrexed, rosuvastatin, sirolimus, streptozocin, pimecrolimus, sertaconazole, tacrolimus, tamoxifen, tegaserod, temozolomide, teniposide, testosterone, tetrahydrocannabinol, thalidomide, thioguanine, thiotepa, tiotropium, topiramate, topotecan, treprostinil, tretinoin, valdecoxib, celecoxib, rofecoxib, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, voriconazole, dolasetron, granisetron, formoterol, fluticasone, leuprolide, midazolam, alprazolam, amphotericin B, podophylotoxins, nucleoside antivirals, aroyl hydrazones, sumatriptan, eletriptan; macrolides such as erythromycin, oleandomycin, troleandomycin, roxithromycin, clarithromycin, davercin, azithromycin, flurithromycin, dirithromycin, josamycin, spiramycin, midecamycin, loratadine, desloratadine, leucomycin, miocamycin, rokitamycin, andazithromycin, and swinolide A; fluoroquinolones such as ciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin, moxifloxicin, norfloxacin, enoxacin, gatifloxacin, gemifloxacin, grepafloxacin, lomefloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin, fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin, clinafloxacin, and sitafloxacin; aminoglycosides such as gentamicin, netilmicin, paramecin, tobramycin, amikacin, kanamycin, neomycin, and streptomycin, vancomycin, teicoplanin, rampolanin, mideplanin, colistin, daptomycin, gramicidin, cohstimethate; polymixins such as polymixin B, capreomycin, bacitracin, penems; penicillins including penicllinase-sensitive agents like penicillin G, penicillin V; penicillinase-resistant agents like methicillin, oxacillin, cloxacillin, dicloxacillin, floxacillin, nafcillin; gram negative microorganism active agents like ampicillin, amoxicillin, and hetacillin, cillin, and galampicillin; antipseudomonal penicillins like carbenicillin, ticarcillin, azlocillin, mezlocillin, and piperacillin; cephalosporins like cefpodoxime, cefprozil, ceftbuten, ceftizoxime, ceftriaxone, cephalothin, cephapirin, cephalexin, cephradrine, cefoxitin, cefamandole, cefazolin, cephaloridine, cefaclor, cefadroxil, cephaloglycin, cefuroxime, ceforanide, cefotaxime, cefatrizine, cephacetrile, cefepime, cefixime, cefonicid, cefoperazone, cefotetan, cefmetazole, ceftazidime, loracarbef, and moxalactam, monobactams like aztreonam; and carbapenems such as imipenem, meropenem, and ertapenem, pentamidine isetionate, albuterol sulfate, lidocaine, metaproterenol sulfate, beclomethasone diprepionate, triamcinolone acetamide, budesonide acetonide, salmeterol, ipratropium bromide, flunisolide, cromolyn sodium, and ergotamine tartrate; taxanes such as paclitaxel; SN-38, and tyrphostines. Bioactive agents may also be selected from the group consisting of aminohippurate sodium, amphotericin B, doxorubicin, am inocaproic acid, aminolevulinic acid, aminosalicylic acid, metaraminol bitartrate, pamidronate disodium, daunorubicin, levothyroxine sodium, lisinopril, cilastatin sodium, mexiletine, cephalexin, deferoxamine, and amifostine in another embodiment.

[0208J Other bioactive agents useful in the present invention include extracellular matrix targeting agents, functional transport moieties and labeling agents. Extracellular matrix targeting agents include, but are not limited to, heparin binding moieties, matrix metalloproteinase binding moieties, lysyl oxidase binding domains, negatively charged moieties or positively charged moieties and hyaluronic acid. Functional transport moieties include, but are not limited to, blood brain barrier transport moieties, intracellular transport moieties, organelle transport moieties, epithelial transport domains and tumor targeting moieties (folate, other). In some embodiments, the targeting agents useful in the present invention target anti-TrkA, anti A-beta (peptide 1 -40, peptide 1 -42, monomeric form, oligomeric form), anti-IGFl -4, agonist RAN -L, anti-ApoE4 or anti-ApoAl , among others. Diagnostic agents

[0209] Diagnostic agents useful in the high MW polymers of the present invention include imaging agents and detection agents such as radiolabels, fluorophores, dyes and contrast agents.

[0210] Imaging agent refers to a label that is attached to the high MW polymer of the present invention for imaging a tumor, organ, or tissue in a subject. The imaging moiety can be covalently or non-covalently attached to the high M W polymer. Examples of imaging moieties suitable for use in the present invention include, without limitation, radionuclides, fluorophores such as fluorescein, rhodamine, Texas Red, Cy2, Cy3, Cy5, Cy5.5, Cy7 and the AlexaFluor (Invitrogen, Carlsbad, CA) range of fluorophores, antibodies, gadolinium, gold, nanomaterials, horseradish peroxidase, alkaline phosphatase, derivatives thereof, and mixtures thereof.

[0211] Radiolabel refers to a nuclide that exhibits radioactivity. A "nuclide" refers to a type of atom specified by its atomic number, atomic mass, and energy state, such as carbon 14 ( ¹⁴ C). "Radioactivity" refers to the radiation, including alpha particles, beta particles, nucleons, electrons, positrons, neutrinos, and gamma rays, emitted by a radioactive substance. Radionuclides suitable for use in the present invention include, but are not limited to, fluorine 18 ( ^{, 8} F), phosphorus 32 ( ³² P), scandium 47 ( ⁴⁷ Sc), cobalt 55 ( ⁵⁵ Co), copper 60 ( ⁶⁰ Cu), copper 61 ( ⁶¹ Cu), copper 62 ( ⁶² Cu), copper 64 ( ⁶⁴ Cu), gallium 66 ( ⁶⁶ Ga), copper 67 ( ⁶⁷ Cu), gallium 67 ( ⁶⁷ Ga), gallium 68 ( ⁶⁸ Ga), rubidium 82 ( ⁸² Rb), yttrium 86 ( ⁸⁶ Y), yttrium 87 ( ⁸⁷ Y), strontium 89 ( ⁸⁹ Sr), yttrium 90 ( ⁹⁰ Y), rhodium 105 ( ^I05 Rh), silver 1 1 1 ( ^{n ,} Ag), indium 1 1 1 (" Ίη), iodine 124 ( ^l24 I), iodine 125 ( ¹²⁵ I), iodine 131 ( ^l3l I), tin 1 17m (" ^7m Sn), technetium 99m ( ^99m Tc), promethium 149 ( ¹⁴⁹ Pm), samarium 153 ( ^{I 53} Sm), holmium 166 ( ¹⁶⁶ Ho), lutetium 177 ( ^,77 Lu), rhenium 186 ( ^{I S6} Re), rhenium 188 ( ^{1 S8} Re), thallium 201 ( ^20, T1), astatine 21 1 ( ^{2l ,} At), and bismuth 212 ( ²¹² Bi). As used herein, the "m" in ^{1 17m} Sn and ^99m Tc stands for meta state. Additionally, naturally occurring radioactive elements such as uranium, radium, and thorium, which typically represent mixtures of radioisotopes, are suitable examples of radionuclides. ⁶⁷ Cu, ^{l 1} I, ^l77 Lu, and ¹⁸⁶ Re are beta- and gamma-emitting radionuclides. ²¹² Bi is an alpha- and beta-emitting radionuclide. ²¹ 'At is an alpha-emitting radionuclide. ³² P, ⁴⁷ Sc, ⁸⁹ Sr, ⁹⁰ Y, ¹⁰⁵ Rh, ^m Ag, ^{, , 7m} Sn, ^,49 Pm, ^{, 53} Sm, ^,66 Ho, and ¹⁸⁸ Re are examples of beta-emitting radionuclides. ⁶⁷ Ga, ^{u l} In, ^99m Tc, and ²⁰¹ T1 are examples of gamma-emitting radionuclides. ⁵⁵ Co, ⁶⁰ Cu, ⁶¹ Cu, ⁶² Cu, ⁶⁶ Ga, ⁶⁸ Ga, ⁸² Rb, and ⁸⁶ Y are examples of positron-emitting radionuclides. ⁶⁴ Cu is a beta- and positron-emitting radionuclide. Imaging and detection agents can also be designed into the polymers of the invention through the addition of naturally occurring isotopes such as deuterium, ¹³ C, or ¹⁵ N during the synthesis of the initiator, linkers, linking groups, comonomers.

[0212] Contrast agents useful in the present invention include, but are not limited to, gadolinium based contrast agents, iron based contrast agents, iodine based contrast agents, barium sulfate, among others. One of skill in the art will appreciate that other contrast agents are useful in the present invention.

Nanoparticles

[0213] The functional agents can also include nanoparticles. Nanoparticles useful in the present invention include particles having a size ranging from 1 to 1000 nm. Nanoparticles can be beads, metallic particles or can in some cases be micelles and in some other be liposomes. Other nanoparticles include carbon nanotubes, quantum dots and colloidal gold. Nanoparticles can be packed with diagnostic and/or therapeutic agents.

[0214] Those skilled in the art will also recognize that the invention can be used to enable coincident detection of more than one agent of the same or different type. Also, the use of flexible linker chemistries can also be used to witness the loss of one fluorescent label, for example as the molecule is taken up into the cell and into a low pH environment.

Conjugates

[0215] The polymers of the present invention can be linked to a variety of functional agents described above to form a conjugate. In some embodiments, the present invention provides a conjugate including at least one polymer having a polymer arm having a plurality of monomers each independently selected from the group consisting of acrylate, methacrylate, acrylamide, methacrylamide, styrene, vinyl-pyridine, vinyl-pyrrolidone and vinyl esters such as vinyl acetate, wherein each monomer includes a hydrophilic group, an initiator fragment linked to a proximal end of the polymer arm, wherein the initator moiety is suitable for radical polymerization, and an end group linked to a distal end of the polymer arm. The conjugate of the present invention also includes at least one functional agent having a bioactive agent or a diagnostic agent, linked to the initiator fragment or the end group.

[0216] The bioactive agent of the conjugate of the present invention can include a drug, an antibody, an antibody fragment, a single domain antibody, an avimer, an adnectin, diabodies, a vitamin, a cofactor, a polysaccharide, a carbohydrate, a steroid, a lipid, a fat, a protein, a peptide, a polypeptide, a nucleotide, an oligonucleotide, a polynucleotide, or a nucleic acid. The diagnostic agent of the conjugate can be a radiolabel, a contrast agent, a fluorophore or a dye. In some embodiments, at least two polymers are linked to the functional agent. In some embodiments, at least two polymers are linked to the functional agent via proximal reactive groups on the functional agent to create a pseudo-branched structure. In other embodiments, the conjugate includes at least two functional agents attached to the polymer.

IV. Preparation of Zwitterion/Phosphorylcholine-Containing High MW polymers

[0217] The high MW polymers of the present invention can be prepared by any means known in the art. In some embodiments, the present invention provides a process for preparing a high MW polymer of the present invention, the process including the step of contacting a mixture of a first monomer and a second monomer with an initiator, I ¹ , under conditions sufficient to prepare a high MW polymer via free radical polymerization, wherein the first monomer comprises a phosphorylcholine, and each of the second monomer and initiator independently comprise at least one of a functional agent or a linking group for linking to the functional agent.

[0218] The mixture for preparing the high MW polymers of the present invention can include a variety of other components. For example, the mixture can also include cataly st, ligand, solvent, and other additives. In some embodiments, the mixture also includes a catalyst and a ligand. Suitable catalysts and ligands are described in more detail below.

[0219] Any suitable monomer can be used in the process of the present invention, such as those described above.

[0220] The high MW polymers of the present invention can be prepared by any suitable polymerization method, such as by living radical polymerization. Living radical polymerization, discussed by Odian, G. in Principles of Polymerization, 4 ^th ,

Wiley-Interscience John Wiley & Sons: New York, 2004, and applied to zwitterionic polymers for example in US 6,852,816. Several different living radical polymerization methodologies can be employed, including Stable Free Radical Polymerization (SFRP), Radical Addition-Fragmentation Transfer (RAFT), and Nitroxide-Mediated Polymerization (NMP). In addition, Atom Transfer Radical Polymerization (ATRP), provides a convenient method for the preparation of the high M W polymers of the invention. [0221] The preparation of polymers via ATRP involves the radical polymerization of monomers beginning with an initiator bearing one or more halogens. The halogenated initiator is activated by a catalyst (or a mixture of catalysts when CuBra is employed) such as a transition metal salt (CuBr) that can be solubilized by a ligand (e.g., bipyridine or

P DETA). RAFT polymerization uses thiocarbonylthio compounds, such as dithioesters, dithiocarbamates, trithiocarbonates, and xanthates, to mediate the polymerization process via a reversible chain-transfer process. Other "living" or controlled radical processes useful in the preparation of the inventive random copolymers include NMP.

Initiators

[0222] Initiators useful for the preparation of the high MW polymers of the present invention include any initiator suitable for polymerization via radical polymerization. In some embodiments, the initiators are suitable for atom transfer radical polymerization (ATRP), such as those described above. Other useful initiators include those for nitroxide mediated radical polymerization (NMP), or reversible addition-fragmentation-tennination (RAFT or MADIX) polymerization. Still other techniques to control a free-radical polymerization process can be used, such as the use of iniferters, degenerative transfer or telomerization process. Moreover, the initiators useful in the present invention include those having at least one branch point, such as those described above. In other embodiments, the initiators are useful for controlled radical polymerization.

[0223] High MW polymers of the present invention having complex architectures including branched compounds having multiple polymer arms including, but not limited to, comb and star structures. Comb architectures can be achieved employing linear initiators bearing three or more halogen atoms, preferably the halogens are chlorine, bromine, or iodine atoms, more preferably the halogens are chlorine or bromine atoms. Star architectures can also be prepared employing compounds bearing multiple halogens on a single carbon atom or cyclic molecules bearing multiple halogens. In some embodiments compounds having star architecture have 3 polymer arms and in other embodiments they have 4 polymer arms. See initiators described above.

Catalysts and Ligands

[0224] Catalysts for use in ATRP or group radical transfer polymerizations may include suitable salts of Cu ¹⁺ , Fe ²⁺ , Fe ³⁺ , Ru ²⁺ , Ru. ³⁺ , Cr ²⁺ , Cr ³ ' , Mo ² ' , Mo. ³⁺ , W ⁺ , W ³⁺ , Mn ² \ Mn ² ', Mn ⁴⁺ , Rh ³⁺ , Rh ⁴⁺ , Re ²⁺ , Re ³⁺ , Co ¹⁺ , Co. ² Co ³⁺ , V ²⁺ , V ³⁺ , Zn. ¹⁺ , Zn ²⁺ , Ni ²⁺ , Ni ⁺ , Au ^,+ , Au ²⁺ , Ag ^,+ and Ag ²⁺ . Suitable salts include, but are not limited to: halogen, C] - Q -alkoxy, sulfates, phosphate, triflate, hexafluorophosphate, methanesulphonate, arylsulphonate salts. In some embodiments the catalyst is a chloride, bromide salts of the above-recited metal ions. In other embodiments the catalyst is CuBr, CuCl or RuCb.

[0225] In some embodiments, the use of one or more ligands to solubilize transition metal catalysts is desirable. Suitable ligands are usefully used in combination with a variety of transition metal catalysts including where copper chloride or bromide, or ruthenium chloride transition metal salts are part of the catalyst. The choice of a ligand affects the function of catalyst as ligands not only aid in solubilizing transition metal catalysts in organic reaction media, but also adjust their redox potential. Selection of a ligand is also based upon the solubility and separability of the catalyst from the product mixture. Where polymerization is to be carried out in a liquid phase soluble ligands/catalyst are generally desirable although immobilized catalysts can be employed. Suitable ligands include those pyridyl groups (including alkyl pyridines e.g., 4.4. dialkyl-2,2' bipyridines) and pyridyl groups bearing an alkyl substituted imino group, where present, longer alkyl groups provide solubility in less polar monomer mixtures and solvent media. Triphenyl phosphines and other phosphorus ligands, in addition to indanyl, or cyclopentadienyl ligands, can also be employed with transition metal catalysts (e.g., Ru ⁺² -halide or Fe ⁺² -halide complexes with

triphenylphosphine, indanyl or cyclopentadienyl ligands).

[0226] An approximately stoichiometric amount of metal compound and ligand in the catalyst, based on the molar ratios of the components when the metal ion is fully complexed, is employed in some embodiments. In other embodiments the ratio between metal compound and ligand is in the range 1 :(0.5 to 2) or in the range 1 :(0.8 to 1 .25).

[0227] Generally, where the catalyst is copper, bidentate or multidentate nitrogen ligands produce more active catalysts. In addition, bridged or cyclic ligands and branched aliphatic poiyamines provide more active catalysts than simple linear ligands. Where bromine is the counter ion, bidentate or one-half tetradentate ligands are needed per Cu ⁺¹ . Where more complex counter ions are employed, such as triflate or hexafluorophosphate, two bidentate or one tetradentate ligand can be employed. The addition of metallic copper can be advantageous in some embodiments particularly where faster polymerization is desired as metallic copper and Cu ⁺² may undergo redox reaction to form Cu ⁺¹ . The addition of some Cu ⁺² at the be ginning of some ATRP reactions can be employed to decrease the amount of normal termination.

[0228] In some embodiments, the amount of catalyst employed in the polymerization reactions is the molar equivalent of the initiator that is present. Since catalyst is not consumed in the reaction, however, it is not essential to include a quantity of catalyst as high as of initiator. The ratio of catalyst to each halogen contained in the initiator, based on transition metal compound in some embodiments is from about 1 :(1 to 50), in other embodiments from about 1 :(1 to 10), in other embodiments from about 1 :(1 to 5), and in other embodiments from 1 : 1.

Polymerization Conditions

[0229] In some embodiments, the living radical polymerization process of the invention is preferably carried out to achieve a degree of polymerization in the range of 3 to about 2000, and in other embodiments from about 5 to about 500. The degree of polymerization in other embodiments is in the range 1 0 to 100, or alternatively in the range of about 10 to about 50. The degree of polymerization in group or atom transfer radical polymerization technique, is directly related to the initial ratio of initiator to monomer. Therefore, in some embodiments the initial ratios of initiator to monomer are in the range of 1 :(3 to about 2,000) or about 1 :(5 to 500), or about 1 :(10 to 100), or about 1 :( 10 to 50).

[0230] Polymerization reactions are typically carried out in the liquid phase, employing a single homogeneous solution. The reaction may, however, be heterogeneous comprising a solid and a liquid phase {e.g., a suspension or aqueous emulsion). In those embodiments where a non-polymerizable solvent is employed, the solvent employed is selected taking into consideration the nature of the zwitterionic monomer, the initiator, the catalyst and its ligand; and in addition, any comonomer that can be employed.

[0231] The solvent may comprise a single compound or a mixture of compounds. In some embodiments the solvent is water, and in other embodiments water is present in an amount from about 10% to about 1 00% by weight, based on the weight of the monomers present in the reaction. In those embodiments where a water insoluble comonomer is to be polymerized with a zwitterionic monomer, it can be desirable to employ a solvent or co-solvent (in conjunction with water) that permits solubilization of all the monomers present. Suitable organic solvents include, without limitation, formamides (e.g., N,N'-dimethylformamide), ethers (e.g., tetrahydrofuran), esters (ethyl acetate) and, most preferably, alcohols. In some embodiments where a mixture of water and organic solvent is to be employed, C1-C4 water miscible alkyl alcohols (methanol, ethanol, propanol, isopropanol, butanol, isobutanol, and tertbutanol) are useful organic solvents. In other embodiments, water and methanol combinations are suitable for conducting polymerization reactions. The reaction may also be conducted in supercritical solvents such as C ⁽ ¾. [0232] As noted above, in some embodiments it is desirable to include water in the polymerization mixture in an amount from about 10% to about 100% by weight based on the weight of monomers to be polymerized. In other embodiments the total non-polymerizable solvent is from about 1 % to about 500% by weight, based on the weight of the monomers present in the reaction mixture. In other embodiments, the total non-polymerizable solvent is from about 10% to about 500% by weight or alternatively from 20% to 400%, based on the weight of the monomers present in the reaction mixture. It is also desirable in some cases to manipulate the solubility of an input reagent, such as initiator or monomer, for example by modifying temperature or solvent or other method so as to modify the reaction conditions in a dynamic fashion.

[0233] In some embodiments, contact time of the zwitterionic monomer and water prior to contact with the initiator and catalyst are minimized by forming a premix comprising all components other than the zwitterionic monomer and for the zwitterionic monomer to be added to the premix last.

[0234] The polymerization reactions can be carried out at any suitable temperature. In some embodiments the temperature can be from about ambient (room temperature) to about 120° C. In other embodiments the polymerizations can be carried out at a temperature elevated from ambient temperature in the range of about 60 ⁰ to 80° C. In other embodiments the reaction is carried out at ambient (room temperature).

[0235] In some embodiments, the compounds of the invention have a polydispersity (of molecular weight) of less than 1.5, as judged by gel permeation chromatography. In other embodiments the polydispersities can be in the range of 1.2 to 1.4. In still other embodiments, the polydispersities can be less than 1.2.

[0236] A number of workup procedures can be used to purify the polymer of interest such as precipitation, fractionation, reprecipitation, membrane separation and freeze-drying of the polymers.

Non-Halogenated Polymer Terminus

[0237] In some embodiments, it can be desirable to replace the halogen, or other initiator fragment Γ, with another functionality. A variety of reactions can be employed for the conversion of the aliphatic halogen. In some embodiments, the conversion of the aliphatic halogen can include reaction to prepare an alkyl, alkoxy, cycloalkyl, aryl, heteroaryl or hydroxy group. Halogens can also be subject to an elimination reaction to give rise to an alkene (double bond). Other methods of modifying the halogenated terminus are described in Matyjaszewski et al. Prog. Polym. Sci. 2001 , 26, 337, incorporated by reference in its entirety herein.

Attachment of Functional agents

[0238] The coupling of functional agents to the high MW polymers of the present invention can be conducted employing chemical conditions and reagents applicable to the reactions being conducted. Exemplary methods are described in Bioconjugate Techniques, Greg T. Hermanson, Academic Press, 2d ed., 2008 (incorporated in its entirety herein). Other bioconj ligation techniques are described in Bertozzi et al. Angewandte Chemie 2009, 48, 6974, and Gauthier et al. Chem. Commun. 2008, 2591 , each incorporated by reference in its entirety herein.

[0239] Where, for example, the coupling requires the formation of an ester or an amide, dehydration reactions between a carboxylic acid and an alcohol or amine may employ a dehydrating agent (e.g., a carbodiimide such as dicyclohexylcarbodimide, DCC, or the water soluble agent l -ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride, EDC).

Alternatively, N-hydroxysuccinimide esters (NHS) can be employed to prepare amides.

Reaction to prepare amides employing NHS esters are typically conducted near neutral pH in phosphate, bicarbonate, borate, HEPES or other non-amine containing buffers at 4° to 25° C. In some embodiments, reactions employing EDC as a dehydrating agent, a pH of 4.5-7.5 can be employed; in other embodiments, a pH of 4.5 to 5 can be employed.

Morpholinoethanesulfonic acid, MES, is an effective carbodiimide reaction buffer.

[0240] Thiol groups can be reacted under a variety of conditions to prepare different products. Where a thiol is reacted with a maleimide to form a thioether bond, the reaction is typically carried out at a pH of 6.5-7.5. Excess maleimide groups can be quenched by adding free thiol reagents such as mercaptoethanol. Where disulfide bonds are present as a linkage, they can be prepared by thiol-disulfide interchange between a sulfhydryl present in the bioactive group and an X functionality which is a disulfide such as a pyridyl disulfide.

Reactions involving pyridyl disulfides can be conducted at pH 4 - pH 5 and the reaction can be monitored at 343 nm to detect the released pyridine-2-thione. Thiol groups may also be reacted with epoxides in aqueous solution to yield hydroxy thioethers. A thiol may also be reacted at slightly alkaline pH with a haloacetate such as iodoacetae to form a thioether bond.

[0241] The reaction of guanido groups (e.g., those of an arginine in a protein or polypeptide of interest) with a glyoxal can be carried out at pH 7.0-8.0. The reaction typically proceeds at 25° C. The derivative, which contains two phenylglyoxal moieties per guanido group, is more stable under mildly acidic conditions (below pH 4) thaii at neutral or alkaline pHs, and permits isolation of the linked materials. At neutral or alkaline pH values, the linkage decomposes slowly. Where an arginine residue of a protein or polypeptide is reacted with a phenylglyoxal reagent, about 80% of the linkage will hydrolyze to regenerate the original arginine residue (in the absence of excess reagent) in approximately 48 hours at 37° at about pH 7.

[0242] Imidoester reactions with amines are typically conducted at pH of 8-10, and preferably at about pH 10. The amidine linkage formed from the reaction of an imidoester with an amine is reversible, particularly at high pH.

[0243] Haloacetals can be reacted with sulfhydryl groups over a broad pH range. To avoid side reactions between histidine residues that can be present, particularly where the sulfhydryl group is present on a protein or polypeptide, the reaction can be conducted at about pH 8.3.

[0244] Aldehydes can be reacted with amines under a variety of conditions to form irnines. Where either the aldehyde or the amine is immediately adjacent to an aryl group the product is a Schiff base that tends to be more stable than where no aryl group is present. Conditions for the reaction of amines with aldehydes to form an imine bond include the use of a basic pH from about pH 9 to about pH 1 1 and a temperature from about 0° C to room temperature, over 1 to 24 hours. Alternatively, where preferential coupling to the N-terminal amine of a protein is desired, lower pHs from about 4-7 can be employed. Buffers including borohydride and tertiary amine containing buffers are often employed for the preparation of imines. Where it is desired imine conjugates, which are hydrolytically susceptible, can be reduced to form an amine bond which is not hydrolytically susceptible. Reduction can be conducted with a variety of suitable reducing agents including sodium borohydride or sodium cyanoborohydride.

[0245] The reaction conditions provided above are intended to provide general guidance to the artisan. The skilled artisan will recognize that reaction conditions can be varied as necessary to promote the attachment of the functional agent to the high MW polymers of the present invention and that guidance for modification of the reactions can be obtained from standard texts in organic chemistry. Additional guidance can be obtained from texts such as Wong, S.S., "Chemistry of Protein Conjugation and Cross-Linking," (CRC Press 1991 ), which discuss related chemical reactions. [0246] Different recombinant proteins have been shown to conjugate successfully to a wide variety of polymers of the present invention of different sizes and architectures via different conjugation chemistries. Many lessons have been learned during the course of process development efforts (conjugation, downstream processing, analytical development) and some unique features of the technology are described below. The conjugate refers exclusively to protein or other therapeutic agents conjugated covalently to the polymers of the present invention.

[0247] In the area of conjugation reactions, low polymer molar excess ratios of 1 - 2 fold are useful in order to obtain good conjugation efficiency. In order to achieve low polymer molar excess and yet maintain good conjugation efficiency (>20%), protein concentration should be much higher than the normally acceptable concentration of 1 - 2 mg/ml. The concentration that can be achieved for any one particular protein used will depend on the stability and biophysical properties of that protein. Exemplary ranges include 5 - 10 mg/ml, 10 - 15 mg/ml, 15 - 20 mg/ml, 20 - 25 mg/ml, 25 - 30 mg/ml, 30 - 50 mg/ml, 50 - 100 mg/mL, >100 mg/ml.

[0248] On the other side of the reaction, a major challenge is the concentration of polymer which is also required to be at a very high level for optimal conjugation efficiencies, a normal concentration being upwards of 100 mg ml. Interestingly, the polymers of this invention demonstrate extreme solubility with low viscosity even at concentrations in excess of 500 mg ml. This feature makes it possible to manipulate the conjugation reaction such as mixing very easily whereas with other polymers such as PEG at such a concentration the solution is too viscous to be handled. The use of a variety of devices to improve mixing further improves the process. For example, an ultrasonic bath with temperature control can be used for initial mixing in order to facilitate polymer solubilization and in turn improve conjugation efficiency. Alternative ultrasonic devices such as VialTweeter from HielscherUltrasonic GmbH improve the efficiency with which ultrasonic energy is delivered compared with an ultrasonic bath. From a theoretical point of view, the ultrasonic wave creates an oscillation wave that facilitates the interaction between polymer and protein. This translates into higher and better conjugation efficiency. The addition of a temperature controlled mechanism such as a cooling system protects heat labile proteins in this system. To scale up such a process to large industrial scale (e.g. kilogram or greater scale), other instrumentation such as the resonant acoustic mixing technology developed by Resodyn is useful. In fact, this type of mixer has been successfully used to solubilize highly viscous polymers and fluids with viscosity over 1 ,000 cP. The polymers of this inv ention at the highest practical concentration are just a fraction of such a viscosity level and therefore render the resonant acoustic mixing technology particularly attractive. Additional advantages of such technology include noninvasive and fully concealable character as well as fast mixing time. These properties make it highly desirable for protein pharmaceutics generally and for combination with the technology of this invention specifically.

[0249] Undesirable poly-PEGylated conjugation byproducts have long been an issue in the industry which increases the cost of goods during manufacturing while also increasing regulatory complexity and product approval hurdles. Interestingly, many different purified conjugates derived from all the polymers of this invention and which have been tested always result in an equal molar ratio between protein and polymer. This is a unique and highly desirable feature as compared to other polymer and conjugation technologies.

[0250] In the area of downstream processing, as described previously, the preferred polymers of this invention are net charge neutral due to their zwitterionic nature. Therefore, they do not interact with anion or cation ion exchange resins under any chromatographic conditions including wide ranges of pH and ionic strength. In other words, the free polymer will flow through any ion exchanger irrespective of pH and ionic strength. However, upon conjugation to different proteins, the chromatographic behavior of the conjugate is dictated by the protein. Due to the presence of the polymer shielding effect and altered charge of the protein during the conjugation chemistry, the interaction of the conjugate with the ion exchange resin is weakened as compared to the native protein. This property is observed for basic and acidic proteins that interact with cation and anion exchanger resins, respectively. These are also highly desirable properties from a manufacturing point of view as they allow for the design of a highly efficient, simple, cost-effective, and orthogonal purification method for separation of conjugate from the product releated contaminants which include: unreactive free polymer, unreacted free proteins and aggregates; and process contaminants such as endotoxin, conjugation reactants and additives. A single ion exchange chromatographic step is sufficient.

[0251] For example, for an acidic protein conjugate where the conjugation reaction is carried out at low ionic strength (e.g. 0-20mM NaCl) with buffer pH higher than the pi of the protein, upon completion of the conjugation reaction, the contents of the conjugation reaction vessel can be applied directly over the anion exchanger resin (e.g. Q type 1EX resin) where the unreacted free polymer will flow through the resin, the column can then be chased and washed with low ionic strength buffer at the same pH similar to the conjugation reaction. The bound fraction can then by eluted stepwise with increasing salt concentrations. The first protein fraction is the pure conjugate as it binds more weakly to the ion exchange resin as compared to the native protein and other contaminants such as aggregates and endotoxin. A step gradient is highly desirable as this minimizes the potential risk that the native protein will leach out from the column. For example, using a strong anion exchange resin, a cytokine polymer conjugate will elute around 30-60mM NaCl at pH 7 while the native cytokine will not elute until l OOmM or higher; under such conditions, the dimeric and aggregated form of the cytokine typically elutes at 200mM NaCl or higher; and finally the endotoxin elutes at an even higher salt concentration.

[0252] For a basic protein conjugate, the separation is accomplished using a cation exchanger (e.g. SP type IEX resin) at low ionic strength (e.g. 0-20mM NaCl) with buffer pH lower than the pi of the protein. In this process, the unreacted free polymer will still be in the flow through fraction together with endotoxin and other negatively charged contaminants while the conjugate and free unreacted protein remain bound to the column. By increasing the ionic strength of the elution buffer, the first protein fraction eluted is the conjugate due to the weaker interaction with the IEX resin as compared to the native protein. A typical Fab' conjugate will elute at 30-60mM NaCl while the native Fab' will elute at 100-200mM NaCl.

[0253] The experience with purifying many different protein conjugates including both acidic protein conjugates (such as cytokines and scaffold-based multi-domain based proteins) and basic protein conjugates (such as Fab') show that the ionic strength required for conjugate elution is largely independent of polymer size (even greater than one million daltons) and architecture (multi-armed architectures). This is a highly desirable feature of the platform technology that enables the design of a generic manufacturing process where major process development efforts are not required with changes in polymers and to some extent therapeutic agents.

[0254] From the manufacturing point of view, the above described downstream purification process has the following advantages:

1. Highly scalable;

2. Amenable to current commercial production processes as the resins are available commercially and the required instrumentation is already at industrial standard;

The sample technique can be used for both In Process Analytics (IPA) as well as scale up production;

4. Development of a generic process is feasible;

5. Cost effective due to its single step nature and orthogonal design;

6. Excellent recovery (current process yields are upwards of 80%). [0255] In the area of analytical development, the zwitterionic nature of the polymers of this invention has two impacts on development of SDS-PAGE analysis of conjugates. Firstly, SDS-PAGE analysis has long been a ubiquitous and convenient method for protein analysis, in that it provides a fast, high resolution, high throughput and low cost method for semiquantitative protein characterization. However, the net charge neutral property and also the large hydrodynamic radius of the polymer means that the polymer migrates poorly or (for very large size polymers) almost not at all into a polyacrylamide matrix even with as low as a 4% gel. Secondly, the polymers of this invention are not stainable by Coomassie Blue type stains, potentially due to their net charge neutral property which prevents the Coomassie Blue dye from interacting with the polymer. However, once the protein is conjugated to the polymer, the conjugate becomes stainable. These are two undesirable properties for most protein biochemists at first glance; however, the combination of these two properties allows for the design of a highly desirable and unique technique that enables quick and easy analysis of conjugation efficiency directly from the reaction mixture without further purification. In this technique, the conjugation reaction mixture is loaded onto the SDS-PAGE gel and separated as per standard protocol. Then the gel is stained with Coomassie Blue and then destained according to the standard protocol. The presence of the conjugate will display Coomassie blue stained bands close to the loading well while the smaller protein migrates at its molecular weight and will display concomitant reduction in band intensity as compared to a control reaction without polymer. It is therefore very easy to distinguish those reactions with inefficient conjugation as the polymer alone will not display any staining at the high molecular weight region of the gel. It should be noted that such a technique for conjugation reaction analysis is impossible for PEGylation reaction as both the PEG polymer and PEGylated proteins stain by Coomassie Blue and migrate at a very similar position in the gel, especially the very large PEG polymers; in addition, PEG polymers display the highly undesirable property of distorting the migration pattern of SDS-PAGE gels. This latter problem is not observed for the polymers of this invention, as the net charge neutral property of the unreacted free polymer renders them unlikely to enter the gel matrix (whereas only the conjugate and unconjugated free protein will do so).

[0256] Another interesting property of the polymers of this invention is that they do not have UV 280nm absorbance due to the absence of an aromatic group. However, they do absorb at 220nm. There is at least 1 Ox lower absorbance for the polymer when compared with an equal mass concentration of protein solution. This is very useful when trying to identify the presence of conjugate in the conjugation reaction mixture using different chromatographic methods such as size exclusion or IEX analysis. By comparing the UV280/UV220 ratio, it is very easy to identify the presence of conjugate as the ratio increases dramatically. The same technique can be used for both analytical scale and production scale monitoring of product elution.

V. Compositions

[0257] The present invention includes and provides for pharmaceutical compositions comprising one or more compounds of the invention and one or more pharmaceutically acceptable excipients. The compounds of the invention may be present as a pharmaceutically acceptable salt, prodrug, metabolite, analog or derivative thereof, in the pharmaceutical compositions of the invention. As used herein, "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

[0258] Pharmaceutically acceptable carriers for use in formulating the high MW polymers of the present invention include, but are not limited to: solid carriers such as lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like; and liquid carriers such as syrups, saline, phosphate buffered saline, water and the like. Carriers may include any time-delay material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate or the like. [0259] Other fillers, excipients, flavorants, and other additives such as are known in the art may also be included in a pharmaceutical composition according to this invention. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions of the present invention.

[0260] The pharmaceutical preparations encompass all types of formulations. In some embodiments they are parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intraperitoneal, intrathecal, intraventricular, intracranial, intraspinal, intracapsular, intraocular including intravitreal, and intraosseous) formulations suited for injection or infusion {e.g., powders or concentrated solutions that can be reconstituted or diluted as well as suspensions and solutions). Where the composition is a solid that requires reconstitution or a concentrate that requires dilution with liquid media, any suitable liquid media may be employed. Preferred examples of liquid media include, but are not limited to, water, saline, phosphate buffered saline, Ringer's solution, Hank's solution, dextrose solution, and 5% human serum albumin.

[0261] Where a compound or pharmaceutical composition comprising a high MW polymer of the present invention is suitable for the treatment of cell proliferative disorders, including but not limited to cancers, the compound or pharmaceutical composition can be administered to a subject through a variety of routes including injection directly into tumors, the blood stream, or body cavities.

[0262] While the pharmaceutical compositions may be liquid solutions, suspensions, or powders that can be reconstituted immediately prior to administration, they may also take other forms. In some embodiments, the pharmaceutical compositions may be prepared as syrups, drenches, boluses, granules, pastes, suspensions, creams, ointments, tablets, capsules (hard or soft) sprays, emulsions, microemulsions, patches, suppositories, powders, and the like. The compositions may also be prepared for routes of administration other than parenteral administration including, but not limited to, topical (including buccal and sublingual), pulmonary, rectal, transdermal, transmucosal, oral, ocular, and so forth. Needle free injection devices can be used to achieve subdermal, subcutaneous and/or intramuscular administration . Such devices can be combined with the polymers and conjugates of this invention to administer low (<20 cP), medium (20 - 50 cP), and high (> 100 cP) viscosity formulations. [0263] In some embodiments, the pharmaceutical compositions of the present invention comprise one or more high MW polymers of the present invention.

[0264] Other pharmaceutical compositions of the present invention may comprise one or more high MW polymers of the present invention that function as biological ligands that are specific to an antigen or target molecule. Such compositions may comprise a high MW polymer of the present invention, where the bioactive agent is a polypeptide that comprises the amino acid sequence of an antibody, or an antibody fragment such as a FAb2 or FAb' fragment or an antibody variable region. Alternatively, the compound may be a high MW polymer and the polypeptide may comprise the antigen binding sequence of a single chain antibody. Where a bioactive agent present in a high MW polymer of the present invention functions as a ligand specific to an antigen or target molecule, those compounds may also be employed as diagnostic and/or imaging reagents and/or in diagnostic assays.

[0265] The amount of a compound in a pharmaceutical composition will vary depending on a number of factors. In one embodiment, it may be a therapeutically effective dose that is suitable for a single dose container (e.g., a vial). In one embodiment, the amount of the compound is an amount suitable for a single use syringe. In yet another embodiment, the amount is suitable for multi-use dispensers (e.g., containers suitable for delivery of drops of formulations when used to deliver topical formulations). A skilled artisan will be able to determine the amount a compound that produces a therapeutically effective dose experimentally by repeated administration of increasing amounts of a pharmaceutical composition to achieve a clinically desired endpoint.

[0266] Generally, a pharmaceutically acceptable excipient will be present in the composition in an amount of about 0.01 % to about 99.999% by weight, or about 1 % to about 99% by weight. Pharmaceutical compositions may contain from about 5% to about 10%, or from about 10% to about 20%, or from about 20% to about 30%, or from about 30% to about 40%, or from about 40% to about 50%, or from about 50% to about 60%, or from about 60% to about 70%, or from about 70% to about 80%, or from about 80% to about 90% excipient by weight. Other suitable ranges of excipients include from about 5% to about 98%, from about from about 15 to about 95%, or from about 20% to about 80% by weight.

[0267] Pharmaceutically acceptable excipients are described in a variety of well-known sources, including but not limited to "Remington: The Science & Practice of Pharmacy", 19 ^th ed., Williams & Williams, (1995) and Kibbe, A. H., Handbook of Pharmaceutical Excipients, 3 ^rd Edition, American Pharmaceutical Association, Washington, D.C., 2000. VI. Methods

[0268] The high MW polymers of the present invention are useful for treating any disease state or condition. The disease state or condition can be acute or chronic.

[0269] Disease states and conditions that can be treated using the high MW polymers of the present invention include, but are not limited to, cancer, autoimmune disorders, genetic disorders, infections, inflammation, neurologic disorders, and metabolic disorders.

[0270] Cancers that can be treated using the high MW polymers of the present invention include, but are not limited to, ovarian cancer, breast cancer, lung cancer, bladder cancer, thyroid cancer, liver cancer, pleural cancer, pancreatic cancer, cervical cancer, testicular cancer, colon cancer, anal cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gall bladder cancer, rectal cancer, appendix cancer, small intestine cancer, stomach (gastric) cancer, renal cancer, cancer of the central nervous system, skin cancer, choriocarcinomas; head and neck cancers, osteogenic sarcomas, fibrosarcoma,

neuroblastoma, glioma, melanoma, leukemia, and lymphoma.

[0271] Autoimmune diseases that can be treated using the high MW polymers of the present invention include, but are not limited to, multiple sclerosis, myasthenia gravis, Crohn's disease, ulcerative colitis, primary biliary cirrhosis, type 1 diabetes mellitus (insulin dependent diabetes mellitus or IDDM), Grave's disease, autoimmune hemolytic anemia, pernicious anemia, autoimmune thrombocytopenia, vasculitides such as Wegener's granulomatosis, Behcet's disease, rheumatoid arthritis, systemic lupus erythematosus (lupus), scleroderma, systemic sclerosis, Guillain-Barre syndromes, Hashimoto's thyroiditis spondyloarthropathies such as ankylosing spondylitis, psoriasis, dermatitis herpetiformis, inflammatory bowel diseases, pemphigus vulgaris and vitiligo.

[0272] Some metabolic disorders treatable by the high MW polymers of the present invention include lysosomal storage disorders, such as mucopolysaccharidosis IV or Morquio Syndrome, Activator Deficiency/GM2 Gangliosidosis, Alpha-mannosidosis,

Aspartylglucosaminuria, Cholesteryl ester storage disease, Chronic Hexosaminidase A Deficiency, Cystinosis, Danon disease, Fabry disease, Farber disease, Fucosidosis,

Galactosialidosis, Gaucher Disease, GM 1 gangliosidosis, hypophosphatasia, I-Cell disease/Mucolipidosis II, Infantile Free Sialic Acid Storage Disease/ISSD, Juvenile Hexosaminidase A Deficiency, Krabbe disease, Metachromatic Leukodystrophy,

Mucopolysaccharidoses disorders such as Pseudo-Hurler polydystrophy/Mucolipidosis IIIA, Hurler Syndrome, Scheie Syndrome, Hurler-Scheie Syndrome, Hunter syndrome, Sanfilippo syndrome, Morquio, Hyaluronidase Deficiency, Maroteaux-Lamy, Sly Syndrome,

Mucolipidosis I/Sialidosis, Mucolipidosis, and Mucolipidosis, Multiple sulfatase deficiency, Niemann-Pick Disease, Neuronal Ceroid Lipofuscinoses, Pompe disease/Glycogen storage disease type II, Pycnodysostosis, Sandhoff disease, Schindler disease, Sal la disease/Sialic Acid Storage Disease, Tay-Sachs/GM2 gangliosidosis and Wolman disease.

[0273] Conjugates of the invention and compositions (e.g., pharmaceutical compositions) containing conjugates of the invention can be used to treat a variety of conditions. For example, there are many conditions for which treatment therapies are known to practitioners of skill in the art in which functional agents, as disclosed herein, are employed. The invention contemplates that the conjugates of the invention (e.g., pbosphorylcholine containing polymers conjugated to a variety of functional agents) and compositions containing the conjugates of the invention can be employed to treat such conditions and that such conjugates provide for an enhanced treatment therapy relative to the same functional agent not coupled to a phosphorylcholine containing polymer.

[0274] Therefore, the invention contemplates the treatment of a condition known to be treatable by a certain bioactive agent by treating the condition using the same certain bioactive agent conjugated to a phosphorylcholine containing polymer.

[0275] Another aspect of the present invention relates to methods of treating a condition responsive to a biological agent comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the invention or of a pharmaceutically acceptable composition of the invention as described above. Dosage and administration are adjusted to provide sufficient levels of the bioactive agent(s) to maintain the desired effect. The appropriate dosage and/or administration protocol for any given subject may vary depending on various factors including the seventy of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.

Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician.

[0276] The pharmaceutical compositions described herein may be administered singly. Alternatively, two or more pharmaceutical compositions may be administered sequentially, or in a cocktail or combination containing two high MW polymers of the present invention or one high MW polymer of the present invention and another bioactive agent. Other uses of bioactive agents set forth herein may be found in standard reference texts such as the Merck Manual of Diagnosis and Therapy, Merck & Co., Inc., Whitehouse Station, NJ and Goodman and Oilman's The Pharmacological Basis of Therapeutics, Pergamon Press, Inc., Elmsford, N.Y., ( 1990).

[0277] This invention describes the modification of hematology related proteins such as Factor VIII, Factor VII, Factor IX, Factor X and proteases such as serine proteases of native sequence or mutein sequence and of native function or altered (for example via phage display, reference Catalyst Biosciences of South San Francisco with technology to alter specificity of binding of an existing enzyme). US Patent 7,632,921 is included in its entirety herein. Modification of the enzyme to allow for site-specific conjugation of a functional ized polymer is disclosed. The use of flexible chemistries between the polymer and the enzyme is disclosed, such that the protein can be released in vivo in the proper setting, for example to enable close to a zero order release profile. A target product profile for a next generation Factor VIII could involve a covalent conjugate of recombinant FVIH or recombinant B- domain deleted FVIII to which an extended form, multi-ami zwitterion-containing polymer of greater than 50 kDa molecular weight is attached to a site-specific amino acid such as a cysteine. The clinical pharmacology of the conjugate would demonstrate unparalled water structuring to shield the conjugate from clearance and immune systems. The conjugate would demonstrate greater than a 50 hour elimination half life in humans (preferably greater than 80 hours). The conjugate would demonstrate a 2x (preferably 4x) increased half-life versus a 60 kDa PEG-BDD FVIII with the same bioactivity. The conjugate as used in patients would show clinically insignificant antibody formation. The biopharmaceutical conjugate would be used both prophylactically (once weekly or less frequent) and for on demand treatment of patients with Hemophilia. It would also be used as rescue therapy for patients with existing FVIII neutralizing antibodies, for example from prior FVIII biopharmaceutical therapy. The drug would enable a liquid formulation for IV and/or subcutaneous administration and with high stability, high concentration, and low viscosity. Active ingredient could be in the range of 250 to 2,000 IU composed of 30 to 250 microgram of polymer drug conjugate in a nom inal volume ideally of 0.4m l. The cost of the polymer would be low, and the conjugation efficiency of the polymer to the FVIII or BDD FVIII protein would be very high, for example upwards of 75%. Such a product and product profile would make use of the extreme biocompatibility of the polymer and as transferred onto the protein. Specifically, the extreme biocompatibility would manifest itself with very tight water binding, extreme solubility, very high concentration, very low viscosity, and extreme stability. Technically, this translates into a >2x (or ideally >4x) increased elimination half- life versus PEGylation or its equivalent technologies, extremely low or no immunogenicity, high concentration, and room temperature stable liquid fonnulations. Product profile benefits include less frequent dosing, lower dose for same Area Under the Curve, effective safe treatment for naive patients, rescue therapy for patients with neutralizing antibodies, at home subcutaneous administration, pre-filled syringe/autoinjector with room temperature storage, higher gauge (lower diameter) syringe needles, lower injection volumes, and longer shelf lives. On the manufacturing front, single pot synthesis, very high polymer molecular weights, complex architectures, and low cost to manufacture are achievable. Furthermore, high efficiency conjugation of polymer to drug is possible. These manufacturing benefits can translate into cheaper, more available medicines and higher gross margins.

[0278] This invention describes attaching high MW zwitterion-containing polymers to multimers of recombinant modified LDL receptor class A domains or relevant consensus sequences as described in US patent application 60/514,391 assigned to Avidia. Those skilled in the art will understand that the avimers can be lysine depleted and then lysines and/or other amino acids added to the N- and/or C- termini for site-specific attachment of a functionalized polymer. An N-terminal lysine is preferably the second amino acid (after methionine) and can drive relative site specific conjugation of an amine-driven initiator such as a functionalized polymer containing an aldehyde or acetal group. Those skilled in the art will also kno the benefit of avimer compositions with relatively hydrophilic amino acids and low pi and high stability, such that pH can be driven very low in the conjugation reaction such as to preferentially conjugate to the amine of the lysine rather than multi-point attachments that also conjugate to N-terminal amine group or other amine groups present in the protein. The therapeutic can have one polymer conjugated to the N- terminus and another conjugated to the C- terminus via a C-terminal lysine (an effective branched structure). Such an avimer can also be made in mammalian systems with an extra N- or C- terminal cysteine added for site specific conjugation with a thiol-reacting functionalized polymer. The polymer's functional group can also contain tissue targeting elements. The chemistry attaching the polymer to the avimer can be flexible such that it breaks in vivo, for example in serum or in a pH responsive manner, etc. Monomers and multimers composed of other domains of interest used similarly include EGF domains, Notch/LNR domains, DSL domains, Anato domains, integrin beta domains or such other domains as described in the referenced patent family.

[0279] This invention also describes the attachment of high MW zwitterion-containing polymers to peptides and synthetic peptides and especially longer synthetic peptides with multiple domains. A big problem with multiple domain peptides is that they are unstable and also have very rapid clearance. The attachment of a highly biocompatible zwitterion- containing polymer such as those described in this invention solves these problems. The polymer increases the stability and also increases the in vivo residence time. This enables simple linear (unstructured) peptides as drugs, for example modules of around twenty amino acids per functional module in series of two, three, four or more modules with the goal to achieve avidity benefit or multifunctionality benefit. Each module could also have a bit of structure ('constrained' peptide like) or each module could actually be a knotted peptide domain such as a cysteine knot or macrocyclic element. The key is they are made synthetically and can be strung together with a site specific moiety for polymer conjugation at N- terminal or C-terminal (or both) or with the polymer conjugation point in the middle, which attachment point can be a site specific amino acid that is a natural amino acid or a non- natural amino acid. In a sense, this is a synthetic avimer with preferential properties. All of the amino acids could be synthetic, as well. Such a peptide plus the polymers of this invention describe a novel and powerful drug format of the future.

[0280] Those skilled in the art will understand that the breadth of application of the high molecular weight polymers of this invention is very broad. A partial list of therapeutic modalities that can benefit from conjugation of such polymers consists of: avimer (LDL receptor A-domain scaffold), adnectin (flbronectin type III scaffold), Ablynx (camelid, llama-ids), NAR's (shark), one-arm and/or single domain antibodies from all species (rat, rabbit, shark, llama, camel, other), diabodies, other multi-domain based proteins such as multimei s of modified flbronectin domains, antibody fragments (scFv monomer, scFv dimers as agonists or antagonists), Fab's, Fab'-2's, soluble extracellular domains (sTNFR l , for example, or soluble cMet receptor fragment), combination with Amunix XTEN which comprises a hydrophilic amino acid string of up to 1 ,500 amino acids made as part of the open reading frame, oligonucleotides such as aptamers, inicroRNA, siRNA, whole antibodies (conjugated to Fc- region ; conjugated to non-Fc regions), Fc-fusions (conjugated to Fc- region; conjugated to fused protein), the use of such polymers as a replacement for the CovX antibody backbone (where high molecular weight polymer is conjugated directly to the peptide itself), more broadly the attachment of the polymers of this invention even to a full- length natural or mutein antibody (CovX body, Peptibody, humanized or other antibody, the new Zyngenia platform from Carlos Barbas where peptides are conjugated to different locations on the antibody to create modular multifunctional drugs on top of an antibody backbone). Also the many domain structures as outlined in US Patent Application 60/514,391 are included in their entirety herein. Of particular interest are conjugates for binding to and inhibiting cell-surface targets, in which setting the large size, extended form architectures, and slow off rates of the polymer conjugates described in this invention can have a particularly advantageous biological effect.

[0281] This invention describes conjugates for ophthalmic and preferentially intravitreal or subconjunctival administration that have an intravitreal mean terminal half live of greater than 10 days as measured by physical presence of active conjugate. The active conjugate can also contain two functional agents, covalently attached proximal ly at one end of the polymer. In this case the two functional agents could be aptamers to VEGF and PDGF for the treatment of wet and dry age-related macular degeneration.

[0282] This invention contemplates conjugation of the high MW polymers of the invention to GLP- 1 , soluble TACI receptor, BAFF as well as inhibitors of BAFF, insulin and its variants, IL- 12 mutein (functional anti-IL-23 equivalent), anti-IL- 17 equivalent, FGF21 and muteins, RANK ligand and its antagonists, factor H and fusion proteins for inhibiton of alternative complement (Taligen), inhibitors of the immune synapse, activators of the immune synapse, inhibitors of T- cell and/or B /cell costimulatory pathways, activators or inhibitors of neuronal cells and/or their supporting matrix cells, extracellular matrix enzymes such as lysyl oxidase or metalloproteinase/metalloproteases, activators or inhibitors of regulatory T cells or antibody producing cells, as protectors of cells from inflammatory or clearance processes such as binding to beta cells of the pancreas and thereby exerting a protective function for the cell to prolong their lifespan in the body (that is, the repairing the biocompatibility by binding to them for cells or tissues or proteins in the body that can benefit from a biocompatibility boost to reduce clearance and/or their involvement in localized or generalized inflammatory processes either active or passive), for treating genetic diseases, to chaperone an existing but mis-folded protein, for stimulating the co-localization of two soluble or cell-surface entities such as bringing together a cell-surface inhibitor module (ITIM) to a cell-surface activating module (1TA ) to inhibit a cell type such as a mast cell.

[0283] This invention contemplates using the polymers of the invention for mediating cell- penetration. For example, conjugation of the polymers of this invention through their initiator structure or end termini to one or more protein-derived peptides and amphipathic peptides either secondary and primary (Current Opinion in Biotechnology, 2006, 17, 638- 642). Those skilled in the art will also recognize the possibility to combine with the stapled peptide technology which adds hydrocarbon moieties to peptides to facilitate cell penetration. [0284] This invention contemplates the combination of these inventions with other drug delivery technologies, such as PLGA. Just as PEG's hydrophilic nature improved a number of PLGA properties, the high MW polymer technology of the current invention should further improve this. For example, increased drug loading as a percent of total mass (current biopharmaceutical state of the art <20% but generally less than 10%), also generally burst % is >5%. Enhanced water binding of the polymers of the current invention drives the solubility and drives higher loading and better in vivo performance of PLGA loaded with biopharmaceutical-polymer conjugate.

[0285] This invention contemplates conjugates that demonstrate lower immunogenicity for a particular drug-polymer conjugate (so lower new incidence of neutralizing antibodies). It also contemplates shielding, masking, or de-immunizing. Not that existing neutralizing antibodies are removed but that the drug-polymer conjugate can be given to patients who already have or have had an antibody response either natively or because the particular patient was previously treated with an immunogenic biopharmaceutical drug and developed antibodies. In this latter patient set, the present invention contemplates the ability to 'rescue' such patients and re-enable them to receive therapy. This is useful, for example, with Factor VIII because patients can be kept on Factor VIII therapy (rather than fail it and then they move to a Factor VII therapy, for example). These immune system shielding aspects of the present technology also enable drugs to be formulated for subcutaneous or needle-free injection where local dendritic and other innate and adaptive immune cell populations increase the incidence of immunogenicity. To the extent that drug-polymer conjugates of the present invention decrease de novo immunogenicity and hide existing neutralizing antibodies, then the technology enables subcutaneous dosing and avoids antibody interactions and therefore expands the eligible patient base and also will decrease incidence of injection related adverse events such as anaphylaxis.

[0286] The present invention allows the possibility to include different populations of polymer conjugate to the same or different therapeutic moieties to be combined into a single formulation. The result is to carefully tailor the desired in vivo and in vitro properties. For example, take a single therapeutic moiety and conjugate to it either in a single pot or separate pots two polymers of different size, architecture. The two populations will behave differently in vivo. One population can be smaller or contain less branched polymers. The second population can be larger, more branched architectures. The conjugate with the smaller polymers will be cleared more quickly. This is great as a loading dose or as a bolus specifically for example to clear existing cytokines (say with the conjugation of an anti-TNF or an anti-IL-6 scFv as the drug moiety) from the serum. The conjugate with the larger polymers will be cleared more slowly and dear ie novo produced TNFa or IL-6, for example. This can be done with different ratios of the populations, for example 1 : 1 or 2: 1 or 10: 1 or 100: 1 , etc. The conjugated therapeutic moiety is the same, but there are different end properties as a result of the different polymers conjugated and is another way to impact biology. Another example would be with insulin or other agonistic proteins where the goal is to have a single injection that has both bolus aspect (quick activity) and also a basal (prolonged) aspect. For Factor VIII, one population of conjugated Factor VIII can have hydrolyzable linker between the polymer and the enzyme and so the enzyme comes off quickly. The second population could have a stable linker and so provide for the longer duration (chronic, prophylaxis) aspect.

[0287] The present invention can create conjugates such that after IV and/or SC injection, a zero order kinetics of release is achieved. The duration of release ( 1 month, 2 months, 3 months, 4 months, 6 months, 12 months) will depend on the size and architecture and linker chemistry of the polymer. This can be functionally equivalent to a medical device or pump that releases a constant amount of drug from a geographically localized reservoir. In the case of this invention, the drug will not be physically contained. Rather it will be in continuous circulation or by virtue of targeting be enriched in a particular tissue, but it is engineered such that onset is similar to or equivalent to zero order kinetics with linear release and minimal burst and equivalent of 100% loading.

[0288] Those skilled in the art will recognize that the present invention allows for the introduction of break points or weak points in the polymers and initiators such that larger polymer structures and/or conjugates will break down over time into smaller pieces that are readily and quickly cleared by the body. First order examples include a sensitive linker between initiator and drug, ester bonds anywhere (initiator, polymer backbone, monomers). Such weak points can break passively (for example by means of hydrolysis) or actively (by means of enzymes). Other approaches to drive breakdown or clearance can involve the use of protecting groups or prodrug chem istries such that over time, a change in exposed chemistry takes place which exposed chemistry drives destruction or targets the conjugate of released polymer to the kidney or liver or other site for destruction or clearance. VII. Examples

Example 1. Preparation of a dihydroxy functionalized nine-arm 2-bromo-2-methyl- propionamide initiator

[0289] Product 1 .1 : (l -(3-tert-butoxycarbonylamino-propionylamino)-2,2-bis[(3-tert - butoxycarbonylamino-propionylamino)-methyl]-2,5,8,l l , 14-pentaoxaheptadec- 16-ene

[0290] A solution of 1 .0 g of the previously described 1 -amino-l 5-allyloxy-2,2- bis(aminomethyl)-4,7, 10,13-tetraoxapentadecane trihydrochloride (from

PCT/US2010/061358) in 40 ml dry acetonitrile was treated with 1 .8 ml (6 eq) triethylamine, followed by 2.06 g of the previously described N-Boc-P-alanine, N-hydroxysuccinimide ester (from PCT/US2010/34252), and the reaction stirred at room temperature for 90 minutes. The reaction was concentrated and the residue partitioned between 50 ml each dichloromethane and I N HCl. The organic layer was washed with 50 ml water, then dried over anhydrous sodium sulfate, filtered, and concentrated. The oily residue was subjected to flash chromatography on silica gel with 0-3% methanol in dichloromethane to give 1 .60 g (85%) of the desired product as a clear oil. Ή NMR (400 MHz, CDC1 ₃ ): δ = 1.43 (s, 27H, CH ₃ ), 2.45 (d, J= 6.1 Hz, 6H, CHjCHjNH), 3.05 (broadened d, J= about 6 Hz, 6H, CCH^NH), 3.23 (s, 2H, OCH ₂ C), 3.41 (q, J= 5.7 Hz, 6H, CH ₂ CH ₂ NH), 3.55-3.65 (m, 16H, OCH ₂ CH ₂ 0), 4.01 (d of t, J= 5.7, 1.4 Hz, 2H, CHCH2O), 5.20 (apparent d of d of q, 2H, CH,=CH), 5.90 (m, 1 H, CH), 7.40 (t, J= 6.4 Hz, 6H, NH).

[0291] Product 1.2: l -(3-amino-propionylamino)-2,2-bis[(3-aminopropionyIamino)- methyl]-2,5,8, l 1 , 14-pentaoxaheptadec- 16-ene trihydrochloride

[0292] 1 .60 g of (l -(3-tert-butoxycarbonylamino-propionylamino)-2,2-bis[(3-tert - butoxycarbonylamino-propionylamino)-methyl]-2,5,8, l 1 , 14-pentaoxaheptadec- 16-ene (product 1.1) was dissolved in 10 ml ethyl acetate and stirred with 10 ml of 3N HC1 for 45 minutes at room temperature, then concentrated. The residue was dissolved in 20 ml water and again concentrated. This residue was dissolved in 20 ml methanol and concentrated a third time, then placed under high vacuum for one hour, to give 1.19 g (95%) of the desired product as a white foam.

[0293] Product 1.3 : 2-( l l-Allyloxy-3,6,9-trioxaundecyloxy)-ethane- 1 , 1 , 1 -tri(methyl-3-[2- (2,2,2-tri(2-bromo-2-methyl-propionylaminomethyl)-ethoxy)-ac etylamino])-propionamide

[0294] A solution of 1.97g of the previously described N-(2-Bromo-2-methylpropionyl)- 5,5-bis[N-(2-bromo-2-methylpropionyl)aminomethyl]-3-oxa-6-am inohexanoic acid, N- hydroxysuccinimidyl ester (from PCT/US2010/34252) and 1.9 ml (5 eq) of triethylamine in 25 ml dry acetonitrile was stirred as a solution of 600 mg of l -(3-amino-propionylamino)- 2,2-bis[(3-aminopropionylamino)-methyl]-2,5,8, l l , 14-pentaoxaheptadec-16-ene trihydrochloride (product 1.2), 1 ml triethylamine, and 5 ml dimethylformamide in 25 ml dry acetonitrile was added dropwise over about 15 minutes. The reaction was stirred at room temperature for three hours, then concentrated. The residue was partitioned between 50 ml each of water and ethyl acetate. The aqueous layer was extracted with another 25 ml of ethyl acetate, then the combined organics were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was subjected to silica gel flash chromatography with 0-3% methanol in dichloromethane to yield the desired product. Ή NMR (400 MHz, CDC1 ₃ ): δ = 1 .99 (s, 54H, CH ₃ ), 2.52 (t, = 6.0 Hz, 6H, CH2CH2NH), 3.10 (d, J= 6.0 Hz, 6H, CCH?NH), 3.14 (d, J= 6.0 Hz, 18H, CCHjNH), 3.25 (s, 6H, OCH ₂ C), 3.31 (s, 2H, OCH ₂ C), 3.55-3.68 (m, 22H, CH2CH ₂ NH and OCH ₂ CH ₂ 0), 4.02 (s, 6H, OCH ₂ C=0), 4.13 (s, 2H, CHCH O), 5.22 (app d of d, 2H, CH2=CH), 5.90 (m, 1 H, CH), 7.39 (t, J= 6.0 Hz, 3H, CCH ₂ NH), 7.82 (t, J= 6.0 Hz, 3H, CH ₂ CH ₂ NH), 8.16 (t, J= 6.0 Hz, 9H, CH ₂ NH).

[0295] Product 1.4: Dihydroxy functionalized nine-arm 2-bromo-2-methyl-propionamide initiator: 2-( 1 1 -[ 1 ,2-dihydroxypropyloxy]-3,6,9-trioxaundecyloxy)-ethane- 1 , 1, 1 -trimethy 1-3- [2-(2,2,2-tri(2-bromo-2-methyl-propionylaminomethyl)-ethoxy) -acetylamino]-propionamide

[0296] A round-bottom flask equipped with a stirbar was charged with 5 ml water, 5 ml t- butanol, 870 mg of 2-(l l -Allyloxy-3,6,9-trioxaundecyloxy)-ethane- 1 , 1 , 1 -tri(methyl-3-[2- (2,2,2-tri(2-bromo-2-methyl-propionylaminomethyl)-ethoxy)-ac etylamino])-propionamide (product 1.3), 360 mg potassium ferricyanide, 1 55 mg potassium carbonate, 50 mg methanesulfonamide, 4.7 mg quinuclidine, and 2.5 mg potassium osmate dihydrate and stirred overnight at room temperature. The reaction mixture was partitioned between 20 ml each of water and dichloromethane. The aqueous layer was extracted thrice more with 10 ml dichloromethane, and the organic layers were combined and concentrated. The residue was subjected to silica gel flash chromatography using 5- 10% methanol in dichloromethane.

Product containing fractions were combined, concentrated, and rechromatographed using 2- 10% methanol in dichloromethane to give 595 mg (67%) of the desired product as a tan, crushable foam. Ή NMR (400 MHz, CDC1 ₃ ): δ = 1.99 (s, 54H, CH ₃ ), 2.54 (broad t, J= 6.0 Hz, 6H, CHoCHzNH), 3.09 (d, J= 5.0 Hz, 6H, CCf NH), 3.14 (d, J= 6.5 Hz, 18H,

CCH ₂ NH), 3.25 (s, 6H, OCH7C), 3.30 (s, 2H, OCH ₂ C), 3.49 (s, 2H, CHjOH), 3.5-3.7 (n 24H, CH ₂ CH ₂ NH and OCH ₂ CH ₂ O and CHCH ₂ O), 3.87 (broad s, 1 H, CH), 4.02 (s, 6H, OCH ₂ C=0), 7.52 (broad s, 3H, CCH ₂ NH), 7.83 (t, J= 5.0 Hz, 3H, CH ₂ CH ₂ NH), 8. 16 (t, J= 6.5 Hz, 9H, CH ₂ NH).

Example 2. Preparation of a protected maleimide functionalized nine-arm 2-bromo-2- methyl-propionamide initiator

[0297] Product 2.1 : 1 , 1 l -tosyl-3,6,9-trioxaundecane

[0298] Into a 2000 ml 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed a solution of 2-[2-[2-(2- hydroxyethoxy)ethoxy]ethoxy]ethan- l -ol (50 g, 257.43 mmol, 1 .00 equiv) in

dichloromethane (500 ml), triethylamine ( 130 g, 1 .28 mol, 4.99 equiv).was at 0~10°C. This was followed by the addition of a solution of 4-methylbenzene- l -sulfonyl chloride (250 g, 1.31 mol, 5.09 equiv) in dichloromethane (400 ml) dropwise with stirring. The resulting solution was stirred for 5 h at 10-20 C. The reaction was then quenched by the addition of 1000 ml of water. The resulting solution was extracted with 3x300 ml of dichloromethane and the organic layers combined. The resulting mixture was washed with 1 300 ml of brine. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica gel column with ethyl acetate/petroleum ether ( 1 : 50—1 : 1 ). This resulted in 106 g (82%) of 1 , 1 l -tosyl-3,6,9-trioxaundecane as yellow oil.

[0299] Product 2.2: 1 -tosyl-l l -(3,5,7-triaza-4,6, 10-triphenyl-adamantan- l -yl methoxy)- 3,6,9-trioxaundecane

[0300] Into a 1000 ml round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed a solution of [2,4,9-tripheny 1- 1 ,3, 5-triazatricyclo[3.3.1 . l ' ⁷ ]decan-7- yl]methanol (30 g, 75.47 mmol, 1 .00 equiv) in THF (300 ml). To this was added sodium hydride (9 g, 225.00 mmol, 2.98 equiv, 60%) at 0-10°C. The mixture was stirred for 30 min at 0—10 C. This was followed by the addition of a solution of 1 , 1 l -tosyl-3,6,9- trioxaundecane (product 2.1 ) (106 g, 210.90 mmol, 2.79 equiv) in THF ( 100 ml) dropwise with stirring. The resulting solution was stirred for 4 h at 60 C in an oil bath. The reaction was then quenched by the addition of 500 ml of water. The resulting mixture was concentrated under vacuum. The resulting solution was extracted with 3x200 ml of ethyl acetate and the organic layers combined. The resulting mixture was washed with 1 x200 ml of brine. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica gel column with ethyl acetate/petroleum ether (1 :50~1 :3). This resulted in 31 g (56%) of 1 -tosyl- l l -(3,5,7-triaza-4,6, 10-triphenyl- adamantan- l -yl methoxy)-3,6,9-trioxaundecane as yellow oil.

[0301] Product 2.3 : 4-[l l -(3,5,7-triaza-4,6,10-triphenyl-adamantan- l -yl methoxy)-3,6,9- trioxaundecyl]- ! 0-oxa-4-aza-tricyclo[5.2.1.0 ^2,6 ]dec-8-ene-3,5-dione

[0302] Into a 500 ml round-bottom flask, was placed a solution of 1 -tosyl-l l -(3,5,7-triaza- 4,6, 10-triphenyl-adamantan- l -yl methoxy)-3,6,9-trioxaundecane (product 2.2) (31 g, 42.59 mmol, 1.00 equiv) in N,N-dimethylformamide (350 ml), K ₂ C0 ₃ (18 g, 129.30 mmol, 3.04 equiv), 10-oxa-4-azatricyclo[5.2.1 ,0 ^A 2,6]dec-8-ene-3,5-dione (14 g, 84.77 mmol, 1 .99 equiv), potassium iodide ( 1 g, 6.02 mmol, 0.14 equiv). The resulting solution was stirred for 1 8 h at 60 C in an oil bath. The reaction was then quenched by the addition of 2500 ml of water. The resulting solution was extracted with 3x500 ml of ethyl acetate and the organic layers combined. The resulting mixture was washed with 1 x500 ml of brine. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. This resulted in 28.5 g (93%) of 4-[l l -(3 _> 5,7-triaza-4,6, 10-triphenyl-adamantan- l -yl methoxy)-3,6,9-trioxaundecyl]- 10-oxa-4-aza-tricyclo[5.2.1.0 ² ' ⁶ ]dec-8-ene-3,5-dione as yellow oil.

[03031 Product 2.4: 4-[l l -(2,2,2-triaminomethyl-ethoxy)-3,6,9-trioxaundecyl]- 10-oxa-4- aza-tricyclo[5.2.1 .0 ² ' ⁶ ]dec-8-ene-3,5-dione trihydrochloride

[0304] Into a 1000 ml round-bottom flask was placed a solution of 4-[l l -(3,5,7-triaza- 4,6, 10-tripheny 1-adamantan- 1 -y 1 methoxy)-3 ,6,9-trioxaundecyl]- 10-oxa-4-aza- tricyclo[5.2.1.0 ² ' ⁶ ]dec-8-ene-3,5-dione (product 2.3) (28.5 g, 39.54 mmol, 1 .00 equiv) in tetrahydrofuraii (300 ml), hydrogen chloride (1N) (125 ml). The resulting solution was stirred for 3 h at 10-20 C. The resulting mixture was concentrated under vacuum. The resulting mixture was washed with 1x200 ml of ethyl acetate. This resulted in 13.36 g (71 %) of 4-[l l -(2,2,2-triaminomethyl-ethoxy)-3,6,9-trioxaundecyl]- 10-oxa-4-aza- tricyclo[5.2.1.0 ^2-6 ]dec-8-ene-3,5-dione trihydrochloride as an off-white solid. LC-MS: (ES, m/z): [M-3HC1+H] ⁺ 457. Ή NMR (400 MHz, CD ₃ OD): δ = 3.00 (s, 2H, CHC=0), 3.40 (s, 8H, CCH ₂ ), 3.6-3.8 (m, 16H, OCH ₂ CH ₂ O), 5.23 (s, 2H, CHO), 6.58 (s, 2H, CH=CH).

[0305] Product 2.5 : (2-{2,2-Bis-[(3-tert-butoxycarbonylamino-propionylamino)-met hyl]- 3-[ l l -(3,5-dioxo-10-oxa-4-aza-tricyclo[5.2.1 .0 ² ' ⁶ ]dec-8-en-4-yl)-3,6,9-trioxaundecyloxy]- propylcarbamoyl}-ethyl)-carbamic acid tert-butyl ester

[0306] Into a 250 ml round-bottom flask, which was purged and maintained with a nitrogen atmosphere, was placed a solution of 4-[l l -(2,2,2-triaminomethyi-ethoxy)-3,6,9- trioxaundecyl]-10-oxa-4-aza-tricyclo[5.2.1.0 ² ' ⁶ ]dec-8-ene-3,5-dionc trihydrochloride (product 2.4) ( 10.0 g, 17.7 mmol, 1 .00 equiv) in dichloromethane (100 ml). Triethylamine (5.40 g,

53.5 mmol, 3.02 equiv) was added to the reaction mixture, and stirred for 30 min at 15—20 C. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (10.2 g, 53.2 mmol, 3.00 equiv), 1 -hydroxybenzotriazole (7.20 g, 53.3 mmol, 3.01 equiv), and 3-[(tert- butoxy)carbonyl]aminopropanoic acid ( 10.0 g, 52.9 mmol, 2.98 equiv) were added to the reaction mixture, and stirred for 16 h at 1 -20 C, before quenched with 1 50 ml of water. The organic materials were extracted with 3x100 ml of dichloromethane, and the combined extracts were washed with 2x100 ml of sodium carbonate (aq.) and 2x 1 00 ml of hydrochloric acid ( I N), then dried over anhydrous sodium sulfate. The solvents were removed under vacuum to yield 12.5 g (69%) of (2-{2,2-Bis-[(3-tert-butoxycarbonylamino-propionylamino)- methyl]-3-[ l l -(3,5-dioxo- 10-oxa-4-aza-tricyclo[5.2.1 .0 ² - ⁶ ]dec-8-en-4-yl)-3 ,6,9- trioxaundecyloxy]-propylcarbamoyl} -ethyl)-carbamic acid tert-butyl ester as a yellow solid. [0307] Product 2.6: 3-Amino-N- {2,2-bis-[(3-amino-propionylamino)-methyl]-3-[l l -(3,5- dioxo- 10-oxa-4-aza-tricyclo[5.2.1.0 ² ]dec-8-en-4-yl)-3,6,9-trioxaundecyloxy]-propyl} - propionamide

[0308] Into a 250 ml round-bottom flask, was placed a solution of (2- {2,2-Bis-[(3-tert- butoxycarbonylamino-propionylamino)-methyl]-3-[ l l -(3,5-dioxo-10-oxa-4-aza- tricyclo[5.2.1 .0 ^2,6 ]dec-8-en-4-yl)-3,6,9-trioxaundecy]oxy]-propylcarbamoy l}-ethyl)-carbamic acid tert-butyl ester (product 2.5) (12.5 g, 12.2 mmol, 1 .00 equiv, 95%) in dichloromethane (150 ml). Trifluoroacetic acid (40 ml) was added to the reaction mixture dropwise, and stirred for 3 h at 10-20 C. Upon completion, the solvent was removed under reduced pressure, and the resulting crude material was dissolved in 200 ml of water. The aqueous solution was washed with ethyl acetate (3x1 50 ml), then concentrated under vacuum to furnish 9.00 g (73%) of 3-Amino-N- {2,2-bis-[(3-am ino-propionylamino)-methyl]-3-[ l 1 -(3,5- dioxo- 10-oxa-4-aza-tricycIo[5.2.1.0 ² ' ⁶ ]dec-8-en-4-yl)-3,6,9-trioxaundecyIoxy]-propyl}- propionamide as a red solid. LC-MS (PH-OGS-006-0): (ES, m/z) 670 [M-3CF ₃ COOH+l ] ⁺ . Ή NMR (400 MHz, CD ₃ OD): δ = 6.57(s, 2H), 5.1 8(s, 2H), 3.61 -3.69(m, 14H), 3.59(s, 5H), 3.40(s, 2H), 3.21 -3.27(m, 14H), 2.96(s, 2H), 2.67-2.70(d, 7H).

[0309] Product 2.7: Protected maleimide functionalized nine-arm 2-bromo-2-methyl- propionamide initiator: 2-( l ] -(3,5-dioxo- 10-oxa-4-aza-tricyclo[5.2.1.0 ^2,6 ]dec-8-en-4-yl)- 3,6,9-trioxaundecyloxy)-ethane- 1 , 1 , 1 -tri(methyl-3-[2-(2,2,2-tri(2-bromo-2-methyl- propiony)aminomethyl)-ethoxy)-acetylamino])-propionamide

[0310] Into a 2000 ml three neck round-bottom flask, which was purged and maintained with a nitrogen atmosphere, was placed a solution of 2-[3-(2-bromo-2-methylpropanamido)- 2,2-bis[(2-bromo-2-methylpropanamido)methyl]propoxy]acetic acid (from

PCT/US2010/34252) (8.80 g, 13.8 mmol, 3.25 equiv) in a solvent mixture of

dichloromethane and DMF (7: 1 , 1 100 mL). HBTU (5.80 g, 15.3 mmol, 3.61 equiv) was added to the reaction mixture, and stirred for 1 .5 h at room temperature. A solution of 3- Amino-N-{2,2-bis-[(3-amino-propionylamino)-methyl]-3-[l l -(3,5-dioxo- 10-oxa-4-aza- tricyclo[5.2.1.0 ² ' ⁶ ]dec-8-en-4-yl)-3,6,9-trioxaundecyloxy]-propyl}-propio namide (product 2.7) (5.50 g, 4.24 mmol, 1 .00 equiv) in a solvent mixture of dichloromethane and DMF (7: 1 , 50 ml) was added to the reaction solution dropwise. The reaction was stirred overnight at room temperature. After completion of the reaction, the volatiles were removed under vacuum. The resulting solution was diluted with 50 ml of ice water, then extracted with 3x100 ml of ethyl acetate. The combined organic layers were washed with 2x100 ml of brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The crude product was purified by Flash-Prep-HPLC to furnish 5.42 g (51 %) of 2-( l l -(3,5-dioxo- 10- oxa-4-aza-tricyclo[5.2.1 .0 ^2,6 ]dec-8-en-4-yl)-3,6,9-trioxaundecyloxy)-ethane- 1 , 1 , 1 -tri(methyl- 3-[2-(2,2,2-tri(2-bromo-2-methyl-propionylaminomethyl)-ethox y)-acetylamino])- propionamide as an off-white solid. Ή NMR (400 MHz, CDC1 ₃ ): δ = 1.99 (s, 54H, CH ₃ ), 2.54 (t, J= 6 Hz, 6H, CH ₂ CH ₂ NH), 2.87 (s, 2H, CHC=0), 3. 13 (d, J= 6.3 Hz, 24H,

CCHjNH), 3.25 (s, 8H, OCH ₂ C(CH ₂ NH) ₃ ), 3.5-3.7 (m, 22H, OCH ₂ CH ₂ O and CH2CH ₂ NH), 4.01 (s, 6H, OCH ₂ C=0), 5.25 (s, 2H, CHO), 6.52 (s, 2H, CH=CH), 7.5 (broadened, 3H, NH), 7.88 (broadened, 3H, NH), 8.14 (t, J= 6.5 Hz, 9H, NHC(=0)C(CH ₃ ) ₂ Br). Example 3. Preparation of a protected maleimide functionalized three-arm 2-bromo-2- methyl-propionic acid ester initiator

[0311] Product 3.1 : 2-(2-Bromo-2-methyl-propionyloxy)-2-methyl-propion ic acid

[0312] A solution of 9 g 2-bromoisobutyryl bromide, 4.5 g a-hydroxyisobutyric acid, and 3.95 g triethylamine in 60 ml tetrahydrofuran and 60 ml dichloromethane was allowed to stir overnight at room temperature. The reaction mixture was partitioned between water and dichloromethane. The organics were dried over anhydrous magnesium sulfate, filtered, and concentrated, then subjected to silica gel flash chromatography with 5% methanol in dichloromethane to yield the desired product. ^l U NMR (400 MHz, CDC1 ₃ ): δ = 1.50 (s, 12H, C¾).

[0313] Product 3.2: 2-(2-Bromo-2-methyl-propionyloxy)-2-methyl-propionic acid, N- hydroxysuccinimidyl ester

[0314] A round-bottom flask equipped with stirbar was charged with 7 g of 2-(2-Bromo-2- methyl-propionyloxy)-2-methyl-propionic acid (product 3.2), 3.76 g of N- hydroxysuccinimide, 5.73 g of N,N-dicyclohexylcarbodiimide, 350 mg of 4- (dimethylamino)pyroidine, and 150 ml of dichloromethane, and allowed to stir for three hours. The mixture was filtered to remove dicyclohexylurea and concentrated. The residue was dissolved in dichloromethane, adsorbed onto silica gel, and reconcentrated. This powder was then subjected to silica gel flash chromatography with 30-70% ethyl acetate in hexane to yield 7.9 g of the desired product as a white powder. Ή NMR (400 MHz, CDC1 ₃ ): δ = 1 .78 (s, 6H, OC(CH ₃ ) ₂ ), 1.96 (s, 6H, BrC(CH ₃ ) ₂ ), 2.83 (s, 4H, CH ₂ ).

[0315] Product 3.3 : Protected maleimide functionalized three-arm 2-bromo-2-methyl- propionic acid ester initiator: 4-[l l -(2,2,2-Tri-{[2-(2-bromo-2-methyl-propionyloxy)-2- methyl-propionylamino]-methyl}-ethoxy)-3,6,9-trioxaundecyl]- 10-oxa-4-aza- tricyclo[5.2.1.0 ^2,6 ]dec-8-ene-3,5-dione

n o

[0316] A round-bottom flask equipped with stirbar was charged with 60 ml dry acetonitrile, 1 .13 g of 4-[ l l -(2,2,2-triaminomethyl-ethoxy)-3,6,9-trioxaundecyl]- 10-oxa-4-aza- tricyclo[5.2.1 .0 ² ' ⁶ ]dec-8 -ene-3,5-dione trihydrochloride (product 2.4), 2.1 g of 2-(2-Bromo-2- methyl-propionyloxy)-2-methyl-propionic acid, N-hydroxysuccinimidyl ester (product 3.2), and 4 ml triethylam ine. The reaction was allowed to stir overnight at room temperature, then concentrated and partitioned between 50 ml each dichloromethane and 0.2N HC1. The aqueous layer was extracted twice with 50 ml dichloromethane. The combined organics were clarified with 1 0 ml brine, then concentrated and subjected to silica gel flash chromatography with 50- 100% ethyl acetate in hexane to yield the desired product as a yellow oil. Ή N R (400 MHz, CDC ): δ = 1 .59 (s, 1 8H, OC(CH ₃ ) ₂ ), 1.96 (s, 1 8H, BrC(CH ₃ ) ₂ ), 2.86 (s, 2H, CHC=0), 3.1 -3.4 (m, 8H, CH ₂ NH and CCH ₂ 0), 3.56-3.66 (m, 16H, OCHbCHjO), 5.26 (s, 2H, CHO), 6.52 (s, 2H, CH=€H), 7.37 (broadened, 2H, Nil), 7.79 (broadened, 1 H, NH).

Example 4. Preparation of a protected maleimide functionalized nine-arm 2-bromo-2- methYl-propionic acid ester initiator

[0317] Product 4.1 : 2,2,2-tribenzyloxymethyl-ethan- l -ol

[0318] Into a 10 L four neck round-bottom flask, which was purged and maintained with a nitrogen atmosphere, was placed a solution of 2,2-bis(hydroxymethyl)propane- l ,3-diol ( 1 10 g, 0.808 mol, 1.00 equiv) in N,N-dimethylformamide (6 L). Sodium hydride (97.0 g, 2.42 mol, 3.00 equiv, 60%) was added to the reaction mixture, and stirred for 40 min at 10-15 C in an ice-water bath. Benzyl bromide (415 g, 2.43 mol, 3.00 equiv) was then added to the reaction solution dropwise, and stirred for another 3 h at 10-15 C. The reaction was quenched by the addition of 30 L of water. The resulting solution was extracted with 3x1500 ml of ethyl acetate. The combined organic layers were washed with 1 1500 ml of brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel chromatography with ethyl acetate/petroleum ether (1 : 100-1 :20) as eluent to furnish 148 g (45%) of 2,2,2-tribenzyloxymethyl-ethan- l-ol as a yellow oil.

[0319] Product 4.2: (2,2,2-tri-benzyloxymethyl-ethoxy)-acetic acid fer/-butyl ester

[0320] Into a 5 L three neck round-bottom flask, which was purged and maintained with a nitrogen atmosphere, was placed a solution of 2,2,2-tribenzyloxymethyl-ethan- l -ol (Product 4.1 ) (148 g, 364 mmol, 1.00 equiv) in N,N-dimethylformamide (3000 ml). Sodium hydride (29.0 g, 725 mmol, 1.99 equiv, 60%) was added to the reaction mixture and stirred for 1 h at

10-15 C in an ice-water bath. This was followed by the addition of /er/-butyl 2-bromoacetate (142 g, 728 mmol, 2.00 equiv) dropwise with stirring. The resulting solution was stirred for 7 h at 10-15 C in an ice-water bath, then quenched with 12000 ml of water. The resulting solution was extracted with 3x2000 ml of ethyl acetate. The combined organic layers were washed with l l 500 ml of brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel chromatography with ethyl acetate/petroleum ether (1 : 100-1 :30) as eluent to furnish 93 g (48%) of (2,2,2-tri- benzyloxymethyl-ethoxy)-acetic acid /er/-butyl ester as a yellow oil.

[0321] Product 4.3: (2,2,2-tri-hydroxymethyl-ethoxy)-acetic acid fer/-butyl ester

[0322] Into a 2000 ml round-bottom flask, which was purged and maintained with a nitrogen atmosphere, was placed a solution of (2,2,2-tri-benzyloxymethyl-ethoxy)-acetic acid tert-buty\ ester (product 4.2) (93.0 g, 162 mmol, 1 .00 equiv, 98%>) and palladium on carbon (36 g) in methanol (1000 ml). The reaction solution was hydrogenated under atmosphere pressure for 3 days at 30 C. After removing the catalyst by filtration, the solution was concentrated under vacuum. This resulted in 34 g (83%) of (2,2,2-tri-hydroxymethyl- ethoxy)-acetic acid tert-butyl ester as a gray solid.

[0323] Product 4.4: (2,2,2-Tri(2-bromo-2-methyl-propionyloxymethyl)-ethoxy)-acet ic acid tert-butyl ester

[0324] Into a 1000 ml three neck round-bottom flask, which was purged and maintained with a nitrogen atmosphere, was placed a solution of (2,2,2-tri-hydroxymethyl-ethoxy)-acetic acid tert-buty\ ester (Product 4.3) (34 g, 133 mmol, 1 .00 equiv, 98%) and triethylamine (1 10 g, 1.09 mol, 8.17 equiv) in tetrahydrofuran (350 ml). 2-Bromo-2-methylpropanoyl bromide (264 g, 1 .1 5 mol, 8.63 equiv) was added to the stirred reaction mixture dropwise. The reaction mixture was stirred for 12 h at 10— 1 5 C in an ice water bath, then quenched with 500 ml of water. The resulting solution was extracted with 3x300 ml of ethyl acetate and the combined organic layers were washed with 1 x300 ml of brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by flash column chromatography (S1O2, eluent: ethyl acetate/petroleum ether ( 1 :50-1 :20)) to afford 96 g (98%) of (2,2,2-Tri(2-bromo-2-methyl-propionyloxymethyl)-ethoxy)-acet ic acid tert-butyl ester as a yellow oil.

[0325] Product 4.5: (2,2,2-Tri(2-bromo-2-methyl-propionyloxymethyl)-ethoxy)-acet ic acid

[0326] Into a 2000 ml three neck round-bottom flask, was placed a solution of (2,2,2-Tri(2- bromo-2-methyl-propionyloxymethyl)-ethoxy)-acetic acid tert-butyl ester (product 4.4) (96.0 g, 131 mmol, 1.00 equiv, 95%) in dichloromethane ( 1000 ml). Trifluoroacetic acid (120 ml) was then added to the reaction mixture dropwise. The resulting solution was stirred for 18 h at 20 C, then quenched with 1 000 ml of water. The resulting solution was extracted with 3x500 ml of dichloromethane and the combined organic layers were washed with 1 x500 ml of brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to afford 86.0 g (97%) of (2,2,2-Tri(2-bromo-2-methyl-propionyloxymethyl)-ethoxy)-acet ic acid as a yellow oi

[0327J Product 4.6: (2,2,2-Tri(2-bromo-2-methyl-propionyloxymethyl)-ethoxy)-acet ic acid n-hydroxysuccinimidyl ester

[0328] Into a 2000 ml three neck round-bottom flask, which was purged and maintained with a nitrogen atmosphere, was placed a solution of (2,2,2-Tri(2-bromo-2-methyl- propionyloxymethyl)-ethoxy)-acetic acid (product 4.5) (86.0 g, 127 mmol, 1 .00 equiv, 95%) in N,N-dirnethylformamide (900 ml). N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (70.0 g, 365 mmol, 2.87 equiv) and N-hydroxysuccinimide (46.0 g, 400 mmol, 3.14 equiv) was added to the reaction mixture and stirred for 12 h at 20 C. The reaction was then quenched by the addition of 5000 ml of water. The resulting solution was extracted with 3x1 500 ml of ethyl acetate and the combined organic layers were washed with 1 x1 500 ml of brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by flash chromatography (S1O2, eluent; ethyl acetate/petroleum ether

(1 :30~1 :2)). The crude product was re-crystallized from a solvent mixture of ethyl acetate and petroleum ether ( 1 .4) to afford 57.0 g (55%) of (2,2,2-Tri(2-bromo-2-methyl- propionyloxymethyl)-ethoxy)-acetic acid n-hydroxysuccinimidyl ester as a white solid. LC- MS (PH-OGS-001 -0): (ES, m/∑): [M+Naf 757. Ή NMR (300 MHz, DMSO-d ₆ ): δ = 1 .90 (s, 1 8H, CH ₃ ), 2.83 (s, 4H, 0=CCH ₂ CH ₂ 00), 3.72 (s, 2H, CCH ₂ OCH ₂ C )), 4.21 (s, 6H, CH ₂ OC=0), 4.66 (s, 2H, CCHzOCH CK)).

[0329] Product 4.7: Protected maleimide functionalized nine-arm 2-bromo-2-methyl- propionic acid ester initiator: 2-(l l -(3,5-dioxo-10-oxa-4-aza-tricyclo[5.2.1 .0 ² ' ⁶ ]dec-8-en-4- yl)-3,6,9-trioxaundecyloxy)-ethane- l , l ,l-tri-(methyl-3-[2-(2,2,2-tri(2-bromo-2-methyl- propionyloxymethyl)-ethoxy)-acetylamino])-propionamide

[0330] Into a 250 ml three neck round-bottom flask, which was purged and maintained with a nitrogen atmosphere, was placed a solution of 3-Amino-N-{2,2-bis-[(3-amino- propionylamino)-methyl]-3-[l l -(3,5-dioxo- 10-oxa-4-aza-tricyclo[5.2.1.0 ² ' ⁶ ]dec-8-en-4-yl)- 3,6,9-trioxaundecyloxy]-propyl}-propionamide (product 2.6) (3.50 g, 3.46 mmol, 1.00 equiv) in dichloromethane (100 ml). Triethylamine (1 .50 g, 14.8 mmol, 4.28 equiv) was added to the reaction mixture. The reaction was stirred for 1 h at 10-20 C followed by the addition of (2,2,2-Tri(2-bromo-2-methyl-propionyloxymethyl)-ethoxy)-acet ic acid n- hydroxysuccinimidyl ester (product 4.6) (8.00 g, 10.8 mmol, 3. 13 equiv). The resulting solution was stirred for 16 h at 10~20 C, then quenched with 200 ml of water. The solution was extracted with 3x200 ml of dichloromethane and the combined organic layers were washed with 1 x200 ml of brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The crude product (10 g) was purified by Prep-HPLC with the following conditions (Gilson Pre-HPLC( ax. pressure:8MPa)): Column, SunFire Prep C I 8, 5um, 19* 100mm; mobile phase, WATER WITH 0.05%TFA and CH3CN (95% CH ₃ CN in 20 min, up to 100% in 20 min, hold 100% in 1 .4 min); Detector, Gilson UV Detector 254nm. This led to 3.75 g (42%) of 2-(l l -(3,5-dioxo-10-oxa-4-aza-tricyclo[5.2.1.0 ² ' ⁶ ]dec-8-en-4-yl)-3,6,9- trioxaundecyloxy)-ethane- 1 , 1 , 1 -tri-(methyl-3-[2-(2,2,2-tri(2-bromo-2-methyl- propionyloxymethyl)-ethoxy)-acetylamino])-propionamide as an off-white solid. ' H NMR (400 MHz, CDC ): 8 = 1.93 (s, 54H, CH ₃ ), 2.50 (t, ./= 6 Hz, 6H, CH ₂ CH ₂ NH), 2.86 (s, 211, CHC=0), 3.08 (d, J= 6 Hz, 6H, CCHjNH), 3.29 (s, 2H, OCH ₂ C(CH ₂ NH) ₃ ), 3.5-3.7 (m, 28H, OCH ₂ CH ₂ O and CH ₂ CH2NH and OCH ₂ C(CH ₂ 0) ₃ ), 3.96 (s, 6H, OCH ₂ C=0), 4.32 (s, 18H, C(CH20) ₃ ), 5.25 (s, 2H, CHO), 6.52 (s, 2H, CH=CH), 7.3 (broadened, 6H, NH). Example 5. Preparation of a dihydroxy functionalized nine-arm 2-bromo-2-methyl- propionic acid ester initiator

[03311 Product 5.1 : 2-(l l -Allyloxy-3,6,9-trioxaundecyloxy)-ethane- 1 , 1 , 1 -tri(methyl-3-[2- (2,2,2-tri(2-bromo-2-methyl-propionyloxymetliyl)-ethoxy)-ace tylamino])-propionamide

[0332] A round-bottom flask equipped with stirbar was charged with 2.95 g of (2,2,2-Tri(2- bromo-2-methyl-propionyloxymethyl)-ethoxy)-acetic acid n-hydroxysuccinimidyl ester (product 4.6) and 30 ml dry acetonitrile, followed by 1 ml triethylamine. A solution of l -(3- amino-propionylamino)-2,2-bis[(3-aminopropionylamino)-methyl ]-2,5,8, l 1 , 14- pentaoxaheptadec- 16-ene trihydrochloride (Product 1 .2) in 20 ml dry acetonitrile was added dropwise and the reaction stirred overnight, then concentrated. The residue was suspended in dichloromethane and filtered, then washed with 10 ml each water and I N HCl. The aqueous layers were then extracted with 20 ml dichloromethane and the combined organics were concentrated. The residue was dissolved in a small amount of 50% ethyl acetate/hexane and subjected to silica gel flash chromatography with 10- 1 00% acetone in hexane, to yield 1.63 g of the desired product as a yellow oil. Ή NMR (400 MHz, CDCI ₃ ): δ = 1.93 (s, 54H, CH ₃ ), 2.51 (t, J= 6 Hz, 6H, CH ₂ CH ₂ NH), 3.07 (d, J= 5.1 Hz, 6H, CCH^NH), 3.26 (s, 2H,

OCH2C(CH ₂ NH) ₃ ), 3.5-3.7 (m, 28H, OCHJCHJO and and OCHjC CI I ₂ 0) ₃ ), 3.97 (s, 6H, OCH ₂ C=0), 4.01 (d, J= 5.5 Hz, 2H, CHCH7O), 4.32 (s, 18H, C(CH ₂ 0) ₃ ), 5.20 (d of d, .7=10.6, 1.2 Hz, 1 H, CH ₂ =CH), 5.29 (d of q, J= 1 7.3, 1 .5 Hz, 1 H, CH ₂ =CH), 5.90 (m, 1 H, CH), 7.30 (broadened, 3H, NH), 7.47 (broadened, 3H, NH).

[0333] Product 5.2: Dihydroxy functionalized nine-arm 2-bromo-2-methyl-propionic acid ester initiator: 2-(l l -[l ,2-dihydroxypropyloxy]-3,6,9-trioxaundecyloxy)-ethane- 1 , 1 , 1 - tri(methyl-3-[2-(2,2,2-tri(2-bromo-2-meth^

propionamide

[0334] A round-bottom flask equipped with a stirbar was charged with 10 ml water, 10 ml t-butanol, 1 .63 mg of 2-( 1 1 -Allyloxy-3,6,9-trioxaundecy loxy)-ethane- 1 , 1 , 1 -tri(methyl-3-[2- (2,2,2-tri(2-bromo-2-methyl-propionyloxymethyl)-ethoxy)-acet lamino])-propionamide (product 5.1 ), 680 mg potassium ferricyanide, 290 mg potassium carbonate, 65.7 mg methanesulfonamide, 6.5 mg quinuclidine, and 3.0 mg potassium osmate dihydrate and stirred for two days at room temperature. The reaction mixture was concentrated, then partitioned between 50 ml each of water and dichloromethane. The aqueous layer was extracted five times more with 50 ml dichloromethane. The organic layers were combined, clarified by washing with 5 ml saturated sodium chloride, and concentrated, then passed over a silica gel plug with acetone and concentrated. The residue was subjected to silica gel flash chromatography using 3.5-5% methanol in dichloromethane. The column was then flushed with 100 ml each 10% methanol in dichloromethane, then pure methanol. Product containing fractions were combined to give the desired product as a clear oil. Ή NMR (400 MHz, CDC1 ₃ ): δ = 1 .93 (s, 54H, CH ₃ ), 2.53 (s, 6H, CH2CH7NH), 3.07 (d, J= 5.1 Hz, 6H,

CCH2NH), 3.26 (s, 2H, OCH ₂ C(CH ₂ NH) ₃ ), 3.5-3.7 (m, 32H, and CH2CH ₂ NH and OCHj,C(CH ₂ 0) ₃ and CHCH2O and CH2OH), 3.94 (s, 1 H.CHOH), 3.96 (s, 6H,

OCH ₂ C=0), 4.32 (s, 18H, C(CH ₂ 0) ₃ ), 7.26 (broadened, 3H, NH), 7.46 (broadened, 3H, NH).

Example 6. Preparation of a protected maleimide functionalized six-arm 2-bromo-2- methyl-propionic acid ester initiator

[0335] Step 1

[0336] Into a 3000 mL three neck round-bottom flask, which was purged and maintained with an inert atmosphere of nitrogen, was placed a solution of 3-hydroxy-2-(hydroxymethyl)- 2-methylpropanoic acid (65.0 g, 485 mmol, 1.00 equiv) and pyridine (100 g, 1.26 mol, 2.61 equiv) in dichloromethane ( 1000 mL). 2-Bromo-2-methylpropanoyl bromide (227 g, 987 mmol, 2.04 equiv) was added drop wise at 0 C in 30 min. The resulting solution was stirred overnight at room temperature, then quenched with 1000 mL of ice water. The aqueous phase was extracted with 2 1000 mL of dichloromethane, and the combined organic layers were dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica gel column with ethyl acetate/hexane ( 1 :50) as eluent. The crude product was purified by re-crystallization from petroleum ether. This resulted in 100 g (48%) of 3- [(2-bromo-2-methy Ipropanoy 1 )oxy] -2- [ [(2-bromo-2-methylpropanoyl)oxy] methy 1] -2- methylpropanoic acid as a white solid.

[0337] Step 2

[0338] Into a 1000 mL round-bottom flask, which was purged and maintained with an inert atmosphere of nitrogen, was placed a solution of 3-[(2-bromo-2-methylpropanoyl)oxy]-2- [[(2-bromo-2-methylpropanoyl)oxy]methyl]-2-methylpropanoic acid (1 1.1 g, 25.7 mmol, 1.00 equiv) in chloroform (250 mL). Thionyl chloride ( 1 1.0 g, 92.5 mmol, 3.60 equiv) and N,N-dimethylformamide (4 drops) were added to the reaction mixture. The resulting solution was stirred overnight at 36°C in an oil bath, then concentrated under vacuum to yield 12.1 g (crude) of 2-[[(2-bromo-2-methylpropanoyl)oxy]methyl]-3-chloro-2-methyl -3-oxopropyl 2- bromo-2-methylpropanoate as a colorless oil. This material was used in the next step reaction directly without isolation.

[0339] Ste 3

[0340] Into a 1000 mL three neck round-bottom flask, which was purged and maintained with an inert atmosphere of nitrogen, was placed a solution of 4-[15-amino- 14,14- bis(aminomethyl)-3,6,9, 12-tetraoxapentadecan- l -yl]-10-oxa-4-azatricyclo[5.2.1.0 ^A [2,6]]dec- 8-ene-3,5-dione (3.55 g, 7.78 mmol, 1.00 equiv) in dichloromethane (450 mL). A solution of 2-[(2-bromo-2-methylpropanoyl)oxy]methyl-3-chloro-2-methyl-3 -oxopropyl 2-bromo-2- methylpropanoate ( 1 1.5 g, 25.4 mmol, 3.27 equiv) in dichloromethane (1 50 mL) was added dropwise at 0 C. To this was added triethylamine (8.50 g, 84.0 mmol, 10.8 equiv). The resulting solution was stirred for 1.5 h at 0°C in an ice bath, then quenched with 30 mL of methanol. The resulting mixture was concentrated under vacuum. The crude product (28 g) was purified by Combiflash with the following conditions (IntelFlash- 1 ): Column, C I 8; mobile phase, CH ₃ CN/H ₂ O=50:50 increased to CH ₃ CN/H ₂ O=100:0 within 50 min; Detector, UV 220 nm. This resulted in 8.00 g (61 %) of the six arm protected maleimide initiator as an light yellow solid. LC-MS: (ES, m/z): [M+H] ⁺ 1699; Ή-NMR: (400MHz, CDC1 ₃ , ppm): δ 7.75(3H, s), 6.52 (2H, s), 5.62(2H, s), 4.39( 12H, m), 3.66(16H, m), 3.31 (2H, d), 2.87(2H, s), 1.92(36H, s), 1.41 (9H, s). Example 7. Preparation of a protected vinyl sulfonamide functionalized six-arm 2- bromo-2-methyl-propioiiic acid ester initiator

[0341] Step 1

[0342] Into a 250 mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed a solution of methyl 3-aminopropanoate hydrochloride (5.0 g, 35.82 mmol, 1 .00 equiv) in dichloromethane (80 mL), triethylamine ( 10.9 g, 1 07.72 mmol, 3.01 equiv). This was followed by the addition of a solution of 7- oxabicyclo[2.2.1]hept-5-ene-2-sulfonyl chloride (8.7 g, 44.70 mmol, 1 .25 equiv) in dichloromethane (20 mL) dropwise with stirring at 0 C. The resulting solution was stirred for 30 min at room temperature. The resulting mixture was washed with 1 x50 mL of brine. The mixture was dried over anhydrous magnesium sulfate and concentrated under vacuum. This resulted in 9.0 g (crude) of methyl 3-[7-oxabicyclo[2.2.1 ]hept-5-ene-2- sulfonamidojpropanoate as red oil.

[0343] Step 2

OMe

[0344] Into a 1 50 mL sealed tube, was placed a solution of methyl 3-[7- oxabicyclo[2.2.1 ]hept-5-ene-2-sulfonamido]propanoate (9 g, 34.44 mmol, 1.00 equiv) in CH3CN ( 100 mL), potassium carbonate (1 1 .9 g, 86.10 mmol, 2.50 equiv), iodomethane (14.6 g, 102.86 mmol, 2.99 equiv). The resulting solution was stirred overnight at 50 C in an oil bath. The solids were filtered out. The organic layer was concentrated under vacuum. The residue was applied onto a silica gel column with ethyl acetate/petroleum ether (1 :3~1 : 1.5). This resulted in 6.02 g (63%) of methyl 3-[N-methyl7-oxabicyclo[2.2. l ]hept-5-ene-2- sulfonamido]propanoate as yellow oil.

[0345] Step 3

Into a 250 mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed a solution of LiAlHL (1 .24 g, 32.67 mmol, 3.35 equiv) in tetrahydrofuran (35 mL). A solution of methyl 3-[N-methyl7-oxabicyclo [2.2.1 ] hept-5-ene- 2-sulfonamido]propanoate (3.0 g, 10.90 mmol, 1.00 equiv) in tetrahydrofuran ( 1 5 mL) was added drop wise with stirring at 0 C. The resulting solution was stirred for 30 min at 0 C in a water/ice bath. The reaction was quenched by the addition of 2 mL of water. The resulting solution was diluted with 3.5 mL of 15%NaOH(aq). The solids were filtered out. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. This resulted in 2.54 g (94%) of 3-hydroxy-N-methyl-S-[7-oxabicyclo[2.2.1 ]hept-5-en-2- yl]propane-l -sulfonamido as yellow oil.

[0346] Step 4

[0347] Into a 250 mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed a solution of 3-hydroxy-N-methyl-S-[7- oxabicyclo[2.2.1 ]hept-5-en-2-yl]propane- l -sulfonamido (2.54 g, 10.27 mmol, 1.00 equiv) in tetrahydrofuran (50 mL). Sodium hydride (700 mg, 17.50 mmol, 1 .70 equiv) was added to reaction mixture at room temperature in 30 min. To this was added a solution of 1 -2,4,9- triphenyl- l ,3,5-triazatricyclo[3.3.1 . l ^A 3,7]decan-7-yl-2,5,8, l l -tetraoxatridecan- 13-yl 4- methylbenzene-l-sulfonate (7.2 g, 9.89 mmol, 0.96 equiv) in tetrahydrofuran (20 mL) drop wise with stirring . The resulting solution was stirred for 1 overnight at 48 C in an oil bath. The reaction was quenched by the addition of 20 mL of water/ice. The resulting solution was extracted with 3x40 mL of ethyl acetate. The combined organic layers were washed with 1 x30 mL of brine, dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was applied onto a silica gel column with ethyl acetate/petroleum ether ( 1 : 1 ). This resulted in 3.0 g (36%) of N-methyl-N-( l -[2,4,9-triphenyl-l ,3,5- triazatricyclo[3.3.1.1 ^A [3,7]]decan-7-yl]-2,5,8, l l , 14-pentaoxaheptadecan- 17-yl)-7- oxabicyclo[2.2. ] ]hept-5-ene-2-sulfonamide as yellow oil.

[0348] Step 5

Into a 250 mL round-bottom flask, was placed a solution of N-methyl-N-(l -[2,4,9-triphenyl- 1 ,3,5-triazatricyclo[3.3.1 .1 ^A [3,7]]decan-7-yl]-2,5,8, 1 1 , 14-pentaoxaheptadecan- 17-yl)-7- oxabicyclo[2.2.1 ]hept-5-ene-2-sulfonamide (3.1 5 g, 3.92 mmol, 1.00 equiv) in

tetrahydrofuran ( 100 mL), IN hydrogen chloride (37 mL). The resulting solution was stirred for 2 h at room temperature. The resulting mixture was concentrated under vacuum. The resulting solution was extracted with 2x20 mL of ethyl acetate. The combined aqueous layers were lyophilized. This resulted in 2.42 g (95%) of N-[ 19-amino- 18, 1 8- bis(aminomethy l)-4,7, 10, 13 , 16-pentaoxanonadecan- 1 -y l]-N-methyl-7-oxabicyclo[2.2.1 ]hept- 5-ene-2-sulfonamide trihydrochloride as a yellow syrup.

[0349] Step 6

[0350] Into a 100 mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed a solution of N-[19-amino-18, 18-bis(aminomethyl)- 4,7, 10, 13, 16-pentaoxanonadecan- 1 -yl]-N-methyl-7-oxabicyclo[2.2.1 ]hept-5-ene-2- sulfonamide trihydrochloride ( 1 .23 g, 1.90 mmol, 1.00 equiv) in dichloromethane (30 mL), triethylamine (4 g, 39.6 mmol, 20.83 equiv). A solution of 3-(2-bromo-l -hydroxy-2- methy]propoxy)-2-[(2-bromo-l -hydroxy-2-methylpropoxy)methyl]- l -chloro-2- methylpropan- l-ol (2.67 g, 5.85 mmol, 3.08 equiv) in dichloromethane (20 mL) was added drop wise with stirring at 0 C to reaction mixture. The resulting solution was stirred for 30 min at 0 C in a water/ice bath. The reaction was quenched by the addition of 10 mL of methanol. The resulting mixture was concentrated under vacuum. The crude product was purified by Flash-Prep-HPLC. This resulted in 0.8 g (24%) of PH-OGS-023-0 as a yellow syrup. LC-MS: (ES, rn/z): [M+H] ⁺ = 1981 ; ' H-NMR:(300MHz,CDCl ₃ , fltw?): 7.687(3H,s), 6.397(l H,m), 5.321 (l H,m), 4.391 ( 1 l H,m), 3.655( 1 8H,m), 3.342(4H,m), 3.164(6H,m), 2.923(3H,m), 1.921 (36H,m), 1.834( l H,m), 1.380(9H,m).

Example 8. Preparation of a protected vinyl sulfonamide functionalized three-arm 2- bromo-2-methyl-propionic acid ester initiator

[0351] Into a 250 mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed a solution ofN-[ 19-amino-18, 18-bis(aminomethyl)- 4,7, 10, 13, 16-pentaoxanonadecan- 1 -yl]-N-methyl-7-oxabicyclo[2.2.1 ]hept-5-ene-2- sulfonamide trihydrochloride ( 1 .6 g, 2.47 mmol, 1.00 equiv) in dichloromethane (100 mL), triethylamine (8.0 g, 79.06 mmol, 32.02 equiv), 4-bromo-2,2,4-trimethyl-3-oxopentanoyl chloride ( 1.82 g, 7.12 mmol, 2.88 equiv). The resulting solution was stirred for 30 min at 0 C in a water/ice bath. The reaction was quenched by the addition of 10 mL of methanol. The resulting mixture was concentrated under vacuum. The crude product ( 1 6 g) was purified by Combiflash with the following conditions (IntelFlash- 1 ): Column, C18; mobile phase,

CH ₃ CN/H ₂ O=50:50 increased to CH ₃ CN/H ₂ 0= 100:0 within 50 min; Detector, UV 220 nm. This resulted in 1.8878 g (61 %) of the title initiator as yellow oil. LC-MS: (ES, m/z): [M+ l ] ⁺ = 1245; Ή-NMR: (COC\3, ppm) 7.368(3H,m), 6.381 (2H,m) , 5.308(1H,

s)5.132( l H,d,J=4.2), 3.607(17H,m), 3.227(4H,s), 3.108(7H,d,J=6), 2.840(3H,m),

2. 132( l H,m), 2.002(19H,m), 1.678( 18H,m), 1.370(2H,m).

Example 9. Preparation of 2-(Acryloyloxyethyl-2 trimethylammonium ethyl phosphate, inner salt (HEA-PC)

[0352] I ^s ' intermediate

[0353] A solution of 1 1 .6 grams of 2-hydroxyethylacrylate and 14.0 ml of triethylamine in 100 ml of dry acetonitrile, under a nitrogen atmosphere, was cooled to -20°C, and a solution of 14. 2 grams of 2-chloro-2-oxo- l ,3,2-dioxaphospholane in 10 ml of dry acetonitrile was added dropwise over about 30 minutes. The reaction was stirred in the cold for 30 minutes, then filtered under a nitrogen atmosphere. The precipitate was washed with 10 ml of cold acetonitrile, and the filtrate was used directly in the next reaction.

[0354] 2-(Acryloyloxyethyl-2 ^, -(trimethylammonium ^' )ethyl phosphate, inner salt

[0355] To the solution from the previous procedure was added 14.0 ml of trimethylamine (condensed using a dry ice-acetone condenser under nitrogen), the reaction mixture was sealed into a pressure vessel, and stirred at 65°C for 4 hours. The reaction mixture was allowed to stir while cooling to room temperature, and as it reached about 30°C, a solid began to form. The vessel was then placed in a 4°C refrigerator overnight. Strictly under a nitrogen atmosphere, the solid was recovered by filtration, washed with 20 ml of cold dry acetonitrile, then dried under a stream of nitrogen for 1 5 minutes. The solid was then dried under high vacuum overnight to give 12.4 grams of product as a white solid. N R (CDCI ₃ ): δ 6.41 (dd, 1 H, .7= 1 .6, 17.2 Hz, vinyl CH), 6.18 (dd, 1 H, J=10.6, 17.2 Hz, vinyl CH), 5.90 (dd, 1 H, J=1.6, 10.4 Hz, vinyl CH), 4.35 (m, 2H), 4.27 (m, 2H), 4.1 1 (m, 2H), 3.63 (m, 2H), 3.22 (s, 9H, N(CH ₃ ) ₃ ).

Example 10. Preparation of high molecular weight zwitterionic polymers

[0356] A representative protocol to produce high molecular weight, tailor-made hydrophilic polymers of the zwitterionic monomer, 2-methacryloyloxyethyl phosphorylcholine (HEMA-PC), using a "living" controlled free radical process, atom transfer radical polymerization (ATRP), is as follows.

[0357] The following initiator was used:

[0358] The initiator and ligand (2,2'-bipyridyl) were introduced into a Schlenk tube. Dimethyl formamide or dimethylsulfoxide was introduced drop wise so that the total weight percent of both initiator and ligand did not exceed 20%. In the event that initiators or ligands were oils, or the quantities involved were below the accuracy limit of the balance, the reagents were introduced as solutions in dimethyl formamide (100 mg/ml). The resultant solution was cooled to -78°C using a dry ice/acetone mixture, and was degassed under vacuum until no further bubbling was seen. The mixture remained homogeneous at this temperature. The tube was refilled under nitrogen and the catalyst (CuBr unless otherwise indicated), kept under nitrogen, was introduced into the Schlenck tube. The solution became dark brown immediately. The Schlenk tube was sealed and kept at -78°C and the solution was purged immediately by applying a vacuum. Care was taken to ensure that the monomer, HEMA-PC, was kept as a dry solid under inert conditions at all times until ready for use. A solution of HEMA-PC was freshly prepared by mixing a defined quantity of monomer, kept under nitrogen, with 200proof degassed ethanol. A degassed solution of CuBr? in dimethyl formamide (100 mg/ml) was added to the solution of HEMA-PC under nitrogen in the ratio of halide/CuBr/CuBr ₂ of 1 /0.9/0.1 for reaction times up to 24 hours and 1 /0.75/0.25 for reaction times longer than 24 hours. The resulting solution was added drop wise into the Schlenk tube and homogenized by light stirring. Unless otherwise indicated, the ratio of monomer (g)/ethanol (ml) was 0.50. The temperature was maintained at -78°C. A thorough vacuum was applied to the reaction mixture for at least 10 to 15 min until bubbling from the solution ceased. The mixture stayed homogeneous at this temperature, i.e. with no precipitation of any reaction ingredients (such as initiator or ligand) thus avoiding premature or unwanted polymerization. The tube was refilled with nitrogen, and the vacuum-nitrogen cycle was repeated twice. The tube was then refilled with nitrogen and warmed to room temperature (25°C). As the polymerization proceeded, the solution became viscous. After some time (defined in the table below), the reaction was quenched by direct exposure to air causing the mixture to become blue-green in color, and was passed through a silica column in order to remove the copper catalyst. The collected solution was concentrated by rotar evaporation and the resulting mixture was purified by careful precipitation into

tetrahydrofuran followed by thorough washing with diethyl ether, or by dialysis against water. Polymer was collected as a white fluffy powder (following freeze drying if dialyzed against water) and placed under vacuum at room temperature.

[0359] Data from several polymerization reactions are shown in the following table.

The ratio of halide/CuBr/CuBr ₂ was 1 /0.9/0.1 for reaction times up to 24 hours and 1/0.75/0.25 for reaction times longer than 24 hours

Monomer (g)/solvent (ml) 0.5

'Monomer (g)/solvent (ml) 0.6

[0360] The peak molecular weight (Mp), number molecular weight (Mn) and

polydispersity (PDI) were determined/derived by multi-angle light scattering.

Example 11. Further preparations of high molecular weight zwitterionic polymers

[0361] A representative protocol to produce high molecular weight, tailor-made hydrophilic polymers of the zwitterionic monomer, 2-methacryloyloxyethyl

phosphorylcholine (HEMA-PC) or the monomer 2-(Acryloyloxyethyl-2'- (trimethylammonium)ethyl phosphate, inner salt (HEA-PC), using a "living" controlled free radical process, atom transfer radical polymerization (ATRP), is as follows.

[0362] The following initiators were used:

PME04M9 (from Example 4)

PME04M3 (from Example 3)

PME04M6 (from Example 6)

PVSAME04M3 (from Example 8)

PVSAME04M6 (from Example 7)

[0363] The initiator and ligand (2,2'-bipyridyl unless otherwise indicated) were introduced into a Schlenk tube. Dimethyl formamide or dimethylsulfoxide was introduced drop wise so that the total weight percent of both initiator and ligand did not exceed 20%. In the event that initiators or ligands were oils, or the quantities involved were below the accuracy limit of the balance, the reagents were introduced as solutions in dimethyl formamide ( 100 mg/ml). The resultant solution was cooled to -78°C using a dry ice/acetone mixture, and was degassed under vacuum until no further bubbling was seen. The mixture remained homogeneous at this temperature. The tube was refilled under nitrogen or argon and the catalyst (CuBr unless otherwise indicated), kept under nitrogen or argon, was introduced into the Schlenck tube. The solution became dark brown immediately. The Schlenk tube was sealed and kept at -78°C and the solution was purged immediately by applying a vacuum. Care was taken to ensure that the monomer, HEMA-PC (or HEA-PC from Example 9), was kept as a dry solid under inert conditions at all times until ready for use. A solution of HEMA-PC (or HEA-PC) was freshly prepared by mixing a defined quantity of monomer, under nitrogen or argon, with 200proof degassed ethanol. In the event HEA-PC was used as monomer, the solution was first passed through an alumina column in order to remove stabilizer prior to introduction into the Schlenk tube. The monomer solution was added drop wise into the Schlenk tube and homogenized by light stirring. Unless otherwise indicated, the ratio of monomer (g)/ethanol (ml) was 0.3. The temperature was maintained at -78°C. A thorough vacuum was applied to the reaction mixture for at least 10 to 15 min until bubbling from the solution ceased. The mixture stayed homogeneous at this temperature, i.e. with no precipitation of any reaction ingredients (such as initiator or ligand) thus avoiding premature or unwanted polymerization. The tube was refilled with nitrogen or argon, and the vacuum-nitrogen/argon cycle was repeated twice. The tube was then refilled with nitrogen or argon and warmed to room temperature (25°C). As the polymerization proceeded, the solution became viscous. After some time (defined in the table below), the reaction was quenched by direct exposure to air causing the mixture to become blue-green in color, and was passed through a silica column in order to remove the copper catalyst. The collected solution was concentrated by rotary evaporation and the resulting mixture was purified by careful precipitation into

tetrahydrofuran followed by thorough washing with diethyl ether, or by cross-flow filtration against ethanol followed by careful precipitation and washing with diethyl ether. Polymer was collected as a white fluffy powder and placed under vacuum at room temperature.

[0364] Data from several polymerization reactions are shown in the following table.

Monomer

Initiator Catalyst Ligand MALS MALS

Monomer Time MALS Conversion

Sample Initiator (10 ^s (10 ^~5 (10 ^"s (Mn (Mp

(PD1) ('HNMR mol) (g) mol) (h)

mol) kDa) kDa)

%)

6 PME04M9 4.44 7.100 40.0 79.9 19 214 215 1.1 1 100

7 PME04 9 1.86 1 1.2 16.8 33.6 21 720 736 1.13 98

8 PME04M9 6.95 1 1.1 62.6 25.3 19 228 237 1.14 99

9 PME04M9 2.25 1 1.25 20.2 40.5 21 640 672 1.17 98

10 PME04M9 2.93 10.8 26.4 52.8 19 370 406 1.09 95

1 1 PME04 9 1.89 13.25 17.0 35.8 17 683 747 1.07 96

12 P E04M9 5.00 35.2 45.3 35.8 17 700 766 1.08 99

13 PME04M9 1.97 14.45 17.9 35.8 21 932 988 1.17 93

14 PME04M9 2.40 14.45 17.9 35.8 21 818 829 1.17 98

15 PME04M9 2.70 14.60 17.9 35.8 22 762 789 1.19 98

16 PME04M9 0.62 1.000 5.58 1 1.2 16 214 221 1.09 98

17 PME04M9 0.62 1.000 5.58 1 1.2 16 226 230 1.1 1 99

18 PME04M9 0.62 1.000 5.58 1 1.2 16 165 1 3 1.10 99

19 PME04M9 7.26 1 1.6 65.4 131 16 21 1 223 1.14 100

20 PME04 3 0.55 0.656 1.65 3.30 21 1 10 145 1.06 95

21 PME04M3 2.54 5.097 7.64 15.2 21 215 250 1.13 97

PME04 3 0.22 0.873 0.67 1.33 21 365 410 1.17 95

23 PME04M3 3.41 14.65 10.2 20.4 22 394 452 1.12 93

24 PMEC M3 4.20 13.00 9.79 19.6 22 275 320 1.08 91

25 PME04M3 3.90 14.00 1 1.1 22.3 21 320 382 1.09 93

26 PME04 6 2.51 5.040 15.1 30.2 21 251 261 1 .04 99

27 PME04M6 5.88 7.000 35.3 70.5 13 150 158 1.02 99

28 PME04M6 1.86 7.180 1 1.2 22.4 21 422 443 1 .04 98

29 PME04M6 1.85 7.150 1 1.1 22.3 21 398 441 1.06 98

30 PME04M6 5.00 10.00 30.0 60.0 21 167 187 1 .02 85

31 PME04 6 5.19 1 1.44 31.2 62.4 21 190 236 1.14 97

32 PME04M6 5.21 1 1.48 31.3 62.6 21 268 285 1 .05 99

33 PME04M6 6.21 1 1.50 37.3 74.5 21 227 240 1.05 99

34 PME04M6 6.02 1 1.14 36.1 72.2 21 228 245 1.06 99

35 PME04M6 9.41 7.100 56.8 1 13 6 91 95 1.04 99

36 PME04M6 10.9 7.120 65.7 131 4 77 81 1.03 97

37 PME04 6 3.00 10.95 18.0 36.0 21 397 428 1 .08 98

38 PME04M6 6.87 13.55 41.3 82.5 20 224 248 1.10 98

39 PME04M6 2.14 15.65 12.9 25.7 19 659 738 1.10 91

40 PME04M6 4.83 35.28 29.0 58.0 19 658 748 1.1 1 87

41 PME04M6 1.96 14.33 1 1.8 23.6 19 623 741 1.13 94

42 PME04M6 6.77 13.34 40.6 81.2 20 218 237 1.08 99 Monomer

Initiator Catalyst Ligand MALS MALS

Monomer Time MALS Conversion

Sample Initiator (10 ^"s (10 ^"s (10 ^"5 (Mn (Mp

(h) (PDI) ('HNMR mol) (g) mol) mol) kDa) kDa)

%)

43 PME04M6 3.82 14.72 22.9 45.9 21 375 419 1.10 96

44 PME04M6 2.26 14.08 13.6 27.2 24 539 642 1.13 93

45 PME04M6 I 0. I 12.00 60.7 122 19 148 158 1.07 99

46 PME04M6 10.9 1 1.55 65.8 132 19 134 141 1.06 99

47 PME04M6 13.3 26.25 80.0 160 20 205 230 1.07 99

48 PME04M6 3.00 10.95 18.0 36.0 19 379 409 1.07 97

49 PME04 6 10.9 12.25 69.8 140 19 120 132 1.05 99

50 PME04M6 3.65 13.38 21.9 43.8 19 375 420 1.10 95

51 PME04M6 5.77 1 1.84 34.7 69.3 20 237 255 1.09 99

52 PME04M6 6.07 1 1.96 36.4 72.8 20 225 241 1.05 99

53 PME04M6 10.2 12.08 61.2 122 20 143 151 1.04 99

54 PME04M6 5.80 1 1.44 34.8 69.7 20 238 250 1.08 99

55 ⁴ PME04M6 2.87 0.508 1.74 1.72 1 79 105 1.18 85

56 ⁴ PME04M6 2.76 0.489 1.67 1.65 4 64 83 1.12 70

57 VSAME04M3 2.51 5.050 7.53 15.1 22 202 232 1.06 95

58 VSAME04M3 6.97 1.100 20.9 38.4 1 ½ 28 29 1.06 96

59 VSAME04M6 2.55 5.104 15.3 30.6 21 268 287 1 .13 99

60 VSAME04M6 2.98 5.076 17.9 35.8 21 245 252 1.10 99

Monomer (g)/solvent (ml) 0.3

'Monomer (g)/solvent (ml) 0.5

2Monomer (g)/solvent (ml) 0.6

HEA-PC monomer (g)/solvent (ml) 0.5

[0365] The peak molecular weight (Mp), number molecular weight (Mn) and

polydispersity (PDI) were determined/derived by multi-angle light scattering.

Example 12. Deprotcction of furan-protected maleimide functionalized polymers using retro Diels-Alder reaction

[0366] Polymers from Example 1 1 were dissolved in ethanol (20 to 50 % w/w) in a round bottom flask. Ethanol was slowly removed by rotary evaporation to make a thin film on the wal l of the flask. The reaction vessel was placed in an oil bath at a temperature of at least 1 10°C for 90 min under vacuum and then cooled to room temperature.

[0367] Deprotection of the maleimide (or phenyl vinyl sulfone) functional group was monitored by 'HNMR (400MHz, d-methanol):

Before deprotection: 5(ppm):5.2 (2H, -CH-0-CH-) and 6.6 (2H, -CH=CH-).

After deprotection: 5(ppm): 6.95 (2H, -CO-CH=CH-CO).

Example 13. Generation of aldehyde functional groups from diol precursors following polymerization of diol functionalized initiators

[0368] A large excess of sodium periodate dissolved in distilled water was added to a solution of diol functionalized polymer (from Example 10) in distilled water (l Owt. %). The reaction was allowed to proceed at room temperature for 90 min in the dark.

water, 90mn RT The reaction was quenched with an aqueous solution of glycerol (1.5X vs. NalC^) to remove any unreacted sodium periodate. The mixture was stirred at room temperature for 15 min and placed in a dialysis bag (MWCO 14 to 25 kDa) and purified by dialysis at room temperature for one day. Water was then removed by lyophilization and the polymer collected as a dry powder. Quantification of aldehyde functionality was by binding of Cy5.5 hydrazide fluorescent dye (GE Healthcare).

Example 14. Conjugation of recombinant human cytokine to aldehyde functionalized polymers

[0369] The following aldehyde functionalized polymers (from Example 10 following oxidation according to Example 13) were used:

[0370] Conjugation of a 22 kDa recombinant human cytokine with a pi of 5.02 was performed in l OmM Hepes buffer at pH 7 containing 40mM sodium cyanoborohydride. The final protein concentration was l - 1.5mg/ml in the presence of 6-7 fold molar excess of polymer dissolved in the conjugation buffer. The reaction was carried out at room temperature or 4°C overnight in the dark with gentle mixing using a rocking table.

[0371] The conjugation efficiency was monitored using two methods: (i) a semiquantitative method using SDS-PAGE analysis and (ii) a quantitative method using analytical size exclusion chromatography (SEC) with a ProPac SEC-10 column, 4x300mm from Dionex Corporation.

[0372] Purification of the resulting cytokine-polymer conjugates was carried out using an anion exchange Q Sepharose HP (QHP) column from GE Healthcare. In general, the conjugation reaction (containing approx. 1 mg protein) was diluted at least 4 fold with QHP wash buffer containing 20 mM Tris pl l 7.5 and loaded onto a 2ml QHP column by gravity flow. The column was washed with at least 10 column volumes (CV) of wash buffer.

Elution of conjugate was achieved by eluting the column with wash buffer containing 40- 50mM NaCI for at least 5 CV. The fractions collected were concentrated with an Amicon Ultrafree concentrator with a 1 0 kDa MW cutoff membrane, buffer exchanged into I xPBS pH 7.4 and further concentrated to a final protein concentration of at least l mg/ml. The final conjugates were sterile filtered with a 0.22 micron filter and stored at 4°C before use. The final protein concentration was determined using OD277nm with the cytokine extinction coefficient of 0.81 (l mg/ml solution in a 10mm pathlength cuvette). The conjugate concentration was then calculated by including the MW of the polymer in addition to the protein.

[0373] Characterization of the cytokine-polymer conjugates was performed with the following assays: (i) MW of the conjugate was analyzed using a Shodex 806MHQ column with a Waters 2695 HPLC system equipped with a 2996 Photodiode Array Detector and a Wyatt miniDAWN Treos multi angle light scattering detector. The PDI and Mp were calculated using the ASTRA Software that was associated with the Wyatt MALS detector and the data are presented in the table above. In addition, in all cases the stoichiometry of the conjugates was shown to be 1 to 1 between protein and polymer; (ii) SDS-PAGE analysis using Coomassie Blue stain. The presence of the high MW conjugate and the lack of free protein under both non-reducing and reducing conditions provided a good indication that the protein was covalently conjugated to the polymers. In addition, there was no sign of non- covalent association between the protein and the polymers nor the presence of inter- molecular disulfide bond mediated protein aggregation in the purified protein-polymer conjugate preparations. Example 15. Conjugation of recombinant human multi-domain protein to maleimide functionalized polymers

[0374] The following maleimide functionalized polymers (from Example 1 1 using HEMA- PC monomer following deprotection according to Example 12) were used:

[0375] Conjugation of maleimide functionalized polymers to recombinant human multi- domain protein was accomplished at polymer to protein molar excess ratio of 3-5x at pH 6. To prepare the conjugation reaction, a polymer stock solution was first prepared in MES buffer pH 6 with 2mM EDTA. The stock solution was degassed and chilled to 4°C, and then mixed with a cold protein stock solution at pH 6. The final protein concentration was at least 2mg/ml. The reaction was allowed to proceed at 4°C in the dark overnight. A conjugation efficiency of over 90% was routinely observed by analytical cation exchange

chromatography. Purification of the conjugate was accomplished with a cation exchange resin at pH 5. The conjugation reaction was first diluted at least 7x in sodium acetate buffer pH 5 and then applied to the cation exchange column. The free polymer remained in the unbound fraction, the conjugate eluted at low salt concentration (50- 100mM NaCl), and the free protein and aggregated protein cluted at a much higher salt concentration (≥150mM NaCl). The conjugate fractions were pooled and buffer exchanged for final analysis. The overall yield was >45%.

[0376] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications can be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.