US 2016O184445A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0184445 A1 Perroth et al. (43) Pub. Date: Jun. 30, 2016

(54) BUTYRYLCHOLINESTERASE Publication Classification ZWITTERONIC POLYMER CONUGATES (51) Int. Cl. (71) Applicant: Kodiak Sciences Inc., Palo Alto, CA A647/48 (2006.01) (US) CI2N 9/18 (2006.01) CI2N 9/96 (2006.01) (72) Inventors: D. Victor Perlroth, Palo Alto, CA (US); A638/46 (2006.01) Stephen A. Charles, Ravenna, OH (US); C07K 7/08 (2006.01) Wayne To, San Mateo, CA (US); (52) U.S. Cl. Xiaojian Huang, Mountain View, CA CPC ...... A61K 47/48 176 (2013.01); A61 K38/465 (US); Martin Linsell, San Mateo, CA (2013.01); C07K 7/08 (2013.01); C12N 9/96 (US); Didier Benoit, San Jose, CA (US) (2013.01); C12N 9/18 (2013.01); C07K 2319/30 (2013.01); C12Y-301/01008 (2013.01) (21) Appl. No.: 14/932,913 (22) Filed: Nov. 4, 2015 (57) ABSTRACT Related U.S. Application Data The present invention provides recombinant butyrylcho (63) Continuation of application No. PCT/US2015/ linesterase fusion proteins, including Fc fusion proteins and 0561 12, filed on Oct. 16, 2015. multi-armed high MW polymers containing hydrophilic (60) Provisional application No. 62/065,471, filed on Oct. groups conjugated to the fusion proteins, and methods of 17, 2014. preparing Such polymers. Patent Application Publication Jun. 30, 2016 Sheet 1 of 5 US 2016/O1844.45 A1

900 800 700 600 500 400 300 200 100

-5 5 15 25 35 TIME (DAYS) FIG. 1 Patent Application Publication Jun. 30, 2016 Sheet 2 of 5 US 2016/O1844.45 A1

800

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500 --R2709 at 37 degree 400 -A-R5743 at 37 degree -HR2709 at 4 degree 300 -A-R5743 at 4 degree

2 12 22 32 TIME (DAYS) FIG.2 Patent Application Publication Jun. 30, 2016 Sheet 3 of 5 US 2016/O1844.45 A1

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O% O 5 10 15 20 25 30 TIME (DAYS) FIG. 4 Patent Application Publication Jun. 30, 2016 Sheet 5 of 5 US 2016/O1844.45 A1

FIG. 5

O CH HC O O R1 O O ouls O HN ul AMAC CH - 1n- N-1\-1N-1 N CH3 o O C MPC O US 2016/01 84445 A1 Jun. 30, 2016

BUTYRYLCHOLINESTERASE serum-derived), the same enzyme which OPs irreversibly ZWITTERONIC POLYMER CONUGATES inactivate, when administered prophylactically was able to protect rhesus monkeys against a challenge of two to five CROSS REFERENCE TO RELATED times the LD50 of 3,3-Dimethylbutan-2-yl methylphospho APPLICATIONS nofluoridate (soman) a highly toxic OP nerve agent designed 0001. The present application is a continuation of PCT for chemical warfare. Wolfe, A. D., et al., (1992) Use of PCT/US2015/0561 12 filed Oct. 16, 2015, which is a nonpro cholinesterases as pretreatment drugs for the protection of visional of 62/065,471 filed Oct. 17, 2014, each incorporated rhesus monkeys against Soman toxicity. Toxicol. Appl Phar by reference in its entirety for all purposes. macol. 117:189-193. 0007 AChE is a member of the carboxyl/cholinesterases (CE/ChEs), a multi-gene family of enzymes that hydrolyze a REFERENCE TO ASEQUENCE LISTING diverse range of carboxylesters. Members of the family 0002 Sequences are provided in an ASCII txt filed desig include AChE, human carboxylesterase 1 (hCE1) and 2 nated 470678 SEQLST.TXT, of 84,727 bytes, created Oct. (hCE2) and butyrylcholinesterase (BuChE). Of these, BChE 16, 2015, incorporated by reference. has generated the greatest interest as a bioscavenger. BuChE is the major cholinesterase in human serum. Although the BACKGROUND closely related enzyme AChE is well described as the primary synaptic regulator of cholinergic transmission, a definitive 1. BioScavengers physiological role for BChE has not yet been demonstrated. 0003 Organophosphorus compounds (OPs) are highly 0008 BuOhE is catalytically promiscuous and hydrolyzes toxic to humans. First developed in the 1930s as insecticides, not only acetylcholine (ACh), but also longer-chain choline the extreme toxicity of OPs to humans eventually led to their esters (e.g., butyrylcholine, its preferred Substrate, and Succi development as nerve agents for warfare. Today, although nylcholine) and a variety of non-choline esters, such as ace generally banned by international law, OPs are considered an tylsalicylic acid (aspirin) and cocaine. Moreover, BuChE ever increasing military and civilian threat. In 1995, the binds most environmentally occurring ChE inhibitors as well Tokyo Subway system was the Subject of a sarin attack, killing as man-made OP pesticides and nerve agents. However, the over a dozen passengers and injuring scores of others. Sarin physiologically available levels of natural BuChE are too low gas has also been reported to have been recently used on to give any meaningful protection with regard to OPs. surface to surface missiles in conflicts in Syria with lethal 0009 BuChE purified from human serum has been used to effect. treat humans Patients successfully received a partially puri 0004 OP nerve agents such as sarin are potent inhibitors fied human BuOhE to counteract the effects of muscle relax of acetylcholinesterase. In vertebrates, acetylcholine is the ants and for OP pesticide poisoning. Cascio, C., et al. (1988) neurotransmitter used to transmit signals from the nervous The use of serum cholinesterase in severe phosphorous poi system to muscle fibers. Normally, acetylcholine is released soning. Minerva Anestesiol (Italian) 54:337-345. However, from a neuron to stimulate a muscle. After stimulation, ace products purified from human serum are highly undesirable tylcholinesterase metabolizes acetylcholine, allowing the due to the threat of contamination by unknown blood borne muscle to relax. OPs such as sarin, however, irreversibly pathogens which of course cannot be tested for. In this regard, inhibit acetylcholinesterase, inhibiting degradation of acetyl for example, FVIII for treatment of hemophilia is produced choline. OP nerve agents are generally lethal at very small today by recombinant DNA technology and is no longer doses with Subjects generally Succumbing to Some type of purified from human serum after thousands of patients asphyxia. Subjects receiving less than lethal doses of OPs became infected with the HIV virus in the late 70s and early may develop permanent neurological damage. In addition, 80s. In addition, production of serum purified BuChE is OP pesticides are still in wide use. Although OP pesticides are severely limited by the volume of serum available. Lockridge, far less toxic than nerve agents, OP pesticide toxicity to O., et al. (2005) J. Med. Chem. Biol. Radiol. Def. humans is still of great concern. 3:nimhS5095. 0005 Antidotes and treatment for OP poising are 0010. To avoid using blood serum purified BuChE, recom extremely limited. There are small molecule treatments for binant BuChE (rBuChE) was produced in Escherichia coli. OP poisoning symptoms (e.g. tremors). Overall, however, Masson, P. (1992) in Multidisciplinary Approaches to Cho these therapies are of very limited benefit. Atropine may be linesterase Functions, eds Shafferman, A., Velan, B. (Plenum, administered for muscarinic ACh receptor blockade and diaz New York) pp. 49-52. The produced protein, however, was epam for symptomatic management of convulsions. Lee E.C. non-functional. rBuChE was also produced in mammalian (2003) Clinical manifestations of sarin nerve gas exposure. J. 293T (Altamirano, C. V. and Lockridge, O. (1999) Chem. Am. Med. Assoc. 290:659-662. Additionally, oxime therapy Biol. Interact. 119-120:53-60) and CHO (Millard, C. B., with 2-pralidoxime (2-PAM) can effectively reactivate some Lockridge, O., and Broomfield, C. A. (1995) Biochemistry but not all OP-AChE adducts. Cannard, K. (2006) The acute 34:15925-15933). The mammalian expression systems, how treatment of nerve agent exposure. J Neurol Sci 249:86-94. ever, failed to produce Sufficient quantities of protein Such Although these therapies can reduce mortality in cases of less that the need for plasma purified BuChE could be eliminated. toxic OPs such as insecticides or very low doses of nerve Given the difficulties experienced with mammalian cellular agents, they are generally seen by clinicians as highly inef expression systems, transgenic goats were created having the fective with regard to possible battlefield levels of exposure to human BuChE gene under the control of the goat B-casein OP nerve agents. promoter, allowing the produced rBuChE to be purified from 0006 For prophylactic anti-OP therapy, it has been found goat milk. Huang, Y. J. et al (2007) Recombinant human that certain enzymes will detoxify OPs such as sarin by acting butyrylcholinesterase from milk of transgenic animals to pro as bioscavengers. Acetylcholinesterase (AChE) (fetal bovine tect against organophosphate poisoning. Proc. Nat. Acad. Sci. US 2016/01 84445 A1 Jun. 30, 2016

104(34): 13603-13608. However, when the transgenic goat which is necessary for reductive animation conjugation. rBuChE was tested in animals, the PK and bioavailability Wang, X. Kumar, S., and Singh, S. (2011) Disulfide Scram results were very disappointing for the enzyme both itself and bling in IgG2 Monoclonal Antibodies: Insights from Molecu the enzyme conjugated to PEG. rBuChE produced in trans lar Dynamics Simulations. Pharm. Res. 28, 3128-3143. genic goats did not apparently have a residence time similar to Moreover, most proteins have several amine residues avail native human BuChE purified from plasma to allow it to be able for reaction. Hence, amine PEG conjugates tend not to be developed as an agent for the prophylaxis of OP poisoning. site specific, resulting in heterogeneous mixtures of different 0.011 Thus there remains a need for recombinant versions PEG-protein conjugates in the final product. Veronese, F. of BuOhE and related bioscavengers which can be produced (2001) Peptide and protein PEGylation a review of problems in large quantities and have good PK properties. and solutions. Biomaterials 22 (5), 405-17; Luxon, B. A., Grace, M., Brassard, D., and Bordens, R. (2002) Pegylated 2. Iodoacetamide Conjugation interferons for the treatment of chronic hepatitis C infection. 0012 Since the first recombinant insulin product was Clin. Ther. 24 (9) 1363-1383. approved in 1982, protein and peptide therapeutics have been 0016. The various drawbacks associated with conjugation successfully used as very efficient drugs for the treatment of of PEG via protein amine groups have led to the development a wide variety of pathophysiological diseases. Development of methods to PEGylate proteins via cysteine. Indeed, poly of these drugs was spurred by advances in recombinant DNA mer conjugation via CyS is one of the main approaches taken technology over the past decades. Protein therapeutics gen by researchers today. Roberts, M. J. and Harris, B. J. M. erally fall into one of two categories: (1) biopharmaceuticals (2002) Chemistry for peptide and protein PEGylation. Adv. that mimic native protein and serve as a replacement, e.g., Drug Del. Rev. 54, 459-576; Stenzel, M. H. (2012) Biocon insulin and (2) monoclonal antibodies that serve as agonists jugation Using Thiols: Old Chemistry Rediscovered to Con or antagonists of various pathways, e.g., AVASTINR). While nect Polymers with Nature's Building Blocks. ACS Macro protein therapeutics enjoy great Success both in terms of Letters 2, 14-18. The number of free cysteines on the surface disease amelioration and sales, it has been observed that of a protein is much less than that of lysine. If a free cysteine many have Suboptimal physiochemical and pharmacokinetic is lacking, one or more free cysteines can be added by genetic properties. The main drawbacks seen with protein therapeu engineering. Goodson, R. J. and Katre, N. V. (1990) Site tics are physicochemical instability, limited solubility, pro directed pegylation of recombinant interleukin-2 at its glyco teolytic instability, short elimination half-life, immunogenic sylation site. Biotechnology 8, 343-46. ity and toxicity. 0017 Various chemistries have been developed for conju 0013. One approach to improving the pharmacokinetic gating polymer to proteins via cysteine, including alkyl properties of protein therapeutics is conjugation of the protein halides, ester acrylates and vinyl Sulfone reagents. But the to a biopolymer. In this regard, a dozen or so polyethylene most prevalent conjugation group is maleimide. Roberts, M. glycol (PEG) conjugates have been approved as of the present J., Bentley, M. D., and Harris, J. M. (2002) Chemistry for date, e.g., Neulasta R. Various benefits have been observed peptide and protein PEGylation. Adv. Drug Del. Rev. 54, with protein PEGylation, including: improved drug solubil 459-76. Maleimide reagents react efficiently with thiol ity, reduced dosage frequency without diminished efficacy groups to provide conjugates at neutral pH, reducing risks and with potentially reduced toxicity, extended circulating associated with protein denaturation. Maleimide conjugation life, increased drug stability and greater resistance to pro has been used in a clinically approved PEG drug. Goel, N. and teolytic degradation. Commercial benefits of PEGylation Stephens, S. (2010) Certolizumab pegol. mAbs 2(2), 137-47. have also been observed, including opportunities for new Maleimide has also been used in some approved non-PEG delivery formats and dosing regimens. Also, since PEGyla conjugates. See, e.g., Gualberto, A. (2012) Brentuximab tion produces a new chemical entity there are opportunities Vedotin (SGN-35), an antibody-drug conjugate for the treat for more patent protection. ment of CD30-positive malignancies. Expert Opin. Invest. 0014. Historically, most PEGylation reagents have been Drugs 21 (2), 205-16. developed to take advantage of the accessible amine function 0018. Although several maleimide based products have alities on proteins Amine groups are present by definition in been approved. Some maleimide-thiol conjugates are known all proteins (i.e. the amino terminus) and in the great majority to be unstable. In this regard, it has been observed the male of proteins there is more than one amino group, e.g., as imide based conjugates can undergo exchange reactions. contributed by Lys Amine conjugated PEG proteins have Baldwin, A. D. and Kiick, K. L. (2011) Tunable Degradation received regulatory approval. Alconel, S. N. S., Baas, A. S. of Maleimide-Thiol Adducts in Reducing Environments. and Maynard, H. D. (2011) FDA-approved poly(ethylene Bioconjugate Chem. 22 (10), 1946-53; Baldwin, A. D. and glycol)-protein conjugate drugs. Polym. Chem. 2 (7), 1442. Kiick, K. L. (2012) Reversible maleimide-thiol adducts yield At least two amine PEG protein conjugates are first line glutathione-sensitive poly(ethylene glycol)-heparin hydro therapies. Foster, G. R. (2010) Pegylated Interferons for the gels. Polym. Chem. 4 (1), 133. Moreover, exchange products Treatment of Chronic Hepatitis C: Pharmacological and have been detected with regard to endogenous proteins. Alley, Clinical Differences between Peginterferon-2a and Peginter S.C., Benjamin, D. R., Jeffrey, S.C., Okeley, N.M., Meyer, feron-2b. Drugs 70 (2), 147-165; Piedmonte, D. M. and Treu D. L., Sanderson, R. J. and Senter, P. D. (2008) Contribution heit, M. J. (2008) Formulation of Neulasta (pegfilgrastim). of linker stability to the activities of anticancer immunocon Adv. Drug Delivery Rev. 60, 50-58. jugates. Bioconjugate Chem. 19, 759-65; Shen, B. Q., et al. 0015. However, there are a number of problems associated (2012) Conjugation site modulates the in vivo stability and with amine conjugation. Reagents specific for amine conju therapeutic activity of antibody-drug conjugates. Nature Bio gation are often inefficient due to competitive hydrolysis technol. 30 (2), 184-89. reactions of the reagent (e.g., PEG-N-hydroxysuccinimide 0019. As maleimide conjugates are known to be unstable ester) or unfavorable equilibria during imine formation, under some circumstances, alternative conjugation strategies US 2016/01 84445 A1 Jun. 30, 2016

have been employed. Roberts, M. J. and Harris, B. J. M. erase protein as fusion protein. Optionally, the non-butyryl (2002) Chemistry for peptide and protein PEGylation. Adv. cholinesterase protein comprises an immunoglobulin (Ig) Drug Del. Rev. 54, 459-576. One of these alternative domain Optionally, the Ig domain is selected from the group approaches is the use of iodoacetamide based conjugation consisting of IgG-Fc, IgG-CH and IgG-CL. Optionally, the agents as shown below: IgG is IgG1. Optionally, the Ig domain is IgG-Fc (SEQ ID NO. 8). Optionally, the Fc domain has amino acid substitu tions in one or more of the positions selected from the group O consisting of E233, L234, L235, G236, G237, A327, A330 and P331. Optionally, the Fc domain has the following sub -- o Her stitutions L234A L235E, G237A, A330S and P331S. mPEG, N --- I HS-R1 H Optionally, the Fc domain further has one of the following substitutions Q347C or L443C. Optionally, the substitution is Q347C. Optionally, the fusion protein further comprises a linker between the butyrylcholinesterase enzyme and the non-butyrylcholinesterase protein. Optionally, the linker is selected from the group consisting of G, GG, GGGGS (SEQ ID NO: 22), GGGS (SEQID NO. 23), and GGGES (SEQID 0020. The PEG-iodoacetamide reacts with free thiols by NO: 24), and oligomers of the foregoing. Optionally, the nucleophilic substitution, creating a stable thioether bond. linker is selected from the group consisting of The thioether link is much more stable than the corresponding GGGSGGGSGGGS (SEQ ID NO: 21) and structure made using maleimide chemistry. The reaction of GGSGGGSGGGS (SEQID NO:25). Optionally the butyryl iodoacetamide with thiol is pH dependent. Thiols are gener cholinesterase enzyme is a carboxyl truncate. Optionally the ally only alkylated in the thiolate anionic form. Lindley, H. butyrylcholinesterase enzyme comprises or consists of amino (1960) A study of the kinetics of the reaction between thiol acids 29-561 of SEQ ID NO. 1. Optionally the butyrylcho compounds and chloroacetamide. Biochem. J. 74, 577-84. linesterase enzyme comprises or consists of amino acids The pK value of the thiol in Cys is generally around 8.5+/- 29-562 of SEQ ID NO. 1. Optionally, the butyrylcholinest 0.5, but it depends on the environment of the Cys in the native erase enzyme has a mutation wherein the C at residue 94 of protein when it is being conjugated to the iodoacetamide SEQ ID NO. 1 is Y. Optionally, the butyrylcholinesterase moiety. Kallis, G. and Holmgren, A. (1980) Differential reac fusion has the amino acid sequence set forth in SEQID NO. tivity of the functional sulfhydryl groups of cysteine-32 and 2 cysteine-35 present in the reduced form of thioredoxin from 0024. In any of the above conjugates, the Zwitterionic Escherichia coli. J. Bio. Chem. 255 (21) 10261-10266. group can comprise phosphorylcholine. Optionally, the at 0021 Kallis and Holmgren observed that Cys residues least one type of monomer unit comprises 2-(acryloyloxy having the normal pK of around 8.5 are not readily acylated ethyl)-2'-(trimethylammoniumethyl) phosphate. Optionally, with iodoacetamide or alkylated with iodoacetic acid at neu at least one type of monomer unit comprises 2-(methacryloy tral pHs. Rapid reaction of these normal thiols was only loxyethyl)-2'-(trimethylammoniumethyl) phosphate observed with elevated pHs. It is well known in the art the (HEMA-PC). Optionally, the polymer has 3 or more arms. exposure of proteins to high pHs often leads to protein dena Optionally, the polymer has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 turation. Denaturation of proteins exposed to basic or high arms. Optionally, the polymer has 3, 6 or 9 arms. Optionally, pHs is highly protein dependent. A few proteins can Survive the polymer portion of the conjugate has a peak molecular exposure to high pH. However, many are permanently dena weight of between 300,000 and 1,750,000 Daltons. Option tured resulting in loss of activity and/or binding capacity. ally, the polymer portion of the conjugate has a peak molecu 0022. There thus remains a need for methods for conju lar weight between 500,000 and 1,000,000 Daltons. Option gating polymers to proteins using iodoacetamide conjugation ally, the polymer portion of the conjugate has a peak at physiological pHs. molecular weight between 600,000 to 800,000 Daltons. Optionally, the polymer is covalently bonded to at least one of SUMMARY OF THE CLAIMED INVENTION an amino group, a hydroxyl group, a Sulfhydryl group and a 0023 The invention provides a conjugate comprising an carboxyl group of the cholinesterase. Optionally, the sulfhy organophosphorus (OP) bioscavenger selected from the dryl group is from a cysteine residue in the cholinesterase. group consisting of a stoichiometric OP bioscavenger and a Optionally, the cysteine residue is at a position at which catalytic OP bioscavenger covalently bound to a Zwitterionic cysteine is not naturally present in the cholinesterase. polymer wherein the polymer comprises one or more types of 0025 Optionally, the cholinesterase is abutyrylcholinest monomer units and wherein at least one type of monomerunit erase fusion corresponding to that of SEQID NO. 2 and the comprises a Zwitterionic group. Optionally, the OP bioscav cysteine residue is that at amino acid 672. Optionally, the enger is a catalytic bioscavenger selected from the group butyrylcholinesterase enzyme comprising or consists of consisting of aryldialkylphosphatases, organophosphate amino acids 29 to 562 of SEQID NO. 1. Optionally, residue hydrolases (OPH), carboxylesterases, triesterases, phospho 94 is tyrosine. Optionally, the butyrylcholinesterase enzyme triesterases, arylesterases, paraoxonases (PON), organophos further comprises a cysteine residue introduced via recombi phate acid anhydrases and diisopropylfluorophosphatases nant DNA engineering. Optionally, the butyrylcholinesterase (DFPases). Optionally, the OP bioscavenger is a stoichiomet enzyme according to claim 41 comprising SEQID NO. 13. ric OP bioscavenger comprising a cholinesterase selected 0026. The invention further provides a butyrylcholinest from the group consisting of acetylcholinesterase (AChE) erase enzyme fusion protein comprising a butyrylcholinest and butyrylcholinesterase (BChE). Optionally, the cholinest erase enzyme and a non-butyrylcholinesterase protein. erase is purified from blood plasma. Optionally, the cholinest Optionally the non-butyrylcholinesterase protein comprises erase is produced by recombinant DNA technology. Option an immunoglobulin (Ig) domain. Optionally, the Ig domain is ally, the cholinesterase is abutyrylcholinesterase. Optionally, selected from the group consisting of IgG-Fc, IgG-CH and the butyrylcholinesterase is fused to a non-butyrylcholinest IgG-CL. Optionally, the IgG is IgG1. Optionally, the IgG1 US 2016/01 84445 A1 Jun. 30, 2016

domain is IgG1-Fc (SEQ ID NO. 8). Optionally, the Fc 0031. The invention further provides an initiator for poly domain has amino acid Substitutions in one or more of the mer synthesis comprising positions selected from the group consisting of E233, L234, R1 L235, G236, G237, A327, A330 and P331. Optionally, the Fc n L2 domain has the following substitutions L234A, L235E, G237A, A330S and P331S. Optionally, the Fc domain further R3 has one of the following substitutions Q347C or L443C. R4 L NH2 Optionally, the substitution is Q347C. Optionally, the fusion L3 protein requires a linker between the butyrylcholinesterase R5 R21 enzyme and the non-butyrylcholinesterase protein. Option ally, the linker is selected from the group consisting of G, GG, wherein L1 is a first linker, L2 is a second linker and L3 is a GGGGS (SEQ ID NO: 22), GGGS (SEQ ID NO. 23), and third linker, wherein L1, L2 and L3 are the same or different GGGES (SEQID NO: 24), and oligomers of the foregoing. and are each selected from the group consisting of C1-C12 Optionally, the linker is selected from the group consisting of alkyl-, - C3-C12 cycloalkyl-, (-(CH2)1-6-O-(CH2)1-6-) GGGSGGGSGGGS (SEQ ID NO: 21) and 1-12-, (-(CH2)1-4-NH-(CH2)1-4)1-12-, -(CH2)1- GGSGGGSGGGS (SEQID NO:25). Optionally, the butyryl 12O (—(CH2)1-4-O-(CH2)1-4)1-12-O-, -(CH2)1- cholinesterase enzyme is a carboxyl truncate. Optionally, the 12-(CO)-O-,-(CH2)1-12-(CO) NH ,-(CH2)1-12 butyrylcholinesterase enzyme comprises or consists of amino O (CO) , -(CH2)1-12-NH (CO) , (-(CH2)1-4- acids 29-561 of SEQID NO. 1. Optionally, the butyrylcho O-(CH2)1-4)1-12-O-(CH2)1-12-, -(CH2)1-12-(CO)– linesterase enzyme comprises or consists of amino acids O-(CH2)1-12-, -(CH2)1-12-(CO) NH-(CH2)1-12-, 29-562 of SEQ ID NO. 1. Optionally, the butyrylcholinest -(CH2)1-12-O-(CO)-(CH2)1-12-, -(CH2)1-12-NH erase enzyme has a mutation wherein the C at residue 94 of (CO)—(CH2)1-12-, -(C3-C12 cycloalkyl)-, —(C1 SEQ ID NO. 1 is Y. Optionally, the fusion protein has the C8alkyl)-(C3-C12 cycloalkyl)-, —(C3-C12 cycloalkyl)-(C1 amino acid sequence set forth in SEQID NO. 2. 8alkyl)-, —(C1-8alkyl)-(C3-C12 cycloalkyl)-(C1-8alkyl)- 0027. The invention further provides a method for treat NH-(CH2CH2-O-)1-20-NH-(CH2CH2-O-)1-20- and ment or prophylaxis of OP poisoning comprising administer —(CH2)0-3-aryl-(CH2)0-3-, a bond and any combination of ing to a patient an effective amount of any of the conjugates or the above; R1 and R2 are the same or different and are fusion proteins in any preceding claim. Optionally, the con selected from the group consisting of H, provided that not jugate is administered by intravenous administration, Subcu both R1 and R2 are H, and one or more basic groups wherein taneous administration, intramuscular administration, a basic group is selected from the group consisting of intralesional administration or intradermal administration. Optionally, the conjugate is administered by intramuscular administration. 0028. The invention further provides a method for effect ing a long term prophylaxis against OP poisoning comprising R6-N administering a conjugate comprising an OP bioscavenger selected from the group consisting of a stoichiometric OP bioscavenger and a catalytic OP bioscavenger covalently bound to a Zwitterionic polymer wherein the polymer com wherein R6, R7, R8, R9 and R10 are the same or different and prises one or more types of monomer units and wherein at are selected from the group consisting of a bond, H, amino, least one type of monomer unit comprises a Zwitterionic aryló-12 and alky11-6: group at least 7 days prior to expected exposure to an OP compound. Optionally, the OP compound is an OP pesticide or an OP nerve agent. Optionally, the conjugate is adminis tered at least 14 days prior to expected exposure to the OP nerve agent. Optionally, the conjugate is administered at least 21 days prior to expected exposure to the OP nerve agent. Optionally, the conjugate is administered at least 30 days prior to expected exposure to the OP nerve agent. wherein R11 and R12 are the same or different and are 0029. The invention further provides a method for effect selected from the group consisting of ing rapid prophylaxis against OP poisoning comprising a bond, H, aryló-12 and alky11-6: administering a conjugate comprising an OP bioscavenger selected from the group consisting of a stoichiometric OP bioscavenger and a catalytic OP bioscavenger covalently R14 R15 bound to a Zwitterionic polymer wherein the polymer com prises one or more types of monomer units and wherein at N N least one type of monomer unit comprises a Zwitterionic group at least 12 hours prior to expected exposure to an OP --- compound. Optionally, the OP compound is an OP pesticide R13 1 YR16 or an OP nerve agent. Optionally, the conjugate is adminis R17 tered at least 24 hours prior to expected exposure to the OP nerve agent. wherein R13, R14, R15, R16 and R17 are the same or differ 0030. Any of the above methods can include administer ent and are selected from the group consisting of a bond, H. ing another pharmaceutical agent selected from among car amino, aryl6-12 and alky11-6 and one or more arginine resi bamates, anti-muscarinics, cholinesterase reactivators and dues; and R3, R4 and R5 are the same or different and are anti-convulsives. selected from the group consisting of US 2016/01 84445 A1 Jun. 30, 2016

0035. Optionally L3 is a bond and R2 is Hand R1 is Y

N-1 CH3, CH O -N NH, O O CH 1N Y 0036) Optionally, L2 is —(CH2)1-6-. Optionally, L2 is N O -(CH2)3-. H CH3 and 0037. The invention further provides a polymerization CH3 process, comprising: O 0.038 polymerizing one or more radically polymeriz able monomers in the presence of an initiator compris O ing a homolytically cleavable bond with a radically HC transferable atom or group; a transition metal com HC Y pound; and a ligand selected from the group consisting CH3 H3C of Y O O O O O CH3 R6 HN N 1N N --> N 1suo O Y f V H H O HCCH

O Y CH wherein R6, R7, R8, R9 and R10 are the same or different and are selected from the group consisting of a bond, H, amino, aryló-12 and alky11-6: wherein Y is NCS, F, Cl, Br or I. 0032. Optionally, Y is Br. Optionally, R3, R4 and R5 are each Y

O N-1 CH3 CH O wherein R11 and R12 are the same or different and are selected from the group consisting of 0033) Optionally R3, R4 and R5 are each 0039 a bond, H, arylo-12 and alky11-6: O O CH 1n N O Y H CH CH3 O

O wherein R13, R14, R15, R16 and R17 are the same or differ HC ent and are selected from the group consisting of a bond, H. HC Y amino, aryl6-12 and alky11-6 and one or more arginine resi dues; wherein the ligand is coordinated to the transition metal compound, and wherein the transition metal compound and 0034. Optionally, R4, R5 and R6 are each the ligand are matched to initiate and propagate polymeriza tion of the monomers. Optionally the ligand is covalently H3C CH3 linked to the initiator to form a unimolecular initiator ligand. Y 0040. Optionally, the unimolecular initiator ligand has the O O Structure:

R1 O O O OO 14 NL, ------Y R3 H H O HCCH R4 -)- L NH2 O Y CH 13 US 2016/01 84445 A1 Jun. 30, 2016

wherein L1 is a first linker, L2 is a second linker and L3 is a -continued third linker, wherein L1, L2 and L3 are the same or different O O and are each selected from the group consisting of C1-C12 CH alkyl-, - C3-C12 cycloalkyl-, (-(CH2)1-6-O-(CH2)1-6-) 1n N O Y 1-12-, (-(CH2)1-4-NH-(CH2)1-4)1-12-, -(CH2)1- H CH 12O (—(CH2)1-4-O-(CH2)1-4)1-12-O-, -(CH2)1- CH 12-(CO)-O-, -(CH2)1-12-(CO) NH ,-(CH2)1-12 O O-(CO) , -(CH2)1-12-NH (CO)-, (-(CH2)1-4- O-(CH2)1-4)1-12-O-(CH2)1-12-, -(CH2)1-12-(CO)– O O-(CH2)1-12-, -(CH2)1-12-(CO) NH-(CH2)1-12-, HC -(CH2)1-12-O-(CO)-(CH2)1-12-, -(CH2)1-12-NH HC Y and (CO) (CH2)1-12-, -(C3-C12 cycloalkyl)-, —(C1 CH3 C8alkyl)-(C3-C12 cycloalkyl)-, —(C3-C12 cycloalkyl)-(C1 3C 8alkyl)-, —(C1-8alkyl)-(C3-C12 cycloalkyl)-(C1-8alkyl)- Y NH-(CH2CH2-O-)1-20-NH-(CH2CH2-O-)1-20- and O O —(CH2)0-3-aryl-(CH2)0-3-, a bond and any combination of O O O CH3 the above; R1 and R2 are the same or different and are selected from the group consisting of H, provided that not 1n N --> N luo O Y both R1 and R2 are H, and one or more basic groups wherein H H O HCCH a basic group is selected from the group consisting of R7 O Y CH wherein Y is NCS, F, Cl, Br or I. N l N1 R8 0041. Optionally Y is Br. Optionally, R3, R4 and R5 are I V each Y wherein R6, R7, R8, R9 and R10 are the same or different and N-1 CH are selected from the group consisting of a bond, H, amino, CH3 aryló-12 and alky11-6: O R12 0042 Optionally R3, R4 and R5 are each O O CH3 1n N O Y H CH3 CH wherein R11 and R12 are the same or different and are O selected from the group consisting of O a bond, H, aryló-12 and alky11-6:

R14 R15 0043. Optionally R4, R5 and R6 are each N N CH3 HC --- Y R13 1 YR16 O O R17 O O O CH O Y wherein R13, R14, R15, R16 and R17 are the same or differ H ent and are selected from the group consisting of a bond, H. --- O HCCH amino, aryl6-12 and alkyl 1-6 and one or more arginine resi dues; and R3, R4 and R5 are the same or different and are O Y CH selected from the group consisting of 0044 Optionally, L3 is a bond and R2 is H and R1 is NH

O -N ls NH. N-1 CH3 H 2 CH 0045. Optionally, L2 is -(CH2)1-6- or -(CH2)3-. Optionally, the radically polymerizable monomers are ole US 2016/01 84445 A1 Jun. 30, 2016 finically unsaturated monomers. Optionally, the unsaturated not limited to, PEGylation, addition of poly(HEMA-PC), monomers comprise HEMA-PC. Optionally, the transition albumination, glycosylation, farnesylation, carboxylation, metal is one of a transition metal in a low Valence state or a hydroxylation, phosphorylation, and other polypeptide modi transition metal coordinated to at least one coordinating non fications known in the art. A truncated cholinesterase is any charged ligand and wherein the transition metal comprises a N- or C-terminal shortened form thereof, particularly forms counterion. Optionally, the transition metal is selected from that are truncated and have OP inactivating activity. the group consisting of copper, iron, nickel or ruthenium. 0054 Acetylcholinesterase” (AChE) refers to enzymes or polypeptides capable of hydrolyzing acetyl esters, includ BRIEF DESCRIPTION OF THE DRAWINGS ing acetylcholine, and whose catalytic activity is inhibited by 0046 FIG. 1 shows the activity over time of plasma the chemical inhibitor BW 284C51. Acetylcholinesterases derived hBuChE versus full length rhBuChE from the milk of include, but are not limited to, plasma derived or recombinant transgenic goats, rBuChE534GGC (C66Y) and rhBuChE534 acetylcholinesterase. Acetylcholinesterases include human (C66Y)-Fc (L234A L235E, G237A, A330S, P331S and and any of non-human origin including, but not limited to, rat, Q347C) stored in cyanomologus monkey plasma over time. mouse, cat, chicken, rabbit, and cow acetylcholinesterases. 0047 FIG. 2 depicts a plot of activity vs. time for Exemplary human acetylcholinesterases include human rhBuChE534 (C66Y)-Fc (L234A L235E, G237A, A330S, AChE set forth in SEQID NO. 3. Acetylcholinesterases can P331S and Q347C) conjugated to 9 arm 750 kDa HEMA-PC be in any form, including, but not limited to, monomer, dimer polymer compared with human plasma derived BuChE at 4 and tetramer forms. C. and 37°C. in cyanomologus monkey plasma. 0055 “Butyrylcholinesterase” (BuChE) refers to 0048 FIG. 3 Activity of unconjugated rhBuChE534 enzymes or polypeptides capable of hydrolyzing acetylcho (C66Y)-Fc (L234A L235E, G237A, A330S, P331S and line and butyrylcholine, and whose catalytic activity is inhib Q347C), conjugated to 3 arm 500 kDa MPC polymer, conju ited by the chemical inhibitor tetraisopropyl-pyrophosphora gated to 9 arm 750 kDa MPC polymer and plasma derived mide (iso-OMPA). Butyrylcholinesterases include, but are hBuChE, normalized activity (unitsx 106) after injection into not limited to, plasma derived or recombinant butyrylcho mice over time (hours). linesterases. Butyrylcholinesterases include any of nonhu 0049 FIG. 4 shows the stability profile for full length man origin including, but not limited to, rat, mouse, cat, rhBuChE from the milk of transgenic goats conjugated via horse, chicken, pig, rabbit, and cow. Butyrylcholinesterases also include butyrylcholinesterases of human origin. Exem iodoacetamide and maleimide to MPC polymers. plary human butyrylcholinesterases include human BuChE 0050 FIG.5 shows 3-arm, 250 kDa, MPC polymers with set forth in SEQID NO. 1. Butyrylcholinesterases can be in modified (R1) iodoacetamide moiety. any form, including, but not limited to, monomer, dimer and DETAILED DESCRIPTION tetramer forms. 0056 “Organophosphorus compound.” “organophos I. General phate compound.” “OP compound,” and “OP” which are used interchangeably herein, refer to chemical compounds that 0051. The present invention provides high molecular contain a phosphoryl center, and further contain one or more weight (MW) polymers having hydrophilic groups or Zwitte esterlinkages. In some aspects, the type of phosphoester bond rions, such as phosphorylcholine. Also provided in accor and/or additional covalent bond at the phosphoryl center clas dance with the present invention are methods and novel start sifies an organophosphorus compound. Organophosphorus ing materials for making the high MW polymers. Also compounds include organophosphorus pesticides and orga provided in accordance with the present invention are conju nophosphorus nerve agents. gates of the high MW polymers and cholinesterases. 0057. “Organophosphorus nerve agent,” “organophos WO2011/130694 and WO/2007/100902 are hereby incorpo phate nerve agent,” or “OP nerve agent” refers to a chemical rated by reference for all purposes. compound that disrupts the functioning of the nervous system of an organism, such as by inhibiting the actions of the II. Definitions enzyme acetylcholinesterase. Nerve agents are generally pre 0052 “Cholinesterase” (ChE) refers to a family of pared by chemical synthesis and are highly toxic if inhaled, enzymes involved in nerve impulse transmissions. Cholinest absorbed, ingested, or otherwise encountered. A nerve agent erases catalyze the hydrolysis of acetylcholine at cholinergic can be any organophosphorus compound, including, but not synapses. Cholinesterases include but are not limited to ace limited to G-type nerve agents and V-type nerve agents. tylcholinesterases and butyrylcholinesterases. Exemplary organophosphorus nerve agents include tabun 0053 Reference to cholinesterases, including acetylcho (GA), sarin (GB), soman (GD), cyclosarin (GF), VX, Russian linesterases and butyrylcholinesterases, includes precursor VX (VR), and classified non-traditional nerve agents (NTAs). cholinesterases and mature cholinesterases (such as those in 0.058 “Organophosphorus pesticide.” “organophosphate which a signal sequence has been removed), truncated forms pesticide' or “OP pesticide” refers to an organophosphorus thereof that have activity, and includes allelic variants and compound that can be used a pesticide or insecticide to species variants, variants encoded by splice variants, and destroy pests and insects. An organophosphorus pesticide can other variants, including polypeptides that have at least 85%, be any organophosphorus pesticide, including, but not limited 90%. 95%, 96%, 97%, 98%, 99% or more sequence identity to, acephate, azinphos-methyl, bensulide, cadusafos, chlore to the polypeptides set forth in the referenced SEQID NOS. thoxyfos, chlorpyrifos, chlorpyrifosmethyl, chlorthiophos, Variants exhibitOP inactivating activity. Cholinesterases also coumaphos, dialiflor, diazinon, diehlorvos (DDVP), dieroto include those that contain chemical or posttranslational modi phos, dimethoate, dioxathion, disulfoton, ethion, ethoprop, fications and those that do not contain chemical or posttrans ethyl parathion, fenamiphos, fenitrothion, fenthion, fonofos, lational modifications. Such modifications include, but are isaZophos methyl, isofenphos, malathion, methamidophos, US 2016/01 84445 A1 Jun. 30, 2016 methidathion, methyl parathion, mevinphos, monocroto drylases and diisopropylfluorophosphatases (DFPases) phos, naled, oxydemeton methyl, phorate, phosalone, phos including recombinant mutations introduced into Such met, phosphamidon, phostebupirim, pirimiphos methyl, pro enzymes to increase their ability to hydrolyze OP com fenofos, propetamphos, Sulfotepp, SulprofoS, temephos, pounds. terbufos, tetraehlorvinphos, tribufos (JDEF) and trichlorfon. 0065 'Aryldialkylphosphatase' refers to naturally occur 0059 “Organophosphorus poisoning or “OP poisoning ring or recombinant enzymes that inactivate or hydrolyze refers to deleterious or undesirable effects to a living creature organophosphorus compounds. Aryldialkylphosphatases exposed to an organophosphorus compound Such as an orga (EC 3.1.8.1) are a class of metal-dependent OP-hydrolases nophosphorus nerve agent oran organophosphorus pesticide. that are capable of hydrolyzing a broad range of organophos 0060 “Cholinergic toxicity' refers to toxicity achieved by phorus compounds. Aryldialkylphosphatases require a diva nerve agent inhibition of acetylcholinesterase, accumulation lent metal ion, such as Zn2+, Mn2+, Co2+, or Cd2+, for of the neurotransmitter acetylcholine, and concomitant enzymatic activity. Aryldialkylphosphatases include, but are effects on the parasympathetic, sympathetic, motor, and cen not limited to, phosphotriesterases or OPhydxolases (PTE or tral nervous systems. Cholinergic toxicity can result in OPH), paraoxon hydrolases or paraoXonases, parathion myopathy, psychosis, general paralysis and death. Symptoms hydrolases (PH), OpdA, carboxylesterases, triesterases, of exposure include twitching, trembling, hypersecretion, phosphotriesterases and arylesterases. Aryldialkylphos paralyzed breathing, convulsions, and ultimately death. Cho phatases include, but are not limited to, organophosphorus linergic toxicity can be monitored by measuring circulating hydrolases from Pseudomonas diminuata MG, Flavobacte (active) cholinesterase levels in the plasma. Generally lethal rium sp., Plesiomonas sp. strain M6, Streptomyces lividans ity occurs only when cholinesterase activity falls below 20% and Agrobacterium radiobacter, parathion hydrolases from of normal levels due to binding by nerve agents. Burkholderia sp. JBA3, Pseudomonas diminuta MG, 0061 “Organophosphorus exposure associated damage' Brevundiomonas diminuta, Flavobacterium sp. strain ATCC refers to short term (e.g., minutes to several hours post-expo 27552, and Sulfolobus acidocaldarius; and methyl parathion Sure) and long term (e.g., one week up to several years post hydrolases (MPH) from Bacillus subtilis WB800 and Plesi exposure) damage, for example, due to cholinergic toxicity, to Omonas sp. Strain M6. Exemplary aryldiakylphosphatases physiological function (e.g., motor and cognitive functions). include, but are not limited to the aryldialkylphosphatases set Organophosphorus exposure associated damage can be mani forth in SEQID NO. 5. fested by the following clinical symptoms including, but not 0.066 Reference to aryldialkylphosphatases includes trun limited to, headache, diffuse muscle cramping, Weakness, cated forms thereof that have activity, and includes allelic excessive secretions, nausea, vomiting, and diarrhea. The variants and species variants, variants encoded by splice vari condition can progress to seizure, coma, paralysis, respira ants, and other variants, including polypeptides that have at tory failure, delayed neuropathy, muscle weakness, tremor, least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, convulsions, permanent brain dismorphology, Social/behav 85%, 90%. 95%, 96%, 97%, 98%, 99% or more sequence ioral deficits and general cholinergic crisis (which can be identity to the polypeptides set forth above and in SEQ ID manifested for instance by exacerbated inflammation and low NO. 5. Variants exhibit OP inactivating activity. blood count). Extreme cases can lead to death of the poisoned 0067 "Paraoxonase” refers to naturally occurring or Subjects. recombinant enzymes that inactivate or hydrolyze organo 0062 “Organophosphorus bioscavenger' or “organo phosphorus compounds. Paraoxonases include, but are not phosphate bioscavenger” or "OP bioscavenger is an enzyme limited to, native or recombinant paraoXonases. Non-human capable of binding to or hydrolyzing an organophosphorus paraoxonases include, but are not limited to, paraoXonases compound, including organophosphorus pesticides and orga from rabbit, mouse, rat, pig, cow, chicken, turkey and dog. nophosphorus nerve agents. Organophosphorus bioscav Exemplary paraoxonases include, but are not limited to, engers include, but are not limited to, cholinesterases, aryl human paraoxonases, including PON1 (SEQID NO. 6). dialkylphosphatases, organophosphate hydrolases (OPH), 0068 Reference to paraoxonases includes truncated carboxylesterases, triesterases, phosphotriesterases, ary forms thereofthat have activity, and includes allelic variants lesterases, paraoXonases (PON), organophosphate acid anhy and species variants, variants encoded by splice variants, and drases, and diisopropylfluorophosphatases (DFPases). Orga other variants, including polypeptides that have at least 85%, nophosphorus bioscavengers can be stoichiometric 90%. 95%, 96%, 97%, 98%, 99% or more sequence identity organophosphorus bioscavengers or catalytic organophos to the polypeptide set forth in SEQID NO. 6. Variants exhibit phorus bioscavengers. OP inactivating activity. Paraoxonases also include those that 0063 “Stoichiometric organophosphorus bioscavenger' contain chemical or posttranslational modifications and those or “stoichiometric OP bioscavenger refers to an enzyme that do not contain chemical or posttranslational modifica where each active site of the enzyme binds to an organophos tions. Such modifications include, but are not limited to, phorus compound in a stoichiometric 1:1 ratio. Stoichiomet PEGylation, albumination, glycosylation, farnesylation, car ric OP bioscavengers include, but are not limited, to cho boxylation, hydroxylation, phosphorylation, and other linesterases, such as acetylcholinesterases and polypeptide modifications known in the art. butyrylcholinesterases. 0069. “Diisopropylfluorophosphatase' refers to naturally 0064 “Catalytic organophosphorus bioscavenger” or occurring or recombinant enzymes that inactivate or hydro “catalytic OP bioscavenger” refers to an enzyme that hydro lyZe organophosphorus compounds. Diisopropylfluorophos lyzes an organophosphorus compound. Catalytic OP biso phatases include, but are not limited to, diisopropylfluoro cavengers include, but are not limited to, aryldialkylphos phosphatases from Loligo vulgaris, Alteromonas sp., phatases, organophosphate hydrolases (OPH), Pseudoalteromonas haloplanktis, Marinomonas mediterra carboxylesterases, triesterases, phosphotriesterases, ary nea, Aplysia Californica, Octopus Vulgaris and rat senescence lesterases, paraoXonases (PON), organophosphate acid anhy marker protein 30; and organophosphate acid anhydrolases US 2016/01 84445 A1 Jun. 30, 2016 from Mycobacterium sp., Amycolatopsis mediterranei, Strep (0079 “Bioavailability” refers to the fraction of an admin tomyces coelicolor; Streptomyces SpAA4, Streptomyces liv istered dose of drug that reaches the systemic circulation. idans TK24, Streptomyces Sviceus, and Streptomyces Bioavailability is a function of the absorption of a drug into griseoaurantiacus M045. Exemplary diisopropylfluorophos the bloodstream. phatases and organophosphate acid anhydrolases include, but 0080) “Dose' refers to the quantity or amount of drug that are not limited to, diisopropylfluorophosphatase from Loligo is administered to a subject for therapeutic or prophylactic vulgaris set forth in any of SEQID NO. 7. purposes. 0070 Reference to diisopropylfluorophosphatases I0081. “Nucleic acids” include DNA, RNA and analogs includes truncated forms thereof that have activity, and thereof, including peptide nucleic acids (PNA) and mixtures includes allelic variants and species variants, variants thereof. Nucleic acids can be single or double stranded. When encoded by splice variants, and other variants, including referring to probes or primers, which are optionally labeled, polypeptides that have at least 85%, 90%. 95%, 96%, 97%, such as with a detectable label, such as a fluorescent or radio 98%, 99% or more sequence identity to the polypeptide set label, single-stranded molecules are contemplated. Such mol forth in SEQ ID NO. 7. Variants exhibit OP inactivating ecules are typically of a length such that their target is statis activity. tically unique or of low copy number (typically less than 5, 0071 "Carbamate” refers to any compound that is a car generally less than 3) for probing or priming a library. Gen bamate inhibitor of cholinesterase. An exemplary carbamate erally a probe or primer contains at least 14, 16 or 30 con is pyridostigmine bromide. tiguous nucleotides of sequence complementary to or identi 0072 “Anti-muscarinic' refers to any compound that is a cal to a gene of interest. Probes and primers can be 10, 20, 30. competitive antagonist to muscarinic receptors, including 50, 100 or more nucleic acids long. muscarinic acetylcholine receptors. An exemplary anti-mus I0082. A "polypeptide linker” or “linker' is a polypeptide carinic is atropine. comprising two or more amino acid residues joined by pep 0073 "Cholinesterase reactivator” refers to any com tide bonds that are used to link two polypeptides (e.g., a VH pound that releases a bound organophosphorus compound and VL domain or a VH domain and an extracellular trap from a cholinesterase. Cholinesterase reactivators include segment). Examples of Such linker polypeptides are well choline-reactivating oXimes, including pyridinium and bispy known in the art (see, e.g., Holliger et al. (1993) Proc. Natl. ridinium aldoximes, including, but not limited to, prali Acad. Sci. USA90:6444-6448; Poljak et al. (1994) Structure doXime, trimedoxime, obidoxime and HI-6, methoxime, and 2:1121-1123). Exemplary linkers include G, GG, GGGGS diazepam. (SEQ ID NO: 22), GGGS (SEQ ID NO. 23), and GGGES 0074 Anti-convulsive' refers to any compound that pro (SEQ ID NO: 24), and oligomers of such linkers (e.g., tects against or reverses seizures. GGGGSGGGGS (SEQ ID NO: 4) and 0075 "Conjugate” refers to a polypeptide linked directly GGGGSGGGGSGGGGSGGGGSG (SEQID NO: 26)). or indirectly to one or more chemical moieties such as poly I0083. “Polymer refers to a series of monomer groups CS. linked together. The high MW polymers are prepared from 0076 “Fusion protein’ refers to a polypeptide encoded by monomers that include, but are not limited to, acrylates, a nucleic acid sequence containing a coding sequence from methacrylates, acrylamides, methacrylamides, styrenes, one nucleic acid molecule and the coding sequence from vinyl-pyridine, vinyl-pyrrolidone, and vinyl esters such as another nucleic acid molecule in which the coding sequences vinyl acetate. Additional monomers are useful in the high are in the same reading frame Such that when the fusion MW polymers of the present invention. When two different construct is transcribed and translated in a host cell, the pro monomer types are used, the two monomer types are called tein is produced containing the two proteins. The two mol “comonomers.” meaning that the different monomers types ecules can be adjacent in the constructor separated by a linker are copolymerized to form a single polymer. The polymer can polypeptide that contains, 1, 2, 3, or more, but typically fewer be linear or branched. When the polymer is branched, each than 25, 20, 15, 10, 9, 8, 7, or 6 amino acids. The protein polymer chain is referred to as a “polymer arm.” The end of product encoded by a fusion construct is referred to as a the polymer arm linked to the initiator moiety is the proximal fusion polypeptide or protein. Fusion proteins may have a end, and the growing-chain end of the polymer arm is the peptide or protein linked between the two proteins. distal end. On the growing chain-end of the polymerarm, the 0077. “Half-life” or “half-life of elimination” or “t/2” polymer arm end group can be the radical scavenger, or refers to the time required for any specified property or activ another group. ity to decrease by half. For example, half-life refers to the time I0084) “Initiator refers to a compound capable of initiat it takes a Substance (e.g. an organophosphorous bioscav ing a polymerization using monomers or comonomers. The enger) to lose half of its activity of its original level. Hence, polymerization can be a conventional free radical polymer half-life can be determined by measuring the activity of a ization or preferably a controlled/living radical polymer Substance in plasma, or it can be determined by measuring the ization, such as Atom Transfer Radical Polymerization plasma level of the Substance in the plasma. For example, (ATRP), Reversible Addition Fragmentation Termination half-life can be determined as the time necessary for the drug (RAFT) polymerization or nitroxide mediated polymeriza to be reduced to half of its original level in the body through tion (NMP). The polymerization can be a “pseudo' controlled various bodily processes. The longer the half-life, the longer polymerization, such as degenerative transfer. When the ini it will take the substance or drug to be purged from the body. tiator is suitable for ATRP, it contains alabile bond which can Units for half-life are generally units of time such as hour, be homolytically cleaved to form an initiator fragment, I, minute or day. being a radical capable of initiating a radical polymerization, 0078 “Absorption” refers to the movement of a drug into and a radical scavenger, I', which reacts with the radical of the the bloodstream. growing polymer chain to reversibly terminate the polymer US 2016/01 84445 A1 Jun. 30, 2016 ization. The radical scavenger I' is typically a halogen, but can 0093 Polymers defined by reference to a specified mono also be an organic moiety, such as a nitrile. mer type can be a homopolymer, i.e., only monomers of the 0085 “Linker refers to a chemical moiety that links two specified type are present, or a copolymer in which case groups together. The linker can be cleavable or non-cleavable. monomers of the specified type are present with other mono Cleavable linkers can be hydrolysable, enzymatically cleav mer type(s). If a co-polymer preferably, at least 50, 75 or 90% able, pH sensitive, photolabile, or disulfide linkers, among of monomer molecules are of the specified type. others. Other linkers include homobifunctional and heterobi (0094) “Molecular weight” in the context of the polymer functional linkers. A "linking group' is a functional group can be expressed as either a number average molecular capable of forming a covalent linkage consisting of one or weight, or a weight average molecular weight, or a peak more bonds to a bioactive agent. Nonlimiting examples molecular weight. Unless otherwise indicated, all references include those illustrated in Table 1. to molecular weight herein refer to the peak molecular I0086. The term “reactive group’ as used herein refers to a weight. These molecular weight determinations, number group that is capable of reacting with another chemical group average (Mn), weight average (Mw), and peak (Mp), can be to form a covalent bond, i.e. is covalently reactive under measured using size exclusion chromatography or other liq Suitable reaction conditions, and generally represents a point uid chromatography techniques. Other methods for measur of attachment for another Substance. The reactive group is a ing molecular weight values can also be used, such as the use moiety, such as maleimide or Succinimidyl ester, on the com of end-group analysis or the measurement of colligative prop pounds of the present invention that is capable of chemically erties (e.g., freezing-point depression, boiling-point eleva reacting with a functional group on a different compound to tion, or osmotic pressure) to determine number average form a covalent linkage. Reactive groups generally include molecular weight, or the use of light scattering techniques, nucleophiles, electrophiles, and photoactivatable groups. ultracentrifugation, or viscometry to determine weight aver 0087 “Functional agent' is defined to include a bioactive age molecular weight. In a preferred embodiment of the agent or a diagnostic agent. A "bioactive agent' is defined to present invention, the molecular weight is measured by SEC include any agent, drug, compound, or mixture thereof that MALS (size exclusion chromatography-multi angle light targets a specific biological location (targeting agent) and/or scattering). The polymeric reagents of the invention are typi provides some local or systemic physiological or pharmaco cally polydisperse (i.e., number average molecular weight logic effect that can be demonstrated in vivo or in vitro. and weight average molecular weight of the polymers are not Non-limiting examples include drugs, vaccines, antibodies, equal), preferably possessing a low polydispersity index antibody fragments, scFVs, diabodies, avimers, vitamins and (PDI) values of for example, less than about 1.5, as judged by cofactors, polysaccharides, carbohydrates, steroids, lipids, gel permeation chromatography. In other embodiments, the fats, proteins, peptides, polypeptides, nucleotides, oligo polydispersities are more preferably in the range of about 1.4 nucleotides, polynucleotides, and nucleic acids (e.g., mRNA, to about 1.2, still more preferably less than about 1.15, and tRNA, snRNA, RNAi, DNA, cDNA, antisense constructs, still more preferably less than about 1.10, yet still more pref ribozymes, etc.). A "diagnostic agent' is defined to include erably less than about 1.05, and most preferably less than any agent that enables the detection or imaging of a tissue or about 1.03. disease. Examples of diagnostic agents include, but are not (0095. The phrase “a” or “an entity as used herein refers to limited to, radiolabels, fluorophores, and dyes. one or more of that entity; for example, a compound refers to 0088 “Therapeutic protein’ refers to peptides or proteins one or more compounds or at least one compound. As such, regardless of length that include an amino acid sequence the terms “a” (or “an”), “one or more', and “at least one' can which in whole or in part makes up a drug and can be used in be used interchangeably herein. human or animal pharmaceutical applications. Numerous 0096. About as used herein means variation one might therapeutic proteins are known including, without limitation, see in measurements taken among different instruments, those disclosed herein. samples, and sample preparations. I0089. “Phosphorylcholine,” also denoted as “PC” refers to the following: (0097) “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 groups vary depending on 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. Suitable protecting groups include those where * denotes the point of attachment. such as found in the treatise by Greene et al., “Protective 0090 “Phosphorylcholine containing polymer is a poly Groups In Organic Synthesis.” 3rd Edition, John Wiley and mer that contains phosphorylcholine. “Zwitterion containing Sons, Inc., New York, 1999. polymer refers to a polymer that contains a Zwitterion. 0.098 “Alkyl refers to a straight or branched, saturated, 0091 Poly(acryloyloxyethyl phosphorylcholine) contain aliphatic radical having the number of carbon atoms indi ing polymer refers to a polymer containing 2-(acryloyloxy) cated. For example, C1-C6 alkyl includes, but is not limited ethyl-2-(trimethylammonium)ethyl phosphate as monomer. to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, 0092 Poly(methacryloyloxyethyl phosphorylcholine) tert-butyl, pentyl, isopentyl, hexyl, etc. Other alkyl groups containing polymer refers to a polymer containing 2-(meth include, but are not limited to heptyl, octyl, nonyl, decyl, etc. acryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate Alkyl can include any number of carbons, such as 1-2, 1-3, aS OOC. 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, US 2016/01 84445 A1 Jun. 30, 2016

3-6, 4-5, 4-6 and 5-6. The alkyl group is typically monovalent, groups is present. When R' and R" are attached to the same but can be divalent, such as when the alkyl group links two nitrogen atom, they can be combined with the nitrogenatom moieties together. to form a 5-, 6-, or 7-membered ring. For example, —NR'R'" 0099. The term “lower referred to above and hereinafter is meant to include, but not be limited to, 1-pyrrolidinyl and in connection with organic radicals or compounds respec 4-morpholinyl. The term “alkyl includes groups including tively defines a compound or radical which can be branched carbon atoms bound to groups other than hydrogen groups, or unbranched with up to and including 7, preferably up to Such as haloalkyl (e.g., -CF3 and —CH2CF3) and acyl (e.g., and including 4 and (as unbranched) one or two carbonatoms. - C(O)CH3, C(O)CF3, C(O)CH2OCH3, and the like). 0100 Alkylene' refers to an alkyl group, as defined 0103 "Alkoxy' refers to alkyl group having an oxygen above, linking at least two other groups, i.e., a divalent hydro atom that either connects the alkoxy group to the point of carbon radical. The two moieties linked to the alkylene can be attachment or is linked to two carbons of the alkoxy group. linked to the same atom or different atoms of the alkylene. For Alkoxy groups include, for example, methoxy, ethoxy, pro instance, a straight chain alkylene can be the bivalent radical poxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, of—(CH2)n, where n is 1, 2, 3, 4, 5 or 6. Alkylene groups tert-butoxy, pentoxy, hexoxy, etc. The alkoxy groups can be include, but are not limited to, methylene, ethylene, propy further substituted with a variety of substituents described lene, isopropylene, butylene, isobutylene, sec-butylene, pen within. For example, the alkoxy groups can be substituted tylene and hexylene. with halogens to form a "halo-alkoxy' group. 0101 Substituents for the alkyl and heteroalkyl radicals 0104 “Carboxyalkyl means an alkyl group (as defined (including those groups often referred to as alkylene, alkenyl, herein) substituted with a carboxy group. The term "carboxy heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocy cycloalkyl means an cycloalkyl group (as defined herein) cloalkyl, cycloalkenyl, and heterocycloalkenyl) can be a vari substituted with a carboxy group. The term alkoxyalkyl ety of groups selected from: OR', —O, —NR", =N OR', means an alkyl group (as defined herein) Substituted with an alkoxy group. The term “carboxy” employed herein refers to carboxylic acids and their esters. 0105. “Haloalkyl refers to alkyl as defined above where NH C(NH2)=NH, NR C(NH2)=NH, NH C some or all of the hydrogenatoms are substituted with halo (NH2)=NR', S(O)R', S(O)2R', S(O)2NR'R'', CN gen atoms. Halogen (halo) preferably represents chloro or and —NO2 in a number ranging from Zero to (2m'+1), where fluoro, but may also be bromo or iodo. For example, haloalkyl m' is the total number of carbon atoms in such radical. R', R" includes trifluoromethyl, fluoromethyl, 1,2,3,4,5-pen and R" each independently refer to hydrogen, unsubstituted tafluoro-phenyl, etc. The term “perfluoro' defines a com (C1-C8)alkyl and heteroalkyl, unsubstituted aryl, aryl substi pound or radical which has all available hydrogens that are tuted with 1-3 halogens, unsubstituted alkyl, alkoxy or thio replaced with fluorine. For example, perfluorophenyl refers to alkoxy groups, oraryl-(C1-C4)alkyl groups. When R' and R" 1,2,3,4,5-pentafluorophenyl, perfluoromethyl refers to 1,1,1- are attached to the same nitrogenatom, they can be combined trifluoromethyl, and perfluoromethoxy refers to 1,1,1-trifluo with the nitrogen atom to form a 5-, 6-, or 7-membered ring. romethoxy. For example, —NR'R" is meant to include 1-pyrrolidinyl and 0106 “Fluoro-substituted alkyl refers to an alkyl group 4-morpholinyl. The term “alkyl includes groups such as where one, Some, or all hydrogen atoms have been replaced haloalkyl (e.g., —CF3 and —CH2CF3) and acyl (e.g., by fluorine. —C(O)CH3, C(O)CF3, —C(O)CH2OCH3, and the like). 0107 “Cycloalkyl refers to a cyclic hydrocarbon group Preferably, the substituted alkyl and heteroalkyl groups have that contains from about 3 to 12, from 3 to 10, or from 3 to 7 from 1 to 4 substituents, more preferably 1, 2 or 3 substitu endocyclic carbon atoms. Cycloalkyl groups include fused, ents. Exceptions are those perhalo alkyl groups (e.g., pen bridged and spiro ring structures. tafluoroethyl and the like) which are also preferred and con 0.108 “Endocyclic” refers to an atom or group of atoms templated by the present invention. which comprise part of a cyclic ring structure. 0102) Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, 0109) “Exocyclic” refers to an atom or group of atoms heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocy which are attached but do not define the cyclic ring structure. cloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or 0110 “Cyclic alkyl ether” refers to a 4 or 5 member cyclic more of a variety of groups selected from, but not limited to: alkyl group having 3 or 4 endocyclic carbon atoms and 1 —OR", =O, —NR', —N OR', NR'R", SR', -halogen, endocyclic oxygen or Sulfur atom (e.g., oxetane, thietane, SiR'R"R", OC(O)R', C(O)R', CO2R', tetrahydrofuran, tetrahydrothiophene); or a 6 to 7 member CONR'R", OC(O)NR'R", NR"C(O)R', NR C(O) cyclic alkyl group having 1 or 2 endocyclic oxygen or Sulfur NR"R", NR"C(O)2R, NR C(NR'R"R") NR", atoms (e.g., tetrahydropyran, 1.3-dioxane, 1,4-dioxane, tet NR C(NR'R")—NR", S(O)R', S(O)2R, S(O) rahydrothiopyran, 1.3-dithiane, 1,4-dithiane, 1,4-oxathiane). 2NR'R", NRSO2R, CN and NO2 in a numberranging 0111 “Alkenyl refers to either a straight chain or from Zero to (2m'+1), where m' is the total number of carbon branched hydrocarbon of 2 to 6 carbon atoms, having at least atoms in such radical. R', R", R" and R' each preferably one double bond. Examples of alkenyl groups include, but are independently refer to hydrogen, substituted or unsubstituted not limited to, vinyl, propenyl, isopropenyl, 1-butenyl, heteroalkyl, Substituted or unsubstituted aryl, e.g., aryl Sub 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, stituted with 1-3 halogens, substituted or unsubstituted alkyl, isopentenyl, 1.3-pentadienyl, 1.4-pentadienyl, 1-hexenyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a 2-hexenyl, 3-hexenyl, 1.3-hexadienyl, 1,4-hexadienyl, 1.5- compound of the invention includes more than one R group, hexadienyl, 2.4-hexadienyl, or 1.3.5-hexatrienyl. Alkenyl for example, each of the R groups is independently selected as groups can also have from 2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5, are each R', R", R" and R' groups when more than one of these 3 to 6, 4 to 5, 4 to 6 and 5 to 6 carbons. The alkenyl group is US 2016/01 84445 A1 Jun. 30, 2016

typically monovalent, but can be divalent, such as when the or naphthyl, preferably phenyl. "Arylene' means a divalent alkenyl group links two moieties together. radical derived from an aryl group. Aryl groups can be mono-, 0112 Alkenylene' refers to an alkenyl group, as defined di- or tri-substituted by one, two or three radicals selected above, linking at least two other groups, i.e., a divalent hydro from alkyl, alkoxy, aryl, hydroxy, halogen, cyano, amino, carbon radical. The two moieties linked to the alkenylene can amino-alkyl, trifluoromethyl, alkylenedioxy and oxy-C2-C3 be linked to the same atom or different atoms of the alk alkylene; all of which are optionally further substituted, for enylene. Alkenylene groups include, but are not limited to, instance as hereinbefore defined; or 1- or 2-naphthyl; or 1- or ethenylene, propenylene, isopropenylene, butenylene, 2-phenanthrenyl. Alkylenedioxy is a divalent substitute isobutenylene, sec-butenylene, pentenylene and hexenylene. attached to two adjacent carbon atoms of phenyl, e.g. meth 0113 Alkynyl refers to either a straight chain or ylenedioxy or ethylenedioxy. Oxy-C2-C3-alkylene is also a branched hydrocarbon of 2 to 6 carbon atoms, having at least divalent Substituent attached to two adjacent carbonatoms of one triple bond. Examples of alkynyl groups include, but are phenyl, e.g. oxyethylene or oxypropylene. An example for not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, oxy-C2-C3-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl. isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, I0120 Preferred as aryl is naphthyl, phenyl or phenyl isopentynyl, 1.3-pentadiynyl, 1.4-pentadiynyl, 1-hexynyl, mono- or disubstituted by alkoxy, phenyl, halogen, alkyl or 2-hexynyl, 3-hexynyl, 1.3-hexadiynyl, 1.4-hexadiynyl, 1.5- trifluoromethyl, especially phenyl or phenyl-mono- or disub hexadiynyl, 2.4-hexadiynyl, or 1.3.5-hexatriynyl. Alkynyl stituted by alkoxy, halogen or trifluoromethyl, and in particu groups can also have from 2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5, lar phenyl. 3 to 6, 4 to 5, 4 to 6 and 5 to 6 carbons. The alkynyl group is 0121 Examples of substituted phenyl groups as Rare, e.g. typically monovalent, but can be divalent, such as when the 4-chlorophen-1-yl, 3,4-dichlorophen-1-yl 4-methoxyphen alkynyl group links two moieties together. 1-yl 4-methylphen-1-yl 4-aminomethylphen-1-yl 4-meth 0114 Alkynylene' refers to an alkynyl group, as defined oxyethylaminomethylphen-1-yl 4-hydroxyethylaminom above, linking at least two other groups, i.e., a divalent hydro ethylphen-1-yl, 4-hydroxyethyl-(methyl)- carbon radical. The two moieties linked to the alkynylene can aminomethylphen-1-yl, 3-aminomethylphen-1-yl 4-N- be linked to the same atom or different atoms of the alky acetylaminomethylphen-1-yl, 4-aminophen-1-yl, nylene. Alkynylene groups include, but are not limited to, 3-aminophen-1-yl, 2-aminophen-1-yl 4-phenyl-phen-1-yl, ethynylene, propynylene, butynylene, Sec-butynylene, penty 4-(imidazol-1-yl)-phenyl, 4-(imidazol-1-ylmethyl)-phen-1- nylene and hexynylene. y1, 4-(morpholin-1-yl)-phen-1-yl 4-(morpholin-1-ylm 0115 "Cycloalkyl” refers to a saturated or partially unsat ethyl)-phen-1-yl 4-(2-methoxyethylaminomethyl)-phen-1- urated, monocyclic, fused bicyclic or bridged polycyclic ring y1 and 4-(pyrrolidin-1-ylmethyl)-phen-1-yl 4-(thiophenyl)- assembly containing from 3 to 12 ring atoms, or the number phen-1-yl, 4-(3-thiophenyl)-phen-1-yl, 4-(4- of atoms indicated. Monocyclic rings include, for example, methylpiperazin-1-yl)-phen-1-yl, and 4-(piperidinyl)-phenyl cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and and 4-(pyridinyl)-phenyl optionally substituted in the hetero cyclooctyl. Bicyclic and polycyclic rings include, for cyclic ring. example, norbornane, decahydronaphthalene and adaman 0.122 "Arylene' refers to an aryl group, as defined above, tane. For example, C3-8cycloalkyl includes cyclopropyl. linking at least two other groups. The two moieties linked to cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and norbor the arylene are linked to different atoms of the arylene. aC. Arylene groups include, but are not limited to, phenylene. 0116 “Cycloalkylene' refers to a cycloalkyl group, as I0123 'Arylene-oxy' refers to an arylene group, as defined defined above, linking at least two other groups, i.e., a diva above, where one of the moieties linked to the arylene is lent hydrocarbon radical. The two moieties linked to the linked through an oxygenatom. Arylene-oxy groups include, cycloalkylene can be linked to the same atom or different but are not limited to, phenylene-oxy. atoms of the cycloalkylene. Cycloalkylene groups include, 0.124 Similarly, substituents for the aryl and heteroaryl but are not limited to, cyclopropylene, cyclobutylene, cyclo groups are varied and are selected from: -halogen, —OR', pentylene, cyclohexylene, and cyclooctylene. 0117 “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 heteroa toms can also be useful, including, but not limited to, B, Al, Si (NH2)=NR', S(O)R', S(O)2R', S(O)2NR'R", N3, and P. The heteroatoms can also be oxidized, such as, but not —CH(Ph)2, perfluoro(C1-C4)alkoxy, and perfluoro(C1-C4) limited to, S(O)—and—S(O)2-. For example, heterocycle alkyl, in a number ranging from Zero to the total number of includes, but is not limited to, tetrahydrofuranyl, tetrahy open Valences on the aromatic ring system; and where R', R" drothiophenyl, morpholino, pyrrolidinyl, pyrrolinyl, imida and R" are independently selected from hydrogen, (C1-C8) Zolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazi alkyl and heteroalkyl, unsubstituted aryland heteroaryl, (un nyl, piperidinyl, indolinyl, quinuclidinyl and 1,4-dioxa-8- substituted aryl)-(C1-C4)alkyl, and (unsubstituted aryl)oxy aza-spiro4.5dec-8-yl. (C1-C4)alkyl. 0118 “Heterocycloalkylene' refers to a heterocyclalkyl 0.125. Two of the substituents on adjacentatoms of the aryl group, as defined above, linking at least two other groups. The or heteroaryl ring may optionally be replaced with a substitu two moieties linked to the heterocycloalkylene can be linked ent of the formula -T-C(O)—(CH2)d-U—, wherein T and U to the same atom or different atoms of the heterocycloalky are independently —NH-, —O— —CH2- or a single bond, lene. and q is an integer of from 0 to 2. Alternatively, two of the 0119 'Aryl refers to a monocyclic or fused bicyclic, tri Substituents on adjacent atoms of the aryl or heteroaryl ring cyclic or greater, aromatic ring assembly containing 6 to 16 may optionally be replaced with a substituent of the formula ring carbon atoms. For example, aryl may be phenyl, benzyl -A-(CH2)r-B wherein A and Bare independently —CH2-, US 2016/01 84445 A1 Jun. 30, 2016

O— —NH —S— —S(O)— —S(O)2-, - S(O) I0132) “Maleimido” refers to a pyrrole-2,5-dione-1-yl 2NR'—or a single bond, and r is an integer of from 1 to 3. One group having the structure: 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 e may optionally be replaced with a substituent of the formula O —(CH2)s-X—(CH2)t-, where sandt are independently inte gers of from 0 to 3, and X is —O— —NR' —S O N —S(O) , S(O)2-, or S(O)2NR -. The substituent R' in —NR'— and —S(O)2NR' is selected from hydrogen or which on reaction with a sulfhydryl (e.g., a thio alkyl) forms unsubstituted (C1-C6)alkyl. an —S-maleimido group having the structure 0126 “Heteroaryl” refers to a monocyclic or fused bicy clic ortricyclic 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, ben Zothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl pyra O ON Zolyl, 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, advanta where “” indicates the point of attachment for the maleimido geously 2- or 3-pyridyl. Thienyl represents 2- or 3-thienyl. group and “a indicates the point of attachment of the sulfur Quinolinyl represents preferably 2-, 3- or 4-quinolinyl. Iso atom the thiol to the remainder of the original sulfhydryl quinolinyl represents preferably 1-, 3- or 4-isoquinolinyl. bearing group. Benzopyranyl, benzothiopyranyl represents preferably 0.133 For the purpose of this disclosure, “naturally occur 3-benzopyranyl or 3-benzothiopyranyl, respectively. Thiaz ring amino acids found in proteins and polypeptides are olyl represents preferably 2- or 4-thiazolyl, and most pre L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cys ferred, 4-thiazolyl. Triazolyl is preferably 1-, 2- or 5-(1,2,4- teine, L-glutamine, L-glutamic acid, L-glycine, L-histidine, triazolyl). Tetrazolyl is preferably 5-tetrazolyl. L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenyla 0127 Preferably, heteroaryl is pyridyl, indolyl, quinoli lanine, L-proline, L-serine, L-threonine, L-tryptophan, L-ty nyl, pyrrolyl, thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyra rosine, and or L-valine. “Non-naturally occurring amino Zolyl, imidazolyl, thienyl, furanyl, benzothiazolyl, benzo acids’ found in proteins are any amino acid other than those furanyl, isoquinolinyl, benzothienyl, oxazolyl, indazolyl, or recited as naturally occurring amino acids. Non-naturally occurring amino acids include, without limitation, the D iso any of the radicals Substituted, especially mono- or di-Substi mers of the naturally occurring amino acids (a mixture would tuted. more accurately be described as some natural and some non 0128. As used herein, the term "heteroalkyl refers to an natural amino acids. Other amino acids, such as 4-hydrox alkyl group having from 1 to 3 heteroatoms such as N, O and yproline, desmosine, isodesmosine, 5-hydroxylysine, epsi S. Additional heteroatoms can also be useful, including, but lon-N-methyllysine, 3-methylhistidine, although found in not limited to, B, Al, Si and P. The heteroatoms can also be naturally occurring proteins, are considered to be non-natu oxidized, such as, but not limited to. —S(O)—and—S(O)2-. rally occurring amino acids found in proteins for the purpose For example, heteroalkyl can include ethers, thioethers, of this disclosure as they are generally introduced by means alkyl-amines and alkyl-thiols. other than ribosomal translation of mRNA. 0129. As used herein, the term "heteroalkylene' refers to a I0134) “Linear in reference to the geometry, architecture heteroalkyl group, as defined above, linking at least two other or overall structure of a polymer, refers to polymer having a groups. The two moieties linked to the heteroalkylene can be single polymer arm. linked to the same atom or different atoms of the heteroalky 0.135 “Branched in reference to the geometry, architec lene. ture or overall structure of a polymer, refers to a polymer 0130 “Electrophile' refers to an ion or atom or collection having 2 or more polymer'arms' extending from an initiator. of atoms, which may be ionic, having an electrophilic center, The initiator may be employed in an atom transfer radical i.e., a center that is electron seeking, capable of reacting with polymerization (ATRP) reaction. A branched polymer may a nucleophile. An electrophile (or electrophilic reagent) is a possess 2 polymer chains (arms), 3 polymer arms, 4 polymer reagent that forms a bond to its reaction partner (the nucleo arms, 5 polymer arms, 6 polymer arms, 7 polymer arms, 8 phile) by accepting both bonding electrons from that reaction polymer arms, 9 polymer arms, 10 polymer arms, 11 polymer partner. arms, 12 polymer arms or more. Each polymer arm extends 0131 “Nucleophile' refers to an ion or atom or collection from a polymer initiation site. Each polymer initiation site is of atoms, which may be ionic, having a nucleophilic center, capable of being a site for the growth of a polymer chain by i.e., a center that is seeking an electrophilic center or capable the addition of monomers. For example and not by way of of reacting with an electrophile. A nucleophile (or nucleo limitation, using ATRP, each site of polymer initiation on an philic reagent) is a reagent that forms a bond to its reaction initiator is typically an organic halide undergoing a reversible partner (the electrophile) by donating both bonding electrons. redox process catalyzed by a transition metal compound Such A “nucleophilic group' refers to a nucleophile after it has as cuprous halide Preferably, the halide is a bromine. reacted with a reactive group. Non limiting examples include 0.136 “Pharmaceutically acceptable' composition or amino, hydroxyl, alkoxy, haloalkoxy and the like. “pharmaceutical composition” refers to a composition com US 2016/01 84445 A1 Jun. 30, 2016 prising a compound of the invention and a pharmaceutically dow). Percentage of sequence identity is calculated by com acceptable excipient or pharmaceutically acceptable excipi paring two optimally aligned sequences over a window of entS. comparison, determining the number of positions at which 0.137 “Pharmaceutically acceptable excipient' and “phar the identical residues occur in both sequences to yield the maceutically acceptable carrier refer to an excipient that can number of matched positions, dividing the number of be included in the compositions of the invention and that matched positions by the total number of positions in the causes no significant adverse toxicological effect on the window of comparison (i.e., the window size), and multiply patient and is approved or approvable by the FDA for thera ing the result by 100 to yield the percentage of sequence peutic use, particularly in humans. Non-limiting examples of identity. pharmaceutically acceptable excipients include water, NaCl, 0143. If a specific SEQ ID NO. is provided for a full normal saline solutions, lactated Ringers, normal Sucrose, length mature protein, residues are numbered consecutively normal glucose and the like. starting at 1 for the first residue of the mature protein (i.e., not 0138 “Patient” or “subject in need thereof refers to a taking into account residues of any signal peptide). Residues living organism Suffering from or prone to a condition that in variants or fragments of a SEQID NO. are numbered by can be prevented or treated by administration of a pharma maximal alignment with the SEQ ID NO. and assigning ceutical composition as provided herein. Non-limiting aligned residues the same number. examples include humans, other mammals and other non 0144. The term “protein’ is used in its broadest sense to mammalian animals. refer to a compound of two or more Subunit amino acids, 0139 Conjugates are preferably provided in isolated amino acid analogs or peptidomimetics. The subunits may be form. Isolated means that an object species has been at least linked by peptide bonds. In another embodiment, the subunit partially separated from contaminants with which it is natu may be linked by other bonds, e.g., ester, ether, etc. A protein rally associated or which are used in its manufacture but does or peptide must contain at least two amino acids and no not necessarily exclude the presence of other components limitation is placed on the maximum number of amino acids intended to act in combination with an isolated species. Such which may comprise a protein's or peptide's sequence. A as a pharmaceutical excipient. Preferably a conjugate is the peptide of three or more amino acids is commonly called an predominant macromolecular species present in a sample oligopeptide if the peptide chain is short. If the peptide chain (i.e., on a molar basis) in a composition and typically com is long, the peptide is commonly called a polypeptide or a prises at least about 50 percent (on a molar basis) of all protein. macromolecular species present. Generally, an isolated con 0145 Non-limiting examples of proteins, include an jugate comprises more than 80, 90, 95 or 99 percent of all enzyme, a cytokine, a neurotropic factor, an antibody, a pep macromolecular species present in a composition. Most pref tide, a hormone, a DNA-binding protein, an aptamer, vac erably, a conjugate is purified to essential homogeneity (i.e., cines, toxins, Interleukin-1. (IL-1 C), IL-1 f, IL-2, IL-3, IL-4, contaminant species cannot be detected in a composition by IL-5, IL-6, IL-11, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, conventional detection methods). Such that the composition IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, consists essentially of a single macromolecular species. Con IL-22, IL-23, IL-24, IL-31, IL-32, IL-33, colony stimulating jugates have the same heavy and light chains are considered to factor-1 (CSF-1), macrophage colony Stimulating factor, glu be the same species notwithstanding there may be variation in cocerobrosidase, thyrotropin, stem cell factor, granulocyte glycosylation on protein moieties and variation in numbers of macrophage colony stimulating factor, granulocyte colony monomers in polymer moieties linked to different molecules stimulating factor (G-CSF), EPO, interferon-alpha. (IFN-C), of the conjugate. consensus interferon-beta. (IFN-B), interferon-(IFN-), inter 0140 “Effective amount” refers to an amount of a conju feron-G2 (IFN-G2), thrombopoietin (TPO), Angiopoietin-1 gated functional agent or of a pharmaceutical composition (Ang-1), Ang-2, Ang-4, Ang-Y, angiopoietin-like polypep useful for treating, ameliorating, or prophylaxis of an identi tide 1 (ANGPTL1), angiopoietin-like polypeptide 2 (AN fied disease or condition, or for exhibiting a detectable thera GPTL2), angiopoietin-like polypeptide 3 (ANGPTL3), peutic or inhibitory effect. The effect can be detected in an angiopoietin-like polypeptide 4 (ANGPTL4), angiopoietin individual patient relative to a baseline measurement before like polypeptide 5 (ANGPTL5), angiopoietin-like polypep treatment or by determining a statistically significant differ tide 6 (ANGPTL6), angiopoietin-like polypeptide 7 (AN ence in outcome between treated and control populations. GPTL7), vitronectin, vascular endothelial growth factor Likewise effective regime, means a combination of amount, (VEGF), angiogenin, activin A, activin B, activin C, bone frequency and route of administration of any conjugation, morphogenic protein-1, bone morphogenic protein-2, bone fusion protein or other agent described herein effective for morphogenic protein-3, bone morphogenic protein-4, bone reducing, inhibiting, or delaying at least one sign or symptom morphogenic protein-5, bone morphogenic protein-6, bone of a disease. morphogenic protein-7, bone morphogenic protein-8, bone 0141. The “biological half-life' of a substance is a phar morphogenic protein-9, bone morphogenic protein-10, bone macokinetic parameter which specifies the time required for morphogenic protein-11, bone morphogenic protein-12, one half of the Substance to be removed from an organism bone morphogenic protein-13, bone morphogenic protein following introduction of the Substance into the organism. 14, bone morphogenic protein-15, bone morphogenic protein 0142 Sequence identity can be determined by aligning receptor IA, bone morphogenic protein receptor IB, bone sequences using algorithms, such as BESTFIT. FASTA, and morphogenic protein receptor II, brain derived neurotrophic TFASTA in the Wisconsin Genetics Software Package factor, cardiotrophin-1, ciliary neutrophic factor, ciliary Release 7.0, Genetics Computer Group, 575 Science Dr. neutrophic factor receptor, cripto, cryptic, cytokine-induced Madison, Wis., using default gap parameters, or by inspec neutrophil chemotactic factor 1, cytokine-induced neutro tion, and the best alignment (i.e., resulting in the highest phil, chemotactic factor 2C, hepatitis B vaccine, hepatitis C percentage of sequence similarity over a comparison win vaccine, drotrecogin alpha., cytokine-induced neutrophil US 2016/01 84445 A1 Jun. 30, 2016

chemotactic factor 23, endothelial cell growth factor, endot thereof, or at least one portion of a binding protein. "Anti helin 1, epidermal growth factor (EGF), epigen, epiregulin, body' also includes both monoclonal antibodies and poly epithelial-derived neutrophilattractant, fibroblast growth fac clonal antibodies. tor 4, fibroblast growth factor 5, fibroblast growth factor 6, 0147 Examples of antibodies include, without limitation, fibroblast growth factor 7, fibroblast growth factor 8, fibro Infliximab, Bevacizumab, Ranibizumab, Cetuximab, Ranibi blast growth factor 8b, fibroblast growth factor 8c, fibroblast Zumab, Palivizumab, Abagovomab, Abciximab, ActoXumab, growth factor 9, fibroblast growth factor 10, fibroblast growth Adalimumab, Afelimomab, AfutuZumab, Alacizumab, Ala factor 11, fibroblast growth factor 12, fibroblast growth factor cizumab pegol, ALD518, Alemtuzumab, Alirocumab, Alem 13, fibroblast growth factor 16, fibroblast growth factor 17, tuZumab. Altumomab, Amatuximab, Anatumomab mafena fibroblast growth factor 19, fibroblast growth factor 20, fibro toX, Anrukinzumab, Apolizumab, Arcitumomab, blast growth factor 21, fibroblast growth factor acidic, fibro Aselizumab, Altinumab, Atlizumab, Atorolimiumab, tocili blast growth factor basic, glial cell line-derived neutrophic Zumab, Bapineuzumab, Basiliximab, Bavituximab, Bectu factor receptor.alpha.1, glial cell line-derived neutrophic fac momab, Belimumab, Benralizumab, Bertilimumab, Besile tor receptor, growth related protein, growth related proteina, Somab, Bevacizumab, Bezlotoxumab, Biciromab, IgG, IgE, IgM, IgA, and Ig|D. C.-galactosidase, B-galactosi Bivatuzumab, Bivatuzumab mertansine, Blinatumomab, dase, DNAse, fetuin, leutinizing hormone, alteplase, estro Blosozumab, Brentuximab vedotin, Briakinumab, gen, insulin, albumin, lipoproteins, fetoprotein, transferrin, Brodalumab, Canakinumab, Cantuzumab mertansine, Can thrombopoietin, urokinase, integrin, thrombin, Factor IX tuZumab mertansine, Caplacizumab, Capromab pendetide, (FIX), Factor VIII (FVIII), Factor Vila (FVIIa), Von Will Carlumab, Catumaxomab, CC49, Cedelizumab, Certoli ebrand Factor (VWF), Factor FV (FV), Factor X (FX), Factor Zumab pegol, Cetuximab, CitatuZumab bogatox, Cixutu mumab, Clazakizumab, Clenoliximab, Clivatuzumab tetrax XI (FXI), Factor XII (FXII), Factor XIII (FXIII), thrombin etan, Conatumumab, Crenezumab, CR6261, Dacetuzumab, (FII), protein C, protein S, tRA, PAI-1, tissue factor (TF), Daclizumab, Dalotuzumab, Daratumumab, Demcizumab, ADAMTS 13 protease, growth related protein.beta., growth Denosumab, Detumomab, Dorlimomab aritox, Drozitumab, related protein, heparin binding epidermal growth factor, Duligotumab, Dupilumab, Ecromeximab, Eculizumab, Edo hepatocyte growth factor, hepatocyte growth factor receptor, bacomab, Edrecolomab, Efalizumab, Efungumab, Elotu hepatoma-derived growth factor, insulin-like growth factor I. Zumab, Elsilimomab, EnavatuZumab, Enlimomab pegol, insulin-like growth factor receptor, insulin-like growth factor Enokizumab, Enokizumab, Enoticumab, Enoticumab, Ensi II, insulin-like growth factor binding protein, keratinocyte tuximab, Epitumomab cituxetan, Epratuzumab, Erlizumab, growth factor, leukemia inhibitory factor, Somatropin, anti Ertumaxomab, Etaracizumab, Etrolizumab, Exbivirumab, hemophiliac factor, pegaspargase, orthoclone OKT 3. Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, adenosine deaminase, alglucerase, imiglucerase, leukemia Fasinumab, FBTA05, Felvizumab, Fezakinumab, Ficlatu inhibitory factor receptor alpha., nerve growth factor nerve Zumab, Figitumumab, FlanVotumab, Fontolizumab, growth factor receptor, neuropoietin, neurotrophin-3, neu Foralumab, Foravirumab, Fresolimumab, Fulranumab, rotrophin-4, oncostatin M (OSM), placenta growth factor, Futuximab, Galiximab, Ganitumab, Gantenerumab, Gavili placenta growth factor 2, platelet-derived endothelial cell momab, Gemtuzumab ozogamicin, Gevokizumab, GirentuX growth factor, platelet derived growth factor, platelet derived imab, Glembatumumab vedotin, Golimumab, Gomiliximab, growth factor A chain, platelet derived growth factor AA, GS6624, Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Igo platelet derived growth factor AB, platelet derived growth Vomab, Imciromab, ImgatuZumab, Inclacumab, Indatuximab factor B chain, platelet derived growth factor BB, platelet ravtansine, Infliximab, Intetumumab, Inolimomab, Inotu derived growth factor receptor alpha., platelet derived Zumab ozogamicin, Ipilimumab, Iratumumab, Itolizumab, growth factor receptor.beta., pre-B cell growth stimulating Ixekizumab, Keliximab, Labetuzumab, Lebrikizumab, factor, stem cell factor (SCF), stem cell factor receptor, TNF, Lemalesomab, Lerdelimumab, Lexatumumab, Libivirumab, TNFO, TNF1, TNF2, transforming growth factor C. hymic Ligelizumab, LintuZumab, Lirilumab, LorVotuZumab mer stromal lymphopoietin (TSLP), tumor necrosis factor recep tansine, Lucatumumab, Lumiliximab, Mapatumumab, tor type I, tumor necrosis factor receptor type II, urokinase Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, type plasminogen activator receptor, phospholipase-activat Metelimumab, Milatuzumab, Minretumomab, Mitumomab, ing protein (PUP), insulin, lectin ricin, prolactin, chorionic Mogamulizumab, Morolimumab, Motavizumab, Moxetu gonadotropin, follicle-stimulating hormone, thyroid-stimu momab pasudotox, Muromonab-CD3, Nacolomab tafenatox. Namilumab, Naptumomab estafenatox. Narnatumab, Natali lating hormone, tissue plasminogen activator (tPA), leptin, Zumab, Nebacumab, Necitumumab, Nerelimomab, Nesvacu Enbrel (etanercept). mab, Nimotuzumab, Nivolumab, Nofetumomab merpentan, 0146 In some embodiments, the protein is an antibody. As Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, used herein, an “antibody' includes, without limitation, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, whole antibodies and any antigen binding fragment, includ Oportuzumab monatox, Oregovomab, Orticumab, Otelixi ing without limitation a Fab, or a single chain thereof. Thus Zumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaxi the term “antibody' includes, without limitation, any protein mab, Palivizumab, Panitumumab, Panobacumab, Parsatu or peptide containing molecule that comprises at least a por Zumab, Pascolizumab, Pateclizumab, Patritumab, tion of an immunoglobulin molecule having biological activ Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pid ity of binding to the antigen. Examples of such may comprise ilizumab, Pintumomab, Placulumab, PoneZumab, Prilix a complementarity determining region (CDR) of a heavy or imab, Pritumumab, PRO140, Quilizumab, Racotumomab, light chain or a ligand binding portion thereof, a heavy chain Radretumab, Rafivirumab, Ramucirumab, Ranibizumab, or light chain variable region, a heavy chain or light chain Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, constant region, a framework (FR) region, or any portion Rituximab, Robatumumab, Roledumab, Romosozumab, US 2016/01 84445 A1 Jun. 30, 2016

Rontalizumab, Rovelizumab, Ruplizumab, Samalizumab, 0153. In preferred embodiments of the present invention, Sarilumab, Satumomab pendetide, Secukinumab, the butyrylcholinesterase enzymes have one or more dele Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Simtu tions and or amino acid substitutions. Preferably, the cysteine Zumab, Siplizumab, Sirukumab, Solanezumab, Solitomab, residue at position 66 (starting from the N-terminus of the Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, mature is changed to some other natural or unnatural amino Suvizumab, Tabalumab, Tacatuzumab tetraxetan, Tadoci acid. More preferably, C66 is converted to tyrosine or isoleu Zumab, Talizumab, Tanezumab, Taplitumomab paptoX, cine. Most preferably, C66 is converted to tyrosine (C66Y). Tefibazumab, Telimomab aritox, Tenatumomab, Tefiba 0154) In accordance with an aspect of the present inven Zumab, Telimomab aritox, Tenatumomab, Teneliximab, tion, C-terminal deletions of rhBuChE are presented. It is Teplizumab, Teprotumumab, TGN1412, tremelimumab, preferred that nor more than 50 amino acids are deleted from Ticilimumab, Tildrakizumab, Tigatuzumab, TNX-650, the C-terminus of rhBuChE. More preferably, 50amino acids Tocilizumab, Toralizumab, Tositumomab, Tralokinumab, are deleted from the C-terminus of rhBuChE resulting in the Trastuzumab, TRBS07, Tregalizumab, Tremelimumab, construct rhBuChE524 (referring to the 524 N-terminal Tucotuzumab celmoleukin, Tuvirumab, Ublituximab, Ure retained amino acids). More preferably, 40 amino acids are lumab, Urtoxazumab, Ustekinumab, Vapaliximab, Vatel deleted from the C-terminus of rhBuChE resulting in the iZumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesen construct rhBuChE534. Preferably, the deletion mutations cumab, Visilizumab, Volocliximab, Vorsetuzumab mafodotin, have one of more of the C66 mutations described above. More Votumumab, Zalutumumab, Zanolimumab, Zatuximab, preferably, the deletion mutations have the C66Y mutation. In Ziralimumab and Zolimomab aritox. For each of the above, a most preferred aspect of the present invention, the protein the present invention contemplates use of a Fab as well as the sequence of the 524 deletion with the C66Y mutation (rh full antibody. BChE524 (C66Y)) is set forth in SEQID NO. 11. In another most preferred aspect of the present invention, the protein III. BioScavenger Conjugates and Constructs sequence of the 534 deletion with the C66Y mutation (rh 0148 BuOhE isolated from human serum is a globular, BChE534 (C66Y)) is set forth in SEQID NO. 10. tetrameric molecule with a molecular weight of 340 kDa. 0.155. In accordance with an aspect of the instant inven Haupt H, Heide K. Zwisler O and Schwick H. G. 1966. Iso tion, a polymer may be conjugated to a BuOhE. Preferably, lierun and Physikalischchemiscle Charakterisierung der polymers are conjugated using maleimide to a Cys residue. Cholinesteraseaus Humanserum. Blut. 14:65-75. Each of the The Cys residue may be a maturally courring cysteine. In four subunits of the enzyme are 574 amino acids in length. other preferred embodiments of the present invention, a cys Each chain has nine ASn-linked carbohydrate chains. Lock teine residue can be added to one of the rhBuChE C-terminal ridge O. Bartels CF, Vaughan TA, Wong CK, Norton SE, deletions so that the protein can be conjugated to a polymer. Johnson L. L. 1987. Complete amino acid sequence of human Preferably, the cysteine is added to the C-terminus of the Serum cholinesterase. J Biol Chem. 262:549-557. deleted rhBuChE. In still more preferred embodiments, the 0149 Various attempts have been made to produce a cysteine is added via a GGC or a GGGC (SEQ ID NO:20) recombinant version of BuChE. However, the dominant tet peptide to the C-terminus. In one of the more preferred ramer form observed in plasma turns out to be just a minor embodiments, the peptide GGGC (SEQID NO: 20) is added component of the protein produced by recombinant DNA to the C-terminal end of the rhBuChE524 (C66Y) enzyme technology. For example, expression of rhBuChE in Chinese (see SEQID NO.12). In another more preferred embodiment, hamster ovary (CHO) cells produces a mixture of proteins: the peptide GGC is added to the C-terminus of the dimers (not stable) (50-55%), monomers (15-40%) and a rhBuChE534 (C66Y) (see SEQID NO. 13). relatively small percentage of tetramers (10-30%). Blong R 0156. In accordance with an aspect of the present inven M. Bedows E. Lockridge O. 1997. Tetramerization domain of tion, abutyrylcholinesterase fusion protein having abutyryl human butyrylcholinesterase is at the C-terminus Biochem J. cholinesterase enzyme segment and a non-butyrylcholinest 327:747-757. erase protein segment is presented. In preferred aspects of the 0150. The C-terminus of hBuChE has been identified as present invention, the butyrylcholinesterase protein segment being responsible for tetramerization. Expression of C-termi is N-terminal to the non-butyrylcholinesterase segment. Pref nally deleted proteins results in exclusive production of erably, the non-butyrylcholinesterase protein is an immuno monomers. In addition, Blong et al. reported that up to 50 globulin (Ig) domain. More preferably the Ig domain is amino acids of the C-terminus can be removed without loss of selected from the group consisting of IgG-Fc, IgG-CH and enzyme activity (as noted above only the monomeric form is IgG-CL. Still more preferably, the Ig domain is from IgG1 detectable). However deletion of 51 amino from the C-termi (SEQID NO. 8). nus results in an inactive version of the protein which is 0157. In other preferred embodiments of the present retained in the cell (i.e. not secreted). invention, the IgG1 Fc sequence can be modified in various 0151. The 602 amino acid sequence of human butyrylcho ways, for example, to modulate complement binding and linesterase (P06276.1) is shown in SEQID NO. 1. The first 28 effector function. Optionally, the IgG1 Fc domain has one or amino acids (MHSKVTIICIRFLFWFLLLCMLIGKSHT more mutations to reduce effector function. Optionally the (SEQ ID NO: 27)) constitute the natural leader sequence of mutations are to one or more of the following amino acid hBuChE. The mature enzyme, thus, begins with the sequence positions (EU numbering): E233, L234, L235, G236, G237, EDDIII (SEQID NO: 28). A327, A330, and P331. Preferably in this regard, the IgG1 Fc 0152. In accordance with an aspect of the present inven has the following substitutions: L234A, L235E, G237A, tion, butyrylcholinesterase (BuChE) enzymes are presented. A330S and P331S. Alysine residue at the C-terminus of an Fc Preferably, the butyrylcholinesterase enzymes are human domain can sometimes be deleted by posttranslation process enzymes (hBuChE). More preferably, the enzymes are ing. Therefore, when a SEQID of an Fc domainterminates in recombinant human enzymes (rhBuChE). lysine, the lysine may or may not be omitted. US 2016/01 84445 A1 Jun. 30, 2016

0158. In accordance with an aspect of the present inven aspect of the present invention, cholinesterases, and the vari tion, modifications of the Fc region may be introduced to ous enzymes associated with catalytic OP bioscavengers, are allow for a site of polymer conjugation. For example, a cys produced using recombinant DNA technology. teine residue may be added which can be the site of polymer 0164. In accordance with certain aspects of the present conjugation. Preferably, the Fc domain has a mutation invention, production of cholinesterases (or the enzymes selected from the group consisting of Q347C and L443C. associated with catalytic OP bioscavengers) includes any More preferably, the mutation is Q347C. method known in the art for (i) the production of recombinant 0159. In accordance with an aspect of the present inven DNA by genetic engineering, (ii) introducing recombinant tion, the fusion protein preferably contains a polypeptide DNA into prokaryotic or eukaryotic cells by, for example and linker between the butyrylcholinesterase enzyme and the without limitation, transfection, electroporation or microin non-butyrylcholinesterase protein. Preferably, the linker is G, jection, (iii) cultivating said transformed cells, (iv) expressing GG, GGGGS (SEQID NO: 22), GGGS (SEQ ID NO. 23), protein, e.g. constitutively or on induction, and (v) isolating and GGGES (SEQID NO: 24), and oligomers of the forego the protein, e.g. from the culture medium or by harvesting the ing. More preferably, the linker is selected from the group transformed cells, in order to (vi) obtain purified protein. consisting of GGGSGGGSGGGS (SEQ ID NO: 21) and GGSGGGSGGGS (SEQID NO:25). 0.165. In preferred aspects of the present invention, protein 0160. In preferred embodiments of the present invention, is produced by expression in a Suitable prokaryotic or eukary the butyrylcholinesterase enzyme of the fusion protein is one otic host system characterized by producing a pharmacologi of the C-terminal truncates described above having the pre cally acceptable protein molecule. Examples of eukaryotic ferred C66Y mutation (rhBuChE524 (C66Y) or cells are mammalian cells, such as CHO, COS, HEK 293, rhBuChE534 (C66Y)) at the N-terminus of the fusion fol BHK, SK-Hip, and HepG2. lowed by a linker and a non-butyrylcholinesterase protein 0166 In still other aspects, a wide variety of vectors are sequence at the C-terminus. In more preferred aspects of the used for the preparation of proteins and are selected from present invention, the fusion protein is composed, starting at eukaryotic and prokaryotic expression vectors. Examples of the N-terminus, of the 524 or 534 C-terminal truncate vectors for prokaryotic expression include plasmids such as, described above, having the C66Y mutation, fused to the and without limitation, preset, pet, and pad, wherein the pro linker GGGSGGGSGGGS (SEQID NO: 21) followed by an moters used in prokaryotic expression vectors include one or IgG1 Fc sequence with the mutations: L234A, L235E, more of, and without limitation, lac, trc, tip, recA, or araBAD. G237A, A330S and P331S. In the most preferred embodi Examples of vectors for eukaryotic expression include: (i) for ments of the invention, the butyrylcholinesterase truncate of expression in yeast, vectors such as, and without limitation, the fusion protein is rhBuChE534 (C66Y). The protein pAO, pPIC, pYES, or pMET, using promoters such as, and sequence of this construct is set forth in SEQID NO. 15. In without limitation, AOX1, GAP, GAL1, or AUG1; (ii) for another highly preferred embodiment of the instant invention, expression in insect cells, vectors such as and without limi the Fc region of the fusion has either the Q347C or L443C tation, pMT, p Ac5, pIB, pMIB, or p3AC, using promoters mutations. Most preferably, the Fc region of the fusion has the such as and without limitation PH, p.10, MT. Ac5, OpIE2. Q347C mutation and L234A, L235E, G237A, A330S and gp64, or polh, and (iii) for expression in mammalian cells, P331S. The protein sequence of this preferred embodiment is vectors such as, and without limitation, pSVL, pCMV, pRc/ shown in SEQID NO. 2. RSV, pcDNA3, or pBPV, and vectors derived from, in one 0161 In accordance with an another aspect of the present aspect, viral systems such as and without limitation vaccinia invention, protein-polymer conjugates are presented having virus, adeno-associated viruses, herpes viruses, or retrovi an OP bioscavenger, either a catalytic OP bioscavenger or a ruses, using promoters such as and without limitation CMV, stoichiometric OP bioscavenger, covalently bound to a poly SV40, EF-1, UbC, RSV. ADV, BPV, and beta-actin. mer. Preferably, the polymer is a Zwitterionic polymer, the (0167 Assays for BuChE are know in the art BuChE effi polymer having one or more monomer units where at least ciently catalyzes the hydrolysis of acetylthiocholine one of the monomer units has a Zwitterion. Catalytic OP (ATCh)—a sulfur analog of the natural substrate of the bioscavengers are typically enzymes selected from the group enzyme. Other thiocholine Substrates can be used, including consisting of aryldialkylphosphatases, organophosphate S-butyrylthiocholine (BTCH) iodide. On hydrolysis, this hydrolases (OPH), carboxylesterases, triesterases, phospho Substrate analog produces acetate and thiocholine. Thiocho triesterases, arylesterases, paraoxonases (PON), organophos line, in the presence of the highly reactive dithiobisnitro phate acid anhydrases, and diisopropylfluorophosphatases benzoate (DTNB) ion, generates a yellow color, which is (DFPases). visible and can be quantitatively monitored by spectrophoto 0162. In an aspect of the present invention, the OP bios metric absorption at 405 nm. Such techniques are described in cavenger is a stoichiometric OPbioscavenger. Preferably, the the following, all of which are incorporated by reference: G. stoichiometric OP scavenger is a cholinesterase selected from L. Ellman, K. D. Courtney, V. Andres, and R. M. Feather the group consisting of acetylcholinesterase (AChE) and stone, "A new and rapid colorimetric determination of ace butyrylcholinesterase. Preferably, the cholinesterase is a tylcholinesterase activity.” Biochemical Pharmacology, Vol. BuChE. 7, no. 2, pp. 88-95, 1961; M.A. Gordon, D. E. Carpenter, H. 0163. In accordance with an aspect of the present inven W. Barrett, and I. B. Wilson, “Determination of the normality tion, cholinesterases, including butyrylcholinesterases, can of cholinesterase solutions.” Analytical Biochemistry, vol. be purified from blood serum according to known techniques. 85, no. 2, pp. 519–527, 1978; and V. Gorun, I. Proinov, V. Thus, if it is desired to prepare a conjugate in accordance with Baltescu, G. Balaban, and O. Barzu, “Modified Ellman pro an aspect of the present invention for administration to cedure for assay of cholinesterases in crude enzymatic prepa humans, human blood serum can be used as a source of the rations.” Analytical Biochemistry, vol. 86, no. 1, pp. 324-326, cholinesterase. Alternatively, in accordance with another 1978. US 2016/01 84445 A1 Jun. 30, 2016

0.168. In accordance with an aspect of the present inven reagent), resulting in conversion of an amine into a thiol. tion, the endogenous leader peptide of hBuChE can be used Reactive thiol groups may be introduced into a protein by for recombinant expression of the protein. More preferably, introducing one, two, three, four, or more cysteine residues however, a leader peptide should be chosen to maximize the (e.g., by preparing variant comprising one or more non-native levels of protein expression. In preferred embodiments, a cysteine amino acid residues). CTLA4 leader is used. Most preferably, the leader peptide set 0.174. In accordance with preferred aspects of the inven forth in SEQID NO. 18 is preferred. tion, the butyrylcholinesterase fusion corresponding to that of 0169. In accordance with preferred aspects of the present SEQID NO. 2 and the Cys residue for conjugation to polymer invention, the Zwitterionic polymer is formed of monomers is that at amino acid 672. having a phosphorylcholine group. Preferably the monomer 0.175. In accordance with an aspect of the present inven is 2-(acryloyloxyethyl)-2'-(trimethylammoniumethyl) phos tion, a method for treatment or prophylaxis of OP poisoning phate. More preferably, the monomer is 2-(methacryloyloxy is presented in which of the present invention conjugates or ethyl)-2'-(trimethylammoniumethyl) phosphate (HEMA fusion proteins are administered to a patient in need thereof in PC). an effective regime. With respect to prophylaxis, a subject 0170 A polymer conjugated to a bioscavenger preferably who will or may be exposed to OP compounds (either OP has at least 2 polymer arms more preferably 3 or more arms. pesticides or OP nerve agents) at a statistically significantly Some polymers have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 arms. higher frequency than a control population of randomly Still more preferably the polymer has 3, 6 or 9 arms. Most selected individuals from the general public would be in need preferably, the polymer has 9 arms. Preferably, the polymer of the instant invention. For example, a soldier or law enforce peak molecular weight is between 300,000 and 1,750,000 Da. ment office who might be exposed to OP nerve agents in a More preferably, the polymer has a peak molecular weight battlefield or criminal context may be in need of the instant between 500,000 and 1,000,000 Da. Still more preferably, the invention. Similarly, a worker involved in applying OP pes polymer has a peak molecular weight between 600,000 to ticides to crops may be in need of the instant inventions as 800,000 Da. Unless otherwise specified, molecular weights would a worker responsible for dealing with accidental leak of a branched polymer refer to the aggregate molecular age of pesticides or nerve agents. Although protective cloth weight of all arms. ing and gas masks might be available to prevent exposure, 0171 Nucleophilic groups on proteins, including antibod Such protective measures can fail to provide protection. In ies, which can be used to conjugate polymer in accordance case of Such failure, the instant prophylactic methods can be with an aspect of the present invention include, but are not applied. limited to: (i) N-terminal amine groups, (ii) side chain amine 0176). In preferred aspects of the instant invention, the groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, conjugate or fusion protein is administered by intravenous and (iv) Sugar hydroxyl or amino groups where the protein is administration, Subcutaneous administration, intramuscular glycosylated Amine, thiol, and hydroxyl groups are nucleo administration, intralesional administration or intradermal philic and capable of reacting to form covalent bonds with administration. Preferably, administered is by intramuscular electrophilic groups on linker moieties and linker reagents administration. attached to the polymer including: (i) active esters such as 0177. In another aspect of the present invention, a method NHS esters, HOBtesters, haloformates, and acid halides; (ii) for effecting a long term prophylaxis against OP poisoning is alkyl and benzyl halides such as haloacetamides; (iii) alde presented in which a conjugate having an OP bioscavenger hydes, ketones, carboxyl, and maleimide groups. Many pro selected from the group consisting of a stoichiometric OP teins, including antibodies, have cysteine thiol groups which bioscavenger and a catalytic OP bioscavenger covalently can potentially be used for conjugation. Preferably, cysteine bound to a Zwitterionic polymer wherein the polymer has one residues are added for conjugation by recombinant DNA or more monomerunits and wherein at least one monomer has technology. Many cysteine residues are in the form of reduc a Zwitterionic group is administered at least 7 days prior to ible interchain disulfides, i.e. cysteine bridges. Cysteine resi expected exposure to an OP compound. In other words, such dues in the form of disulfides are generally not available to a conjugate administered prophylactically is effective for at react with reagents such as maleimide. Cysteine residues may least seven days against possible exposure to OP poisoning. also be free or unpaired. However, free cysteine residues are 0178. In preferred aspects of the present invention, the frequently found to be “capped by one or more reagents in conjugate is administered at least 14 days prior to expected various media and are also not available for conjugation. exposure to the OP nerve agent. More preferably, the conju Cysteine residues may be made reactive for conjugation with gate is administered at least 21 days prior to expected expo linker reagents such as maleimide by treatment with a reduc sure to the OP nerve agent. Still more preferably, the conju ing agent such as DTT (dithiothreitol) or tricarbonyleth gate is administered at least 30 days prior to expected ylphosphine (TCEP), such that the protein is fully or partially exposure to the OP nerve agent. Or put another way, the reduced. Each cysteine bridge will thus form, theoretically, conjugate is effective in providing protecting for at least 14, two reactive thiol nucleophiles. In the case of free cysteine, 21 or 30 days against possible exposure to an OP. one thiol nucleophile is formed by reduction. 0179. In another aspect of the present invention, a method 0172 Depending on the conditions employed, reduction for effecting rapid prophylaxis against OP poisoning is pre by TCEP or DTT can result in the loss of proper protein sented in which a conjugate having an OP bioscavenger folding with concomitant loss of activity. However, activity selected from the group consisting of a stoichiometric OP may be recovered by allowing protein refolding under the bioscavenger and a catalytic OP bioscavenger covalently appropriate conditions. bound to a Zwitterionic polymer wherein the polymer has one 0173 Additional nucleophilic groups can be introduced or more monomerunits and wherein at least one monomer has into antibodies through modification of lysine residues, e.g., a Zwitterionic group is administered to a subject in need by reacting lysine residues with 2-iminothiolane (Traut’s thereof at least 12 hours prior to expected exposure to an OP US 2016/01 84445 A1 Jun. 30, 2016 compound. More preferably, the conjugate is administered at positions of the invention may be employed in combination least 24 hours prior to expected exposure to the OP com with pharmaceutically acceptable diluents, adjuvants, or car pound. In other words, the conjugate remains effective in riers. Such excipients may include, but are not limited to, providing protection for at least 12 or 24hr. saline, buffered saline (such as phosphate buffered saline), 0180. The pharmaceutical composition of the invention dextrose, liposomes, water, glycerol, ethanol and combina may further comprise a pharmaceutically acceptable diluent, tions thereof. adjuvant or carrier. 0185. The pharmaceutical compositions may be adminis 0181 Pharmaceutical compositions adapted for oral tered in any effective, convenient manner effective for treat administration may be presented as discrete units such as ing a patient’s disease including, for instance, administration capsules, as solutions, syrups or Suspensions (in aqueous or by oral, intravenous, Subcutaneous, intramuscular, non-aqueous liquids; or as edible foams or whips; or as emul intraosseous, intranasal routes, among others. In therapy or as sions). Suitable excipients fortablets or hard gelatin capsules a prophylactic, the active agent may be administered to an include lactose, maize starch or derivatives thereof, Stearic individual as an injectable composition, for example as a acid or salts thereof. Suitable excipients for use with soft sterile aqueous dispersion, preferably isotonic. gelatin capsules include for example vegetable oils, waxes, fats, semi-solid, or liquid polyols, etc. For the preparation of 0186 For administration to mammals, and particularly Solutions and syrups, excipients which may be used include humans, it is expected that the daily dosage of the active agent for example water, polyols and Sugars. For the preparation of will be from 0.01 mg/kg body weight, typically around 1 Suspensions oils (e.g. vegetable oils) may be used to provide mg/kg. The physician in any event will determine the actual dosage which will be most suitable for an individual which oil-in-water or water in oil Suspensions. will be dependent on factors including the age, weight, sex 0182 Pharmaceutical compositions adapted for nasal administration wherein the carrier is a solid include a coarse and response of the individual. The above dosages are exem powder having a particle size for example in the range 20 to plary of the average case. There can, of course, be instances 500 microns which is administered in the manner in which where higher or lower dosages are merited, and Such are Snuff is taken, i.e. by rapid inhalation through the nasal pas within the scope of this invention. sage from a container of the powder held close up to the nose. 0187 Dosages of the substance of the present invention Suitable compositions wherein the carrier is a liquid, for can vary between wide limits, depending on the disease or administration as a nasal spray or as nasal drops, include disorder to be treated, the age and condition of the individual aqueous or oil solutions of the active ingredient. Pharmaceu to be treated, and so forth, and a physician will ultimately tical compositions adapted for administration by inhalation determine appropriate dosages. include fine particle dusts or mists which may be generated by 0188 This dosage may be repeated as often as appropriate. means of various types of metered dose pressurized aerosols, If side effects develop, the amount and/or frequency of the nebulizers or insufflators. dosage can be reduced, in accordance with normal clinical 0183 Pharmaceutical compositions adapted for practice. In one embodiment, the pharmaceutical composi parenteral administration include aqueous and non-aqueous tion may be administered once every one to thirty days. sterile injection Solutions which may contain anti-oxidants, 0189 The pharmaceutical compositions of the invention buffers, bacteriostats and solutes which render the formula may be employed alone or in conjunction with other com tion substantially isotonic with the blood of the intended pounds, such as therapeutic compounds or molecules, e.g. recipient; and aqueous and non-aqueous sterile Suspensions carbamates, anti-muscarinics, cholinesterase reactivators and which may include Suspending agents and thickening agents. anti-convulsives. Such administration with other compounds Excipients which may be used for injectable solutions include may be simultaneous, separate or sequential. The compo water, alcohols, polyols, glycerin and vegetable oils, for nents may be prepared in the form of a kit which may com example. The compositions may be presented in unit-dose or prise instructions as appropriate. multi-dose containers, for example sealed ampoules and 0.190 Preferably, the pharmaceutical compositions of the vials, and may be stored in a freeze-dried (lyophilized) con invention and the other therapeutic compounds are directly dition requiring only the addition of the sterile liquid carried, (as opposed to what?) administered to a patient in need for example water for injections, immediately prior to use. thereof. Extemporaneous injection Solutions and Suspensions may be 0191 The invention also provides a kit of parts comprising prepared from sterile powders, granules and tablets. Pharma a pharmaceutical composition of invention, and an adminis ceutical compositions can be substantially isotonic, implying tration vehicle including, but not limited to, capsules for oral an osmolality of about 250-350 mOsm/kg water. administration, inhalers for lung administration, and inject 0184 In general, the pharmaceutical compositions may able solutions for parenteral administration. contain preserving agents, solubilizing agents, stabilizing 0.192 As used herein, the term “treatment includes any agents, wetting agents, emulsifiers, Sweeteners, colorants, regime that can benefit a human or a non-human animal. The odorants, salts (Substances of the present invention may them treatment of “non-human animals' extends to the treatment selves be provided in the form of a pharmaceutically accept of domestic animals, including horses and companion ani able salt), buffers, coating agents or antioxidants. They may mals (e.g. cats and dogs) and farm/agricultural animals also contain therapeutically active agents in addition to the including members of the ovine, canine, porcine, bovine and Substance of the present invention. The pharmaceutical com equine families. The treatment may be in respect of any US 2016/01 84445 A1 Jun. 30, 2016 20 existing condition or disorder, or may be prophylactic (pre wherein R8 and R9 are the same or different and are selected ventive treatment). The treatment may be of an inherited oran from the group consisting of a bond, H, aryló-12, and alkyll acquired disease. The treatment may be of an acute or chronic 6; condition. 0193 In accordance with an aspect of the present inven tion, a method is presented of preparing a conjugate having the step of contacting a polymeric reagent with a biologically active agent comprising a free Sulfhydryl residue under Suit able conditions to thereby provide the conjugate, wherein the polymeric reagent has the following structure:

wherein R10, R11, R12, R13 and R14 are the same or differ ent and are selected from the group consisting of a bond, H. amino, aryl6-12 and alky11-6 and one or more arginine resi dues. 0194 Preferably, the biologically active agent is selected from the group consisting of a therapeutic protein and an aptamer. More preferably, the biologically active agent is a therapeutic protein. The therapeutic protein is preferably a wherein P is a branched or unbranched water soluble poly mer; L1 is a first linker, L2 is a second linker and L3 is a third cytokine, an enzyme, an antibody and an antibody fragment. linker, wherein L1, L2 and L3 are the same or different and are Still more preferably, the therapeutic protein is a Fab. Most each selected from the group consisting of C1-C12 alkyl-, preferably, the Fab is IgG1. —C3-C12 cycloalkyl-, (-(CH2)1-6-O-(CH2)1-6-)1-12-, 0.195. Where the biologically active agent is a therapeutic protein, the free sulfhydryl group is preferably contributed by a cysteine residue of the protein. In some aspects of the present invention, the cysteine residue is a naturally occurring cysteine residue. Alternatively, the cysteine residue is intro O-(CH2)1-4)1-12-O-(CH2)1-12-, -(CH2)1-12-(CO)– O-(CH2)1-12-, -(CH2)1-12-(CO) NH-(CH2)1-12-, duced via recombinant DNA technology. -(CH2)1-12-O-(CO)-(CH2)1-12-, -(CH2)1-12-NH (0196. P is preferably poly(alkylene oxide), poly(MPC), (CO) (CH2)1-12-, -(C3-C12 cycloalkyl)-, —(C1 poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline C8alkyl)-(C3-C12 cycloalkyl)-, —(C3-C12 cycloalkyl)-(C1 or poly(N-acryloylmorpholine). More preferably, P is poly 8alkyl)-, —(C1-8alkyl)-(C3-C12 cycloalkyl)-(C1-8alkyl)- (alkylene oxide). Still more preferably, P is poly(ethylene NH-(CH2CH2-O-)0-20-NH-(CH2CH2-O-)0-20- and oxide) more commonly known as polyethylene glycol or —(CH2)0-3-aryl-(CH2)0-3-, a bond and any combination of PEG. Where P is PEG, the PEG is preferably branched. Pref. the above; X is selected from the group consisting of F, C1, Br erably, the branched PEG has two arms. Each arm is prefer and I; R1 and R2 are the same or different and are selected ably between about 5 to about 40 kDa. More preferably, each from the group consisting of H, provided that not both R1 and R2 are H, and one or more basic groups wherein a basic group arm has a molecular weight of PEG of about 20 kDa. is selected from the group consisting of 0.197 With respect to the water-soluble polymer, the poly meric reagents of the invention also comprise at least one water-soluble polymer segment. Water-soluble polymers that R6 are nonpeptidic and water-soluble, with from 2 to about 2000 A monomer units, are particularly useful in the invention. Examples of suitable water-soluble polymers include, but are R7-N - N1it not limited to, poly(alkylene glycols). Such as poly(ethylene M V glycol) (“PEG'), copolymers of ethylene glycol and propy R5 R4 lene glycol having water-solubility, poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), wherein R3, R4, R5, R6 and R7 are the same or different and poly(hydroxyalkylmethacrylate), poly(saccharides), poly(C- are selected from the group consisting of a bond, H, amino, hydroxy acid), poly(vinyl alcohol), polyphosphaZene, poly aryló-12 and alky11-6: oxazoline, poly(MPC) and poly(N-acryloylmorpholine), such as described in U.S. Pat. No. 5,629,384, incorporated herein by reference. In some applications where relatively high water solubility is desired, the water-soluble polymer is not poly(propylene oxide). 0.198. In another aspect of the present invention, P is poly (MPC). Poly(MPC) preferably has 2,3,4,5,6,7,8,9, 10, 11 or 12 arms. More preferably, polyMPC has 3, 6 or 9 arms. Most preferably, the poly(MPC) has 9 arms. US 2016/01 84445 A1 Jun. 30, 2016

0199 According to an aspect of the present invention, poly(MPC) preferably has a peak molecular weight of between 300,000 and 1,750,000 daltons, more preferably between 500,000 and 1,000,000 daltons and still more pref erably between 600,000 to 800,000 daltons. 0200. According to a preferred aspect of the present inven tion, L3 is a bond, R2 is Hand R1 is wherein R6, R7, R8, R9 and R10 are the same or different and are selected from the group consisting of a bond, H, amino, NH aryló-12 and alky11-6:

-N ls NH. H 2

0201 Preferably, L2 is-(CH2)1-6-. More preferably, L2 is —(CH2)3-. wherein R11 and R12 are the same or different and are 0202 In another aspect of the present invention, an initia selected from the group consisting of tor for polymer synthesis is presented having the following a bond, H, aryló-12 and alky11-6: Structure: R14 R15

N N

R3 --- R13 1. YR16 R -1 NH R17 R5 / wherein R13, R14, R15, R16 and R17 are the same or differ ent and are selected from the group consisting of a bond, H. amino, aryl6-12 and alky11-6 and one or more arginine resi dues; and R3, R4 and R5 are the same or different and are selected from the group consisting of wherein L1 is a first linker, L2 is a second linker and L3 is a third linker, wherein L1, L2 and L3 are the same or different and are each selected from the group consisting of C1-C12 Y alkyl-, - C3-C12 cycloalkyl-, (-(CH2)1-6-O-(CH2)1-6-) 1-12-, (-(CH2)1-4-NH-(CH2)1-4(1-12-, -(CH2)1- N-1 CH3, 12O (—(CH2)1-4-O-(CH2)1-4)1-12-O-, -(CH2)1- CH 12-(CO)-O-, -(CH2)1-12-(CO) NH ,-(CH2)1-12 O O-(CO) , -(CH2)1-12-NH (CO)-, (-(CH2)1-4- O O O-(CH2)1-4)1-12-O-(CH2)1-12-, -(CH2)1-12-(CO)– CH O-(CH2)1-12-, -(CH2)1-12-(CO) NH-(CH2)1-12-, 1N N O Y -(CH2)1-12-O-(CO)-(CH2)1-12-, -(CH2)1-12-NH H CH3 and (CO) (CH2)1-12-, -(C3-C12 cycloalkyl)-, —(C1 CH3 C8alkyl)-(C3-C12 cycloalkyl)-, —(C3-C12 cycloalkyl)-(C1 8alkyl)-, —(C1-8alkyl)-(C3-C12 cycloalkyl)-(C1-8alkyl)- O NH-(CH2CH2-O-)1-20-NH-(CH2CH2-O-)1-20- and —(CH2)0-3-aryl-(CH2)0-3-, a bond and any combination of O the above; R1 and R2 are the same or different and are HC selected from the group consisting of H, provided that not HC Y both R1 and R2 are H, and one or more basic groups wherein CH3 a basic group is selected from the group consisting of H3C Y O O R17 O O O CH

N 1n N --> N --- O Y 1N1N1 YR18, H H O HCCH

O Y CH wherein R17 and R18 are C1-6 alkyl, preferably wherein R17 and R18 are both methyl, wherein Y is NCS, F, Cl, Br or I. US 2016/01 84445 A1 Jun. 30, 2016 22

0203 Preferably, Y is Br. R3, R4 and R5 are preferably protein sequence of rhBuChE524GGGC (C66Y) is shown in each SEQID NO. 12 (“GGGC disclosed as SEQID NO: 20). 0209 Another bioscavenger was made by truncating hBuChE at position 534 (referring to the mature protein). The Y sequence of rhBuChE534 (C66Y) is shown in SEQ ID NO. O 10. For conjugation to a polymer using cysteine, GGC is N-1 CH3. optionally added to the end. The protein sequence of CH3 O rhBuChE534GGC (C66Y) is SEQID NO. 13. Example 2 0204 More preferably, R3, R4 and R5 are each rBuChE Fusion Proteins

O O 0210 A rhBuChE-Fc fusion protein was made by recom CH3 binant genetic engineering by using the 534 truncate Y 1N O described above and fusing it to the Fc region of IgG1 with a H CH3. GS linker (GGGSGGGSGGGS) (SEQID NO:21) in between CH3 the two protein segments. The sequence rhBuChE534-Fc is O shown in SEQID NO. 16. The fusion construct with the C66Y mutation (rhBuChE534 (C66Y)-Fc) is shown in SEQID NO. O 17. In another version, various effector function mutations are introduced: L234A, L235E, G237A, A330S and P331S. The HC hBuChE534 (C66Y)-Fc(L234A, L235E, G237A, A330S and HC Y P331S) fusion is shown in SEQID NO. 15. Another mutation which can be engineered into the Fc portion of the protein is 0205 Still more preferably, R4, R5 and R6 are each Q347C which can be used to conjugate polymers such as POLY(MPC) to the protein. The protein sequence of rhBuChE534 (C66Y)-Fc(L234A L235E, G237A, A330S, H3C CH P331S and Q347C) is shown in SEQID NO. 2. 0211. The various protein constructs were expressed in Y CHO-K1 cells using appropriate DNA constructs for eukary O O otic gene expression. While the endogenous hBuChE leader sequence can be used for recombinant expression of the con -----O O O OO 14 Y. structs (see SEQ ID NO. 1), a CTLA4 leader was preferred H H O H3C CH3 the sequence of which is shown in SEQID NO. 18. The DNA sequence used to express rhBuChE534 (C66Y)-Fc(L234A, L235E, G237A, A330S, P331S and Q347C) is set forth in O Y CH SEQID NO. 9. 0206. In an aspect of the present invention, preferably L3 Example 3 is a bond, R2 is H and R1 is Transient Expression of Constructs 0212. A transient expression analysis was performed in NH Expi293 Expression System (Gibco). The expression of a number of rhBuChE524 truncates was examined. In general, -N l NH. the rhBuChE524 constructs either were not expressed or were H 2 not expressed as well as the corresponding rhBuChE534 con structs. With regard to the rhBuChE534 constructs, it was 0207 L2 is preferably -(CH2)1-6-. More preferably, L2 found that the C66Y constructs were in generally better is —(CH2)3-. expressed than constructs without this change A Summary of transient expression data for the 534 constructs is shown in IV. Examples the table below:

Example 1 TABLE 1 Ellman's Assay test rBuChE Truncations Construct (units/mL) 0208. An OP bioscavenger was made by truncating the rhBuChE534 15 hBuChE enzyme at position 524 (referring to the mature rhBCES34GGC 7.5 protein) (rhBuChE524). In addition, the mutation C66Y was rhBuChE534GGC (C66Y) 32 rhBuChE534 (C66Y) - Fic (L234A, 27 introduced. The protein sequence of rhBuChE524 (C66Y) is L235E, G237A, A330S, P331S and shown in SEQ ID NO. 10. If it is desirable to conjugate a Q347C) polymer to the rhBuChE524 (C66Y) a GGC or GGGC sequence (SEQID NO: 20) can be added on to the end. The US 2016/01 84445 A1 Jun. 30, 2016

Example 4 Example 5 Stable Transfectants Conjugation of BuChE-Fc Fusion Protein to MPC Polymer 0213 Based on the results of the transient expression analysis, stable transformants were made of the 0214 Human BuChE-Fc fusion protein according to SEQ rhBuChE534GGC (C66Y) and rhBuChE534 (C66Y)-Fc ID NO. 2 was denatured (as set forth in Example 6 below) (L234A, L235E, G237A, A330S, P331S and Q347C). The with TCEP to provide a free cysteine thiol at position 347 of stable transfectant for the Fc fusion employs the CTLA4 the Fc chain, followed by limited renaturation. TCEP treated leader which is shown in SEQID NO. 18 fused at the N-ter BuChE-Fc was then conjugated to a 750 kDa polymer using minus of the protein shown in SEQ ID NO. 2. The DNA a 15 to 50 fold excess of polymer to protein to produce a sequence for this construct is set forth in SEQID NO. 9. conjugate as shown below.

O p- H3C MPC O O O ^c

Example 6 Decapping and Refolding of BuOhE-Fc 0215. The BuChE-Fc fusion was reduced at room tem perature with 30 fold molar excess of TCEP for several hours at room temperature in the presence of EDTA. Following this reduction, the protein was subjected to a step of TCEP wash ing in UF/DF to reduce the TCEP concentration to about an 8 fold molar excess. Further UF/DF and dilution was per formed to reduce TCEP to less than 1 M. Reduced BuChE Fc was then allowed to renature and the progress of renatur ation (seen by disappearance of monomeric BuChE-Fc and the appearance of dimeric BuChE-Fc) was followed by SDS PAGE. Following renaturation, conjugation of the protein to polymer was performed as described above. US 2016/01 84445 A1 Jun. 30, 2016 24

Example 7 Synthesis of Polymer R5743 0216) Polymer R5743, shown below, was synthesized as follows:

MPC

O MPC CH3 CH3 O O MPC HC HC

MPC US 2016/01 84445 A1 Jun. 30, 2016 25

0217. A TFA/amine salt initiator (Compound L) having the structure below was synthesized as follows.

Compound L.

Br H3C O CH 3 CH Br O H3C O O O O O CH3 Br H3C O HN Br

H3C O H3C O O O Br HN O CH3 CH3 O NH NH O

4 HN r O O TFA O

O O Br H3C H3C

NH

O HC O H3C CH3 O O O O Br CH3 O O CH3 CH Br CH3 US 2016/01 84445 A1 Jun. 30, 2016 26

0218 First, Compound K, having the following structure, was synthesized:

Compound K

Br HC O CH 3 CH Br O H3C O O O O O CH3 Br H3C O HN Br

H3C O H3C O O O Br HN CH CH3 O O 3 HC O CH O NH NH

H3C O NH 4 r O O O

O O Br H3C H3C

NH

O HC

O Ot CH3 O O O Br CH3 O O CH3 Dr. Br CH3 US 2016/01 84445 A1 Jun. 30, 2016 27

0219 Into a 200 mL round bottom flask under nitrogen was placed Compound J (1.9 g, 2.67 mmol. 3.3 equiv)

Compound J HC HC Br O O

O O HO--> O NH ---O -- skCH3 CHBr O O H3C O H3C Br and Compound E (0.525 g, 0.81 mmol. 1.0 equiv) (see below)

Compound E CH vis 3 HBir

followed by dimethylformamide (10 mL) then diisopropyl 37 (m, 8H, CH2), 3.47-3.55 (m, 12H, CH2), 3.58 (s, 6H, ethylamine (2.5 mL, 14.6 mmol. 18 equiv). The flask was OCH2C), 3.87 (s, 6H, d at room temperature for 30 minutes. cooled to 0°C. using an ice bath. To this was added propy The reaction was concentrated under a vacuum. The reaction lphosphonic anhydride solution (50 wt.% in ethyl acetate, 2.5 was diluted using dichloromethane (10 mL) and concentrated mL, 4.04 mmol. 5 equiv) over ~6 minutes. under a vacuum. The residue was dissolved using acetonitrile (10 mL), filtered through a syringe filter (Acrodisc CR25, PN 0220. The reaction was warmed to room temperature and 4225T) and loaded onto a preparatory HPLC column and stirred for 15 minutes. The reaction was quenched by adding eluted with 60% acetonitrile in water (with 0.1% trifluoro water (20 mL), Saturated aqueous sodium bicarbonate (20 acetic acid) up to 98% acetonitrile (with 0.1% trifluoroacetic mL), and ethyl acetate (100 mL). The organic layer was acid). The tubes containing product were pooled, concen separated and the aqueous layer extracted with ethyl acetate trated under vacuum, frozen and placed on a lyophilizer. This (75 mL). The combined organic layers were washed with resulted in 990 mgs (0.4 mmol. 50% over 2 steps) Compound saturated aqueous sodium bicarbonate (30 mL), 0.5 M aque L as a white powder. ous citric acid (40 mL), water (25 mL), and saturated aqueous 0222 1H NMR (400 MHz DMSO-d6): 8=1.90 (s, 54H, sodium chloride (40 mL), then dried (sodium sulfate), filtered CC(CH3)2Br), 2.31 (t, J–72 Hz, 6H, CCH2CH2NH), 2.97 and concentrated under vacuum. The residue which was used 3.0 (m, 8H, CCH2NH and OCH2CH2NH), 3.17 (s. 2H, without further purification resulted in 2.0 g (0.80 mmol. OCH2C), 3.3 (q, 6H, CH2CH2NHC=O), 3.4-3.59 (m, 20H, 99%) of Compound K. CH2), 3.87 (s, 6H, O—CCH2O), 4.27 (s, 18H, 0221 1H NMR (400 MHz DMSO-d6): 8=1.36 (s, 9H, CCH2OC=O), 7.69-7.84 (m, 9H, both CH2NHC=O and OCCH3), 1.90 (s.54H, CC(CH3)2Br), 2.31 (t, J=7.2 Hz, 6H, NH3+). CCH2CH2NH), 2.98 (d. J=5.6 Hz, 6H, CCH2NH), 3.04 (q, 0223 LC-MS (ES, m/z): (M+2H)/2+ Calcd for J=6.0 Hz, 2H, OCH2CH2NH), 3.18 (s. 2H, OCH2C), 3.3-3. (C84H136Br9N7O33+2H)/2=1196.6. Found 11974. US 2016/01 84445 A1 Jun. 30, 2016 28

0224 ATFA/amine salt initiator (Compound P) having the structure below was synthesized as follows:

Compound P Br HC O CH 3 CH Br O H3C O O O O O CH3 Br HC O HN Br

H3C O HC O TFA O O Br HN O O CH CH HN O NH NH O

O 2 NH 4 ^- O O O

O Br O HC HC

NH

H3C

O H3 O O O O Br CH O O CH Br C.CH H3

0225 Into a 20 mL vial was placed Compound L (430 mg. bonate and ethyl acetate. The organic layer was separated and 0.172 mmol. 1.0 equiv) (see above) and alpha-t-Butyloxycar the aqueous layer extracted with ethyl acetate. The combined bonylamino-omega-carboxy dodecadethylene glycol) (154 organic layers were washed with Saturated aqueous sodium mg, 0.215 mmol. 1.25 equiv) followed by N.N-dimethylfor bicarbonate, 0.5 M aqueous citric acid, water, and Saturated mamide (2 mL) then N,N-diisopropylethylamine (0.18 mL, aqueous Sodium chloride, thendried (sodium Sulfate), filtered 1.03 mmol. 6 equiv). The flask was cooled to 0°C. using an ice bath. To this was added propylphosphonic anhydride solu and concentrated under vacuum. The residue which was used tion (50 wt.% in ethyl acetate, 0.215 mL, 0.343 mmol. 2 without further purification resulted in 0.6 g (0.194 mmol) of equiv) over 1 minute. The reaction was warmed to room Compound N. shown below. temperature and stirred for 30 minutes. The reaction was 0226 LC-MS (ES, m/z): (M+2H-boc)/2+ Calcd for quenched by adding water, Saturated aqueous sodium bicar (C111H189Br9N8O46+2H-Boc)/2=1496.3. Found 1497.2. US 2016/01 84445 A1 Jun. 30, 2016 29

Compound N

Br HC CH CH3 O 3 Br O H3C O O O O

O St.Br HC O HN Br

HC O H3C O O O Br HN O O CH CH HC O NH- O hus NH -N- NH Nis?. DrH3C S1O 2 4 Sr-"O S,

O ityH3C HC

NH 1. H3C O H3C CH3 O O O O Br CH O. O

CH Br Dr.CH3

0227 Into a 100 mL round bottom under nitrogen was CH2CH2C=O), 3.0 (m, 8H, CCH2NH and OCH2CH2NH), added Compound N (0.6 g), dichloromethane (4 mL) fol 3.1-3.6 (m,84H, OCH2C), 3.87 (s, 6H, O—CCH2O), 4.27 (s, lowed by trifluoroacetic acid (3 mL). The reaction stirred at 18H, CCH2OC=O), 7.6-7.8 (m, 10H, both CH2NHC–O room temperature for 15 minutes. The reaction was concen and NH3+). trated under a vacuum. The residue was dissolved using 0229. LC-MS (ES, m/z): (M+2H)/2+ Calcd for acetonitrile (3 mL), filtered through a syringe filter (Acrodisc (C106H181Br9N8O44+2H)/2=1496.3. Found 1496.6. CR25, PN 4225T) and loaded onto a preparatory HPLC col umn and eluted with 50% acetonitrile (with 0.1% trifluoro Example 8 acetic acid) in 50% water (with 0.1% trifluoroacetic acid) up to 90% acetonitrile (with 0.1% trifluoroacetic acid). The tubes containing product were pooled, concentrated under Preparation of Zwitterionic Polymers vacuum, frozen and placed on a lyophilizer. This resulted in 200 mgs (0.064 mmol, 37% over 2 steps) Compound P. 0230. Initiator is typically prepared as a stock solution in 0228 1H NMR (400 MHz DMSO-d6): 8=1.90 (s, 54H, DMF of about 100 mg/mL. The initiator and the ligand (2,2'- CC(CH3)2Br), 2.3 (br t, 8H, CCH2CH2NH and bipyridyl) were introduced into a Schlenk tube. The resultant US 2016/01 84445 A1 Jun. 30, 2016 30

Solution was cooled to -78°C. using a dry ice/acetone mix- TABLE 2-continued ture, and was degassed under vacuum for 10 min. The tube was refilled under Argon and the catalyst (CuBrunless oth- Theor. erwise indicated), kept under Argon, was introduced into the MW Polymer ID Initiator Mn Mp Schlenck tube (the Molar ratio of atom bromine on the ini- (kDa) No. (see Table 3) (kDa) (kDa) PDI tiator/catalyst (CuBr)/ligand was kept at 1/1/2). The solution became dark brown immediately. The Schlenk tube was 250 8O B5 117 137 1.1 sealed and immediately purged by applying a short cycle wR3350 vacuum/Argon. A solution of HEMA-PC was prepared by dR3324 mixing a defined quantity of monomer, prepared in a glove 250 90 B6 235 258 1.1 box kept under nitrogen, with 200 proof degassed ethanol. wR3373 The monomer solution was added drop wise into the Schlenk dR3372 tube (via canula). The temperature was maintained at -78°C. 250 1OO B 242 259 1.1 A thorough vacuum was applied to the reaction mixture for at wR346OI) least 10 to 15 min until bubbling from the solution ceased. wR3461M) The tube was then refilled with Argon and warmed to room dR3418 temperature. The solution was stirred, and as the polymeriza- 250 110 F1 245 270 1.1 tion proceeded, the solution became viscous. After 3 to 8 wR3482I hours or overnight, the reaction was quenched by direct expo- wR3483M sure to air in order to oxidize Cu (I) to Cu (II), the mixture R3463 became blue-green in color, and was passed through a silica 500 2O F1 490 557 column in order to remove the copper catalyst. The collected wR3763 Solution was concentrated by rotary evaporation and the R3662 resulting mixture was either precipitated with tetrahydrofu- 500 30 L 490 530 ran or dialyzed against water followed by freeze drying to wR3758 yield a free-flowing white powder. Table 2 sets forth exem- R3706 plary polymers made in accordance with the present inven- 750 40 F1 586 695 tion. wR3764 R3667 750 50 L 645 750 TABLE 2 wR3759 R3707 Theor. MW Polymer ID Initiator Mn Mp 750 60 O 656 740 (kDa) No. (see Table 3) (kDa) (kDa) PDI wR3836 150 60 B5 1SO 158 1.05 R3804 w2199 750 70 P 785 900 d2191 wR3835 250 70 B4 2OS 230 1.05 wR2765 R3808 dR2731)

TABLE 3

B Br CH3 O CH O O

CH orkTFA O O CH3 CH3 O CH Br

US 2016/01 84445 A1 Jun. 30, 2016 32

TABLE 3-continued

F1 Br

O CH3 CH3 O Br CH3 NH O CH3 TFA O O CH3 O O CH3 O O CH3 HN1\1 N-no-n-N-no O NH O CH3 CH Br HC O O NH CH3 HC CH Br O 3 O CH HC Br O

Br

B CH3

US 2016/01 84445 A1 Jun. 30, 2016 34

Example 9 Preparation of Maleimide 9-Arm Conjugatable Polymer 0231. A maleimide conjugatable 9-arm polymer (Q) hav ing the following structure was synthesized as follows:

Polymer Q Polymer HC O CH

Polymer O HC O

Br

HC O H3C O O O Polymer O 7 O O CH3 CH3 N NH O NH NH O n-r OT, NH 4. -n- O O O O O

O O Polymer HC HC

HC es H3C Polymer O O O O O Polymer CH3 O O CH3 Polymer CH

0232 Conjugatable polymer Q was prepared as follows: procedure was repeated 5 more times, after which the reten into a 20 mL vial was placed Polymer ID No. 160 (Table 2) tate was removed and placed into a vial. The Amicon mem (540 mg, 0.0007 mmol. 1.0 equiv) and dissolved using water brane tubes were rinsed with water (2x-2 mL each tube) and (4 mL). To this was added 0.5 M aqueous sodium phosphate this combined with the retentate. The retentate solution was dibasic (0.4 mL). In a separate vial was dissolved 3-maleimi filtered through a syringe filter (Acrodisc Supor, PN 4612), dopropionic acid, NHS ester (0.93 mg, 0.0035 mmol. 5 equiv) frozen and placed on a lyophilizer. This resulted in 508 mgs in tetrahydrofuran (1 mL). The NHS ester solution was added (94%) Polymer Qas a white powder. to the polymer Solution over ~2 minutes at room temperature and the resulting solution was stirred for 30 minutes. The Example 10 reaction was diluted with water (~15 mL), filtered through a syringe filter (Acrodisc Supor, PN 4.612) and placed evenly Preparation of Maleimide 9-Arm Conjugatable into 3 Amicon centrifuge membrane dialysis tubes (30,000 Polymer mwco). The tubes were diluted and mixed with water (-5 mL each), placed into centrifuge (rpm 3000) for 30 minutes. The 0233. A maleimide conjugatable 9-arm polymer (R) hav filtrate was removed for analysis while the retentate was ing the following structure was synthesized using the same diluted and mixed with water (~10 mL/tube). The centrifuge techniques as describe for Conjugatable polymer Q: US 2016/01 84445 A1 Jun. 30, 2016 35

Polymer R Polymer H3C O CH3 CH Polymer O H3C O O O O CH Polymer HC y O HN

O O O Polymer HN O / O O CH3 CH N NH O NH NH O n-n OT, NH 4 ^- O O O O O

O O Polymer HC

NH

H3C es H3C Polymer O O O O O Polymer CH3 O O C H3 CH3 Polymer CH3

Example 11 maintained good activity. Moreover, activity was well main tained after conjugation to polymers. For the Fc construct, Activity of Conjugates activity loss after conjugation to MPC polymer was only 5%. 0234 BuChE enzyme activity was measured for conju gated and unconjugated protein as set forth in Table 1 below. Example 12 In Table 1, rBuChE534 (C66Y) is rhBuChE534GGC and rBuChE534Fc is rhBuChE534 (C66Y)-Fc (L234A, L235E, Plasma Stability of BuChE G237A, A330S, P331S and Q347C): 0236. To determine if the rBuChE534GGC (C66Y) (SEQ ID NO. 13) and rhBuChE534 (C66Y)-Fc (L234A L235E, TABLE 4 G237A, A330S, P331S and Q347C (SEQ ID NO. 2) had Enzyme Activity S.D. overcome the stability problems associated with transgenic Sample (EU/mg) (%) A Activity goat rBuChE, we tested the stability of these constructs ver sus human plasma derived BuChE, and transgenic goat rBuChE534 (CHO-K1) 724 4 rBuChE534Fc (CHO-K1) 788 5 rhBuChE (full length) in cynomolgus monkey (cyno) plasma. rBCES34-9A7SOKHEMA-PC 62O 4 14% Samples were introduced into commercially available cyno rBCES34Fc-9A7SOKHEMA-PC 748 3 59 plasma and aliquots were withdrawn over time and enzyme BuChE (human plasma) (control) 640 4 activity determined via Ellman's assay. FIG. 1 shows the rBuChE (goat milk) (control) 423 5 activity of both the rBuChE534GGC (C66Y)) and rhBuChE534 (C66Y)-Fc (L234A L235E, G237A, A330S, 0235 rBuChE (goat milk) refers to rhBuChE produced in P331S and Q347C) were well maintained over nearly 35 days transgenic goats. In Summary, the BuChE-Fc fusion protein storage at 37° C. in cyno plasma. Both of these constructs US 2016/01 84445 A1 Jun. 30, 2016 36 performed better than the gold standard human plasma puri Toxicity Studies (2011) Chem. Res. Toxicol. 24(11) 1891 fied BuChE. In contrast, the activity of the transgenic goat 1898. These ES1 KO mice are completely deficient in plasma enzyme dropped off rapidly over time, approaching less than carboxylesterase activity which has been reported as boosting 5% towards the end of the study. 0237. It was also determined whether conjugation to poly the LD50 of nerve agents in mice relative to humans. Duysen mer affected enzyme stability. FIG. 2 shows a plot of activity et al. vs. time for protein rhBuChE534 (C66Y)-Fc (L234A, 0241 Serum stability of the various constructs and conju L235E, G237A, A330S, P331S and Q347C conjugated to a 9 gates described below was tested in mouse serum (C57BL/6) arm 750 kDa HEMA-PC polymer compared with human as described above for monkey serum. The results were simi plasma derived BuChE at 4° C. and 37° C. Although the lar (data not shown). The various constructs and conjugates activity of polymer conjugated protein declines somewhat were injected intravenously into groups of 4-6 mice at time 0 over time, the decline is no more than that seen with human (Zero). Aliquots of blood were then withdrawn from the ani plasma derived enzyme. mals at various times after injection and the activity of BuOhE Example 13 measured as described above. Below is a summary of test articles injected into KO mice. In Vitro Inhibition of BuChE by Nerve Agents TABLE 6 0238. To determine whether the rBuChEs of the instant invention were active against nerve agents, various BuChE batches, conjugated and unconjugated, were assayed using Design of InVivo pK Study in KO Mice the Ellman's assay described above in the presence of OP agents: GA (Tabun (Ethyl dimethylamidocyanophosphate)), Specific Activity Concentration Delivered Dose GB (Sarin (Propan-2-yl methylphosphonofluoridate)), GD Test Article (Umg) (protein) (mg/Kg) (Soman (3.3-Dimethylbutan-2-ylmethylphosphonofluori date)), GF (Cyclosarin (Cyclohexyl methylphoshonofluori BuChE (Plasma 641 22 5 60.6 date)), VX (V Series nerve agent (Ethyl (2-bis(propan-2- derived) yl)aminoethylsulfanyl)(methyl)phosphinate), and VR (V rhBuChE534 534 14 5.58 17 series nerve agent (N,N-diethyl-2-(methyl-(2-methylpro poxy)phosphoryl)sulfanylethanamine)). Table 2 shows the (C66Y) - Fc inhibition constant Ki for human plasma rBuChE, (L234A, L235E, rBuChE534=rBuChE534GGC (C66Y), rBuChE534 G237A, A330S, Fc-rhBuChE534 (C66Y)-Fc (L234A, L235E, G237A, P331S and Q347C) A330S, P331S and Q347C), and rBuChE534-Fc 3A500K-conjugate SO39 5.17 17.2 polymer rhBuChE534 (C66Y)-Fc (L234A, L235E, G237A, A330S, P331S and Q347C)-9arm 750 kDa HEMA-PC poly mer with the various nerve agents. BuChE enzyme activity was monitored in the withdrawn blood samples and normalized based on the above activity TABLE 5 data. pK data for the various constructs set forth in Table 1 is shown below in FIG. 3. ki (M-lmin-1) of Nerve Agents Plasma rBuChE534 Agent BuChE rBuChE534 rBuChE534-Fc Fc-polymer Example 15 GA 4.93 x 106 4.75 x 106 3.25 x 106 8.89 x 106 GB 8.62 x 106 8.48 x 106 7.19 x 106 6.87 x 106 Buffer Stability Comparison or BuChE Conjugates GD 8.52 x 107 4.76 x 107 3.74 x 107 S.61 x 107 at 37° C. GF 1.61 x 108 9.43 x 107 6.81 x 107 1.27 x 108 VX 6.92 x 106 3.12 x 106 2.93 x 106 9.06 x 106 VR 108 x 108 4.34 x 107 4.53 x 107 4.53 x 107 0242 Full length rhBuChe purified from the milk of trans genic goats was conjugated via a native cysteine to a polymer using malemide or iodoacetamide. The stability of these two 0239. As can be seen from the table, each of the constructs, different conjugates was compared below in FIG. 4. To assess including the conjugate, is very close to the standard human conjugate Stability, the maleimide and iodoacetamide based plasma BuOhE. conjugates were incubated in phosphate buffered saline at 37° Example 14 C., pH 7.0 for 30 days. Aliquots were withdrawn and free protein assessed by SDS PAGE. The results can be seen in In Vivo Pharmacokinetics of BuOhE Constructs FIG. 4. Iodoacetamide conjugates were very stable over the 30 days with the line remaining near the base line set at time 0240. To determine the in vivo pharmacokinetic properties 0. In contrast, the maleimide based conjugates rapidly of the rhBuChE534 (C66Y)-Fc (L234A L235E, G237A, jumped off the base line set at time 0. Hence, in accordance A330S, P331S and Q347C) (and corresponding conjugates) with the present invention, it can be seen that iodoacetamide of the present invention, constructs were injected adminis based conjugates are Substantially more stable for some pro tered to homozygous carboxylesterase ES1 knockout mice teins than maleimide conjugates. The instability of maleimite (-/-) on a C57BL/6 background. Duysen, E. G. et al. Pro conjugates is protein dependent. Many malemide conjugates duction of ES1 Plasma Carboxylesterase Knockout Mice for are quite stable. US 2016/01 84445 A1 Jun. 30, 2016 37

Example 16 Maleimide 750 kDa MPC Polymer Conjugated to Goat Derived rhBuChE 0243

Compound AA Br

O O Br O O O

O Br O O NH H )-(-

I ------'NH O (- O

Br

Br

Br

0244. For maleimide conjugation, a heat deportectable treated BuChE in Tris buffered saline, pH 7.5 at 4° C. over maleimide initiator was employed to conjugate MPC poly night. Conjugation was assayed by SDS PAGE. Conjugates merto BuChE. An initiator, Compound AA, having the struc were purified from free protein and polymer via DEAE chro ture above was synthesized as set out in PCT/US2012/060301 matography. (see Example 6): 0245 Synthesis of MPC polymer was carried out as Example 17 described in Example 11 of PCT/US2012/060301) to pro duce an approximately 750 kDa 6-arm MPC polymer. The Iodoacetamide 3 Arm, 250 kDa MPC Polymer polymer was prepared for conjugation by heat deprotection as Conjugated to Goat Derived rhBuChE described in U.S. provisional app. no. U.S. 61/875,099. The 0246 ATFA/amine salt initiator (Compound B) having resulting heat deprotected polymer was conjugated to TCEP the structure below was synthesized as follows.

Compound B

O Br

O CH Br O HC O O O NH H3C N- 1N1 N-1 no 1n- '2 H -- CH F

F F

HO O US 2016/01 84445 A1 Jun. 30, 2016

-continued NH2 HC HC H CH 3 O N N N O HC CH CH3 6 - Compound B O OHne-HCl, O O CH RXXX3

0247 First, a BOC protected 3-arm initiator, Compound A, having the following structure: Compound A

Br CH3 CH CH3 O O O HC 1n-N-11-n-N Br H3C O NH O r O O CH O CH3 O CH3 CH Br was prepared as follows: into a 25 mL round bottom flask acid, water, then dried (sodium sulfate), filtered and concen under nitrogen was placed tert-butyl 2-2-(2-aminoethoxy) trated under vacuum. The residue was applied onto a silica gel ethoxyethylcarbamate (66 mg, 0.26 mmol. 1.2 equiv) and column (60 mL) and eluted with 70% ethyl acetate with 30% (2.2.2-Tri(2-bromo-2-methyl-propionyloxymethyl)- hexanes. The tubes containing product was pooled and con ethoxy)-acetic acid (prepared as described in PCT/US2012/ centrated under vacuum, which resulted in 150 mg (0.17 060301 for Product 4.5, which is incorporated herein by mmol. 77%) of Compound A. reference) (142 mg, 0.22 mmol. 1.0 equiv) followed by N.N- 0248 Compound A was de-protected to yield Compound dimethylformamide (2 mL) and then N,N-diisopropylethy B as follows: into a 20 mL round bottom under nitrogen was lamine (0.19 mL, 1.1 mmol. 5.0 equiv). The flask was cooled added Compound A (120 mg, 0.14 mmol. 1 equiv), dichlo to 0°C. using an ice bath. To this was added propylphospho romethane (2 mL) followed by trifluoroacetic acid (2 mL, nic anhydride solution (50 wt.% in ethyl acetate, 0.16 mL, 26.9 mmol. 192 equiv). The reaction stirred at room tempera 0.26 mmol. 1.2 equiv) over 1 minute. The reaction was ture for 30 minutes. The reaction was diluted using hexanes warmed to room temperature and stirred for 1.5 hours. The dichloromethane (20 mL) and concentrated under a vacuum. reaction was quenched by adding water, then partitioned The reaction was diluted using hexanes (50 mL) and concen using water and ethyl acetate. The organic layer was sepa trated under vacuum (twice), which resulted in 2.2 g (2.73 rated and the aqueous layer extracted with ethyl acetate. The mmol, (with residual dichloromethane)) of compound B. combined organic layers were washed with water, Saturated 0249 Polymer B2, having the following structure, was aqueous Sodium bicarbonate, water, 0.5 M aqueous citric prepared as follows. Polymer B2 Polymer CH3 O CH3 O O O Null. O NH Polymer NH1N1 N-1N1\-1 ro -k CH3 O CH3 O CH3 CH3 Polymer US 2016/01 84445 A1 Jun. 30, 2016 39

(0250 Into a 20 mL vial was placed Polymer 103 (280 mg, TABLE 7 0.00123 mmol. 1.0 equiv) and dissolved using water (2 mL). To this was added 0.5 M aqueous sodium phosphate dibasic Conjugation to rhBuChE from goat milk (0.2 mL). In a separate vial was dissolved iodoacetic acid N-hydroxysuccinimide ester (1.6 mg, 0.00548 mmol. 4.5 Rise FIG. 5) for structure Poss Coniugation equiv) in tetrahydrofuran (0.5 mL). The NHS ester solution reR1 excess pH A.incy was added to the polymer Solution over ~2 minutes at room temperature and the resulting solution was stirred for 75 R4341R1 = CH3 10 x 7.5 11% minutes. The reaction was diluted with 4:1 water:tertahydro- R4118. R1 = (CH2)3-guanidine 10 x 7.5 24% furan (4 mL) and placed into a Amicon centrifuge membrane R4440 R1 = CH2-guanidine 10 x 7.5 1796 dialysis tube (30,000 mwco) and the tube placed into centri- R4442 R1 = (CH2)2-guanidine 10 x 7.5 3. fuge (rpm 3000) for 30 minutes. The filtrate is removed for t s th 3-guanidine s analysis while the retentate is diluted and mixed with 4:1 R4440 R1 = CH2-guanidine 10 x 8.5 40% water:tertahydrofuran (6 mL) and the tube placed into centri- R4442 R1 = (CH2)2-guanidine 10 x 8.5 64% fuge (rpm 3000) for 30 minutes. The filtrate is removed for R4341R1 = CH3 10 x 9.5 74% analysis while the retentate is diluted and mixed with water (8 R4342 R1 = 10 x 8.5 22% mL), placed into centrifuge (rpm 3000) for 30 minutes. The filtrate is removed for analysis while the retentate is diluted HC and mixed with water (8 mL). The centrifuge procedure Y repeated 3 more times, after which the retentate is removed -CH -( and placed into avial. The Amicon membrane tube was rinsed with water (2x-2 mL) and this combined with the retentate, N which was frozenO and placed on a lyophilizer. This resulted in R4439 R1 = (CH2)3. N(CH3)2 10 x 8.5 43% 270 mgs (96%) Polymer B2 as a white powder. R4441 R1 = (CH2)2- N(CH3)2 10 x 8.5 6% 0251 Polymer B2 was conjugated to TCEP treated R4320 R1 = (CH2)4 N(CH3)2 10 x 8.5 42% rhBuChE, prepared as described above. Approximately 5x R4438. R1 = CH2 N(CH3)2 10 x 8.5 4% molar excess ration of Polymer B2 to rhBuChE was used in R4451 R1 = (CH2)4-guanidine 10 x 8.5 51% approximately pH 8.5 sodium carbonate buffer at room tem- R4118. R1 = (CH2)3-guanidine 2Ox 8.5 70% perature overnight. Conjugate was purified from free polymer R4451 R1 = (CH2)4-guanidine 2Ox 8.5 75% and protein via DEAE chromatography. Example 18 Example 19 Enhanced Conjugation of 3A 250 kDa Iodoacetamide MPC Polymer Via Basic Groups Synthesis of Polymer R4341 0252 Various basic groups were positioned near the iodoacetamide functionality and their ability to enhance con- 0253 First, R4163, having the following structure was jugation to rBuChE was assayed. synthesized:

Compound R4163

O CH

OX--- CH O CH3 H3C ls O Br CH O N 1N1 N-1 no 1N1 O O 3 H3C H O CH CH3 O O

B-ÖH, KO US 2016/01 84445 A1 Jun. 30, 2016 40

0254 Compound R3329, structure shown below, was pur (0255 6 g of R3329 was mixed with 2.8g of N BOC-2, chased from JStar Research. 2'-(ethylenedioxy)diethylamine in THF at room temperature. Compound R3329 To this was added 8.2 ml of diisopropylethylamine, followed by 7.7 ml of propylphosphonic anhydride solution (1.6 M). Br Following incubation at room temperature for 30 minutes, the reaction mixture was partitioned by EtOAcMTBE and water. Br CH3 This was followed by washing with 0.5 M citric acid x1, saturated sodium bicarbonate and saturated NaCl. The prod uct was dried over magnesium sulfate. R4163 was confirmed by LCMS. O (0256 Next the BOC group was removed from R4613. 9.4 HC g of R4163 was dissolved in 60 ml of dichloromethane. To O )—on this was added 60 ml of trifluoroacetic acid. After 10 minutes O incubation at room temperature. The Solution was dried Br x's CH3 down. The solid material was dissolved in waterfacetonitrile and R4180, shown below, was purified by RP-HPLC. Compound R4180

O CH3 O ) He HC O os-4. 1N-1 O n1n 1\-1 NH2. CH3 H F CH3 O F F Br CH3 O HO O

0257 Next, compound R4203, having the following struc ture, was produced: Compound R4203

O CH3

O O)-- ÖH, H3C Null H Br HC x" N 1N1 N-----> NX CH3. CH3 ) H3 O CH3 CH3 O O Br ÖH, O

(0258 200 mg of R4180 was mixed with 43 mg of BOC L-Ala-OH in 2 ml of acetonitrile. To this was added 200 ul of diisopropylethylamine, followed by 175 ul of propylphos phonic anhydride solution (1.6 M). Following incubation at room temperature for 20 minutes, water was added and the solution stirred for 5 minutes. The solution was then acidified by acetic acid and diluted in methanol. R4203 was purified by RVHPLC. 0259. The initiator was prepared for polymer synthesis by removing the BOC functionality via trifluoro acetic acid to produce a TFA salt. 171 mg of R4203 was dissolved in 500 ul of trifluoroacetic acid and incubated at room temperature for a few minutes. R4235, shown below, was purified by RV HPLC and confirmed by LCMS and NMR. US 2016/01 84445 A1 Jun. 30, 2016 41

Compound R4235 R4235 Br O CH O CH H3C O O CH3

Br O Nulls 1N1'N-1\1\-O NH2 CH H CH3 O O H3C - { TFA Salt Br O

0260 R4235 was then used to initiate MPC polymer syn thesis to produce compound R4258, shown below: Compound R4258

R4258 MPC O CH3

O CH3 HC O O CH H MPC O ouls N 1-1'N-1N O 1N-N NH2. CH3 H CH3 O O

He-MPC KO

0261. In a 25 mL Schlenk tube was loaded 4.8 mg of (100 mg) was dissolved in water to which phosphate buffer 2,2'-bipyridyl, then 47.7 uL of a stock solution of initiator was added. To this was added 100 ul of a 6.5 mg/ml solution (R4235) in DMF (6.3 mg of initiator in 63 uL of DMF). of iodoacetic acid N-hydroxysuccinimide ester in THF. After De-gas the stock Solution then add 2.8 mg of CuBr, de-gas, 5 minutes another 100 ul of the ester was added and the then add the stock solution of monomer (2-hydroxyethyl reaction mixture incubated at room temperature for 30 min methacrylate (HEMA) phosphorylcholine (PC)) in ethanol (1 utes. Dilute to ~10 mL with water. Filter. Centrifuge dialyze g in 4 mL of ethanol) dropwise. Degas the reaction mixture (30 kDa cut-off). Re-dialyze x4. Dilute to ~10 mL with water, for one hour at least and seal under 3 psi of Ar. Allow the filter and lyophilize: Product: Mp: 221.9 kDa, PDI 1.10. reaction to proceed for 20hat RT. Brown coloration: quench ing, sampling for conversion and MW analysis. Purification Example 20 by dialysis (MWCO is 1 kDa) 4 washings before freeze drying. Synthesis of Polymer R4118 0262 Finally, R4341 was synthesized by putting an iodoacetate group on the lone primary amine. Polymer R4258 0263 Polymer R4118 is shown below: Compound R4118

R4118 I O MPC

HC O .. H H MPC CH --~~~~."O NH tic-CH { MPC US 2016/01 84445 A1 Jun. 30, 2016 42

0264. First, R4180 was synthesized as described above. R4180 was then used in the following reaction to produce RXXX1:

O Br

H3C O ouls 1N1 O N-1\1\-1 NH2 CH H F -- CH3 O

Br O HO O R418O

HC HC O NH 1. ul x" H3C X N N O CH3 CH O O OHne-HCl, O O CH3

H3Cy-ch, CH3 O Br O

Br O HC O HN H3C 1N-1 N-1 no-1N 2 CH3 H CH3 O O N O HC O Br O HC HC CH Compound RXXX1

0265 200 mg of R4180 and 107 mg of RXXX1 were 0266 The BOC protective groups were removed with TFA dissolved inacetonitrile (2 ml). To this was added 200 microL to produce a TFA salt. 140 mg of RXXX1 was dissolved in 1.5 of N,N'-Diisopropylethylamine and 175 microL of propy- ml of dichloromethane. To this 1.5 ml of TFA was added. This lphophonic anhydride (50% in ethylacetate). This reaction Solution was incubated at room temperature for about 2 hours mixture was incubated for about 3 hours at room temperature. and 45 minutes. The TFA and dichloromethane were vacu 3 ml of water was added and the solution stirred for 5 minutes. umed off and the solid material was dissolved in waterfaceto The solution was acidified with acetic acid and diluted with 1 nitrile. Compound R4037, shown below, was purified by RV ml of methanol. RXXX1 was purified by RV-HPLC. HPLC and confirmed by LCMS. US 2016/01 84445 A1 Jun. 30, 2016 43

Compound R4037 R4037 O Br

O O 2 H H HC O ouls 1N1 O N-1S-1N1 N N NH2. CH3 H CH3 O O NH he-H( F O Br O r) -o F 2 0267 Next, an MPC polymer was produced using R4037 as an initiator. In a 10 mL Schlenk tube was loaded 4.8 mg of 2,2'-bipyridyl, then 57.7 uL of a stock solution of initiator (R4037) in DMF (6.2 mg of initiator in 62 uL of DMF). Degas the stock solution then add 2.9 mg of Cu(I).Br. degas, then add the stock solution of monomer (2-hydroxyethyl methacrylate (HEMA) phosphorylcholine (PC)) in ethanol (1 g in 4 mL of ethanol) dropwise. 0268 Degas the reaction mixture for one hour at least and seal under 3 psi of Ar. Allow the reaction to proceed overnight at RT (20 hours). Polymer R4053, shown below, was then subjected to SEC/MALS and was found to have a PDI of 1.073 and an Mp of 213.7 kDa. Compound R4053 R4053 O MPC

O ) O ch H H MPC O ouls 1n-1'N-1O no-1\- N N NH2. CH H CH3 O O NH

tic-MPC {O 0269 Compound R4118 was then produced by reacting 0270 Dissolve supernatant in ~10 mL water, filter and R4053 with Iodoacetic acid N-hydroxysuccinimide ester. 150 lyophilize: SEC/MALS: Mp215.3 kDa, PDI 1.1. mg of the polymer was stirred in water until it dissolved and phosphate buffer was added. 3 mg of Iodoacetic acid N-hy Example 21 droxysuccinimide ester was dissolved in 450 ul of THF. Add two 150 uL aliquots of NHS ester solution 5 minutes apart. Synthesis of Polymer R4440 Stir at room temperature for 30 minutes. Dilute to ~10 mL with water and centrifuge and dialyze (30K cut off). Re 0271 First, R4180, see above, was reacted as shown dialyze 5x with ~10 mL water. below to produce a BOC protected initiator: O CH

H3C O ouls 1N-1 O N-1N1)n- NH2 CH3 H F -- CH3 O F F

He- { HO Br O R418O Compound R4180 US 2016/01 84445 A1 Jun. 30, 2016 44

-continued CH

CH3"Y HC O "Y O HN O H3C 1. O N NH OH NSO N. CH3

HC R4247 Compound R4247

HC CH O Br x" NH 2 "A CH HC CH3 r6 YNH 6 H3C CH H3C O HC Q HC Br O ouls N O x" CH ~. H l CH tic-CH3 {O O Br O

Compound R4302

(0272 R4180 (200 mg) and R4247 (111 mg) were dis (0274) 173 mg of R4302 was dissolved in 500 ul of TFA solved in 2 ml of acetonitrile. 200 ul of N,N'-Disopropylethy and incubated at room temperature for 80 minutes. The solu tion was diluted with waterfacetonitrile and the product puri lamine was added followed by 175 ul of propylphosphonic fied by RP-HPLC. Identity of the product was confirmed with anhydride. This solution was incubated at room temperature LCMS and NMR. for 25 minutes. Add water, followed by acetic acid then 0275 Compound 4315 was used as an initiator for poly methanol. R4302 was purified by RP-HPLC. Identity was mer synthesis to produce MPC polymer (R4367, shown confirmed by LCMS. below). In a 25 mL Schlenk tube was loaded 4.8 mg of (0273) Next, BOC groups were removed from R4302 via 2,2'-bipyridyl, then 56.2 uL of a stock solution of initiator TFA to produce R4315, shown below. (R4315) in DMF (6.8 mg of initiator in 68 uL of DMF). Degas

Compound 4315 R4315 HN NH O Br Y O NH O) H.C. ch O O O Br 1N1 NH2 CH CH3 O O H3C - { O Br O F 2 US 2016/01 84445 A1 Jun. 30, 2016

the stock solution then add 2.8 mg of Cu(I).Br. degas, then add the stock solution of monomer ({2-(2-methylprop-2-enoyl) oxyethoxy-2-(trimethylamino)ethoxylphosphinic acid) in ethanol (1 g in 4 mL of ethanol) dropwise. Degas the reaction mixture for one hour at least and seal under 3 psi of Ar. Allow the reaction to proceed for 20 h at RT. Brown coloration: quenching, sampling for conversion and MW analysis. Puri fication by dialysis (MWCO is 1 kDa) 4 washings before freeze-drying. The resulting polymer was analyzed by SEC/ MALS: PDI 1.1 and Mp 200.7 kDa. Compound R4367 R4367 O MPC HN NH

NH HC O) HC O ch H MPC O ouls N 1N1'Nu-1N O 1n-N NH2 CH3 H CH3 O O

He-MPC KO 0276 Finally, compound R4440, shown below, was pro cinimide ester. Incubate at room temperature for 5 minutes, duced by coupling Iodoacetic acid N-hydroxySuccinimide then add a further 100 uL and incubated a further 30 minutes ester to R4367. 100 mg of R4367 was dissolved in 800 ul of at room temperature. Dilute with water to ~10 mL, filter, water. To this was added 80 ul of 0.5 M pH 7.4 sodium centrifuge dialyze (30 kDa cut-off). Re-dialyze from water phosphate buffer. To the buffered solution was added 100 L x4. Dilute to ~10 mL, filter and lyophilize. SEC/MALS of a 6.5 mg/mL solution of Iodoacetic acid N-hydroxysuc analysis: Mp 206 kDa, PDI 1.1. Compound R4440

R4440 NH O MPC

O C "Ne NH ) O ch O HC O H3C H MPC O ouls 1N1 O N-1S-1N-1 N I CH3 H CH O

ic-MPC (O Example 22 Synthesis of Polymer R4342 (0277 First, R4180 (see above) was reacted with BOC-His (3-Me)-OH to produce R4176 as shown in the reaction scheme below: O Br

O CH HC O H. C O O O O NH Br CH - N----H N-1-1-N-" -- He-H(CH3 O oTFA Br O R4180 (actually R4167) Compound R418OBOC-His(3-Me)-OH US 2016/01 84445 A1 Jun. 30, 2016 46

-continued CH3 N NH \ p Hos H3CX O CGO H3C CH3 O HO Boc-His(3-Me)-OH CH3

H3CDX O Br O

O CH3 o N HC O HC NH N Br O O 1N1O N-1 no-1N-1 N CH H V CH3 tic-CH3 {O O Br O R4176 Compound R4176

(0278 165 mg of R4180 and 50 mg of BOC-His(3-Me)- 2.8 mg of CuBr, degas, then add the stock solution of mono OH were suspended in acetonitrile. The R4180 dissolved but mer2-hydroxyethyl methacrylate phosphorylcholine in etha the BOC-His(3-Me)-OH did not. 165ul of N,N-Diisopropy nol (1 g in 4 mL of ethanol) dropwise. Degas the reaction lethylamine was added and the acid dissolved. Following, mixture for one hour at least and seal under 3 psi of Ar. Allow 144 ul of polyphosphonic acid anhydride Solution was added the reaction to proceed for 20 h at RT. Brown coloration: and the reaction mixture incubated for about an hour. Another quenching, sampling for conversion and MW analysis. Puri 80 ul of polyphosphonic anhydride solution was added and fication by dialysis (MWCO is 1 kDa) 4 washings before the reaction mixture allowed to stand for another 45 minutes. freeze-drying. SEC/MALS: PDI-1.1 and Mp=225.7 kDa. The reaction mixture was then acidified with acetic acid and 0281 Finally, compound R4259 was reacted with diluted with methanol. R4176 was purified by RP-HPLC. Iodoacetic acid N-hyroxySuccinimide ester to produce com (0279. Next, the BOC group of R4176 was removed via pound R4342, shown below. 100 mg of polymer R4259 was TFA to produce compound R4182, shown below. 117 mg of dissolved 800 ul of water. To this 50 ul of 0.5 M pH 7.4 R4176 was dissolved in 500 ul TFA and incubated for 10 sodium phosphate buffer was added. Add 100 uL of a 6.5 minutes at room temperature. The reaction mixture was then mg/mL Solution of Iodoacetic acid N-hydroxySuccinimide diluted with waterfacetonitrile and R4182 purified by RV ester in THF was added. This solution was incubated in room HPLC. temperature for 5 minutes. Add an additional 100 uL of the

Compound R4182 Compound R4182 O Br

HC O HC O NH2 N O O Br CH3 --~~---->O He-CH3 {O CH3 Br O

0280 Compound R4182 was used as a substrate for MPC 6.5 mg/mL NHS ester/THF solution. Incubate for 30 more polymer synthesis to produce compound R4259. In a 25 mL minutes at room temperature. Dilute to ~10 mL with water. Schlenk tube was loaded 4.8 mg of 2,2'-Bipyridyl, then 57.1 Filter. Centrifuge dialyze (30 kDa cut-off). Re-dialyze x4. uL of a stock solution of initiator (R4182) in DMF (6.8 mg of Dilute to ~10 mL with water, filter and lyophilize SEC/ initiator in 68 uL of DMF). Degas the stock solution then add MALS: Mp 227.7 kDa, PDI 1.1. US 2016/01 84445 A1 Jun. 30, 2016 47

Polymer R4342 R4342 O MPC I

H3C O O) HC O ch O1. NH N

MPC O ouls N 1-1'N-1a O -- W N y CH H CH3 O O CH

He-H(MPC O

Example 23 Synthesis of Polymer R4439 0282 First, R4180, see above, was reacted with 2-(tert butoxy)carbonyl)amino-5-(dimethylamino)pentanoic acid trifluoroacetic acid salt, as shown below, to produce com pound R4301.

CH3

H.C.3 ' HO O HC1 F CH3 O O OH 9 F B r is O O HN CH, F O r n-1\1\-1 N1SNH, O CH3 H3C O O O oTFA CH3 O HC Br H3C

R418O Compound R4180 Br HC CH O O B CH O O CH r O r N-1 no-1N-1 n-n N1 3 H3C O O HN CH3 O oTFA H3C O )=O O H3C Br HC x" CH3

R4301 Compound R4301

0283 200 mg of R4180 was dissolved in 2 ml of acetoni- tion mixture was allowed to stand at room temperature for trile. This solution was added to 130 mg of 2-(tert-butoxy) about 30 minutes. An additional 200 ul of DIPEA and 100 ul carbonyl)amino-5-(dimethylamino)pentanoic acid trifluo of T3P were added and the reaction mixture allowed to stand roacetic acid salt which was dissolved. 300 ul of N.N- for another 15 minutes. The mixture was diluted with water, Diisopropylethylamine (DIPEA) was added as was 175ul of acetic acid and methanol. R4301 was purified by RP-HPLC propylphosphonic acid anhydride (T3P solution). The reac and confirmed by LCMS. US 2016/01 84445 A1 Jun. 30, 2016 48

0284. Next the BOC protective moiety was removed from quenching, sampling for conversion and MW analysis. Puri R4301 via TFA to produce compound R4314. 93 mg of fication by dialysis (MWCO is 1 kDa) 4 washings before R4301 was dissolved in 500 ul of TFA. The reaction mixture freeze-drying. SEC/MALS: PDI-1.1, Mp=233.4 kDa. 0286 Finally, R4366 was reacted with Iodoacetic acid was allowed to stand for 20 minutes at room temperature. N-hydroxysuccinimide ester to produce R4439 (shown Thereafter the reaction mixture was diluted with waterfaceto below). 100 mg of polymer R4366 was dissolved in 800 ul of nitrile and R4314 was purified by RV-HPLC. water. To this was added 80 ul of 0.5 M pH 7.4 sodium 0285 R4314 was then used a substrate for MPC polymer phosphate buffer. To this 100 ul of 6.5 mg/ml solution of production to produce R4366. In a 25 mL Schlenk tube was Iodoacetic acid N-hydroxysuccinimide ester in THF. The loaded 4.8 mg of 2,2'-Bipyridyl, then 56.9 uL of a stock reaction mixture was allowed to stand at room temperature solution of initiator (R4314) in DMF (9.2 mg of initiator in 92 for 5 minutes at which point another 100 ul of the ester was uL of DMF). Degas the stock solution then add 2.8 mg of added. The reaction mixture was allowed to stand at room CuBr, degas, then add the stock Solution of monomer in temperature for another 30 minutes. Dilute with water to ~10 ethanol (1 g in 4 mL of ethanol) dropwise. Degas the reaction mL, filter, centrifuge dialyze (30 kDa cut-off). Re-dialyze mixture for one hour at least and seal under 3 psi of Ar. Allow from water x4. Dilute to ~10 mL, filter and lyophilize: SEC/ the reaction to proceed for 20 h at RT. Brown coloration: MALS: Mp 233.4 kDa, PDI 1.1. Polymer R4439 R4439 MPC H3C CH O 3 O Mpc. " O O CH O r N-1S-1N1 n-n N1 3 H3C O O O NH CH3 O O HC MPC I HC

Example 24 Synthesis of R4441 (0287 First, R4108 (see above) was reacted with BOC Dab(Me2)-OH, TFA salt to produce R4308 as shown in the scheme below:

O CH3

O ouls O NH2 + HC CH3 H 1n-N-1- 1N1 CH3 O F F

HO O B-HCH, O R418O HO Y O / O F HN F F O --- HO O CH3 Boc-Dab(Me2)-OH, TFA salt Compound R4108BOC-Dab(Me2)-OH, TFA US 2016/01 84445 A1 Jun. 30, 2016 49

-continued CH3 HC CH3 O O O HN NN-O O CH3 F N-\, 1Nul O Br -N HCN,3 F F HC3 3 O O Br HO O O CH CH3 "F- O CH3 HC ,

R4308

Compound R4308

0288 200 mg of 4180 was dissolved in 2 ml of acetonitrile. degas, then add the stock Solution of monomer in ethanol (1g The solution was then used to dissolve 106 mg of BOC-Dab in 4 mL of ethanol) dropwise. Degas the reaction mixture for (Me2)-OH, TFA salt. 200 ul of N,N-Diisopropylethylamine one hour at least and seal under 3 psi of Ar and allow the (DIPEA) and 265 ul of propylphosphonic anhydride (T3P) reaction to proceed for 20 h at RT when a brown coloration. was added. Incubate at room temperature for 15 minutes. Add The reaction was then quenched for sampling, conversion and 200 more of DIPEA and 175 ul T3P and incubate for 15 MW analysis. Purification by dialysis (MWCO is 1 kDa) 4 minutes more. Add water, thenacetic acid. Dilute with metha washings before freeze-drying. SEC/MALS analysis: PDI nol. Purify R4308 via RV-HPLC. Confirm by LCMS. 1.1, Mp 256.6 kDa. 0291 Finally, R4368 was reacted with Iodoacetic acid 0289 Next, the BOC protective group was removed to N-Hydroxysuccinimide ester to provide R4441, shown provide R4331 via TFA. 135 mg of R4308 was dissolved in below. 100 mg of R4368 was dissolved in 800 ul of water. 80 500 ul of TFA. The reaction mixture was allowed to stand for n1 of 0.5M pH 7.4 sodium phosphate buffer was added. Add 15 minutes. R4331 was purified by RV-HPLC. 100 uL of a 6.5 mg/mL solution of NHS ester. Leave at room 0290 R4331 was then used as an initiator for polymer temperature for 5 minutes, then add a further 100 uL. Allow to synthesis to provide polymer R4368. In a 25 mL Schlenk tube sit for 30 minutes. Dilute with water to ~10 mL, filter, centri was loaded 4.8 mg of 2,2'-Bipyridyl, then 56.2 uL of a stock fuge dialyze (30 kDa cut-off). Re-dialyze from water x4. solution of initiator in DMF (7.1 mg of initiator in 71 uL of Dilute to ~10 mL, filter and lyophilize SEC/MALS R4441 DMF). Degas the stock solution then add 2.8 mg of CuBr, Mp 256.6 kDa, PDI 1.1.

Compound R4441

R4441 O MPC HC n1 CH3

H3C O Hid O H O O MPC N 1 CH --~~~ H CH3 O O

ic-H(MPC O US 2016/01 84445 A1 Jun. 30, 2016 50

Example 25 Synthesis of Polymer R4320 0292 First, R4180, see above, was reacted with BOC-Lys (Me2)-OH to produce R4196.

O CH O ) Br H3CQ HO O Br O HC O N O H3C HC O ouls N 1N1'N-1a O 1N-NH2 HC HN CH3 CH H O F C H3 O CH F F Br Boc-L-Lys(Me2)-OH CH O HO O R418O Compound R418OBOC-L-Lys(Me2)-OH OX--- CH3 CH3 O O CH O Br H3C 1. N 1N1'N1\1\1O O O O NH O CH3 N CH O O HC

HC x" Br O 3 CH3 CH3

R41.96 Compound R4196

0293 200 mg of R4180 and 74 mg of BOC-L-Lys(Me2)- acetic acid and diluted with methanol. R4196 was purified by OH were dissolved in 2 ml of acetonitrile. To this was added RV-HPLC and the product confirmed by LCMS. 0294 Next the BOC group on R4196 was removed via 200ul of N,N-Diisopropylethylamine (DIPEA) and 204 ul of TFA to produce R4211, compound shown below. 153 mg of Propylphosphonic acid anhydride (T3P). The reaction mix R4196 was dissolved in 500 ul of TFA. Solution allowed to sit ture was allowed to stand for 15 minutes at room temperature. at room temperature for 15 minutes. Dilute by waterfaceto The reaction mixture was then diluted with water and stirred nitrile. Purify by RV-HPLC. Structure confirmed by LCMS for 5 minutes. Finally, the reaction mixture was acidified with and NMR.

Compound R4211

R4211 OX--- CH O CH

N O Br CH 3 HC1 1- n-n-n-rro O NH2 O CH3 F F CH3 O O F F F F B- { ÖH O HO O HO O US 2016/01 84445 A1 Jun. 30, 2016

0295) Next, R4211 was used as a substrate for MPC poly 0296 Finally, polymer R4320 (shown below) was pro mer production to produce polymer R4255. In a 25 mL duced by coupling polymer R4255 to Iodoacetic acid N-Hy Schlenk tube was loaded 4.8 mg of 2,2'-Bipyridyl, then 57.7 uL of a stock solution of initiator in DMF (8 mg of initiator in droxysuccinimide ester. 100 mg of R4255 was dissolved in 80 uL of DMF). Degas the stock solution then add 2.8 mg of 800 ul of water. To this was added 80 ul of 0.5 M pH 7.4 CuBr, degas, then add the stock Solution of monomer in sodium phosphate buffer. Add 100 uL of a 6.5 mg/mL solu ethanol (1 g in 4 mL of ethanol) dropwise. Degas the reaction tion of NHS ester in THF. Stir 5 minutes, then add a further mixture for one hour at least and seal under 3 psi of Ar. Allow 100 uL of NHS ester solution. Let stand at room temperature the reaction to proceed for 20 h at RT. Brown coloration: quenching, sampling for conversion and MW analysis. Puri for 30 minutes. Dilute with water to ~10 mL volume, filter, fication by dialysis (molecular weight cutoff is 1 kDa) 4 centrifuge and dialyze (30 kDa MW cutoff). Dilute to ~10 mL washings before freeze-drying. SEC/MALS: PDI 1.076, Mp and re-dialyze 4 times. Dilute to ~10 mL, filter and lyophilize 205.3. SEC/MALS: Mp: 225.7 kDa, PDI: 1.1. Polymer R4320

R4320 O CH I

O O)--stic CH HC O O1. NH H3C O ouls 1N1 O n-1\1\-1 N1 CH3 MPC O H O O CH CH MPC CH Example 26 Synthesis of Polymer R4438 0297 First, R4180 (see above) was reacted with BOC Dap(Me2)-OH TFA salt to produce compound R4300 as shown below. O CH3 O Br O HO O Br O HC CH O H3C F O O 1n 1'n-1\1\1O NH'2 -N >{ CH F H3C N O HC3 N O CH H H CH3 HO O C H3 O F Boc-Dap(Me2)-OH TFA salt B-H F F CH O HO O R418O Compound R418OBOC-Dap(Me2)-OH, TFA HC O H O

H3C Y-'. O O NN-O N-\ H N-CH3 O X-y Br I 1Nul O HC N, 3 O Br O CH CH3 "F- ; CH, HC3 O R43OO Compound R4300 US 2016/01 84445 A1 Jun. 30, 2016 52

0298 200 mg of R4180 and 86 mg of BOC-Dap(Me2)- OHTFA salt were dissolved in 2 ml of acetonitrile. 200 ul of N,N-Diisopropylethylamine (DIPEA) and 175 ul of propy lphosphonic anhydride solution (T3P) were added and the solution incubated at room temperature for about 15 minutes. 40 mg more of the BOC-Dap(Me2)-OHTFA salt, 200ul more of DIPEA and 100 ul more of T3P were added, followed by another 10 minutes incubation at room temperature. Dilute with water, acidify with acetic acid then dilute with methanol. R4300 purified by RV-HPLC and confirmed by LCMS. 0299 Next, the BOC group on R4300 was removed via TFA to produce R4307 as shown below:

HC O H O

H3C CH3 Y-'. 0. N O O CH N-\ul IN e 3 O 1NullH O X-y Br HC HC TFA O -e- O Br O CH3 F CH O 4 CH, F F HC "f-3 O HO O Compound R4300 O) CH3His O O CH3 HC NN ~-N-N-N- r- "\ CH, CH NH N O Oro NXCH CH O F F 3 O F F F F Br ( CH O HO O HO O Compound R4307

0300 118 mg of R4300 was dissolved in 500 ul of TFA and MW analysis. Purification by dialysis (MWCO is 1 kDa) and the reaction mixture allowed to stand for 20 minutes. and washing before freeze-drying procedure. SEC/MALS: Waterfacetonitrile were added and compound 4307 was puri Mp 230.8 kDa, PDI 1.1. fied by RF-HPLC and confirmed by LCMS. 0302 Finally, iodoacetamide functionality was added to R4307 to produce compound R4438 (shown below). 100 mg 0301 Next, R4307 was used as an initiator for MPC poly of R4307 was dissolved in 800 ul of water. To this was added mer synthesis. In a 25 mL Schlenk tube was loaded 4.8 mg of 80 ul of 0.5M pH 7.4 sodium phosphate buffer. Then add 100 2.2 Bipyridiyl, then 55.5ul of a stock solution of initiator in ul of a 6.5 mg/ml solution of the NHS ester was added, DMF (6.8 mg of initiator in 68 uL of DMF). Degas the stock followed by incubation at room temperature for 5 minutes. Solution then add 2.8 mg of CuBr, degas, then add the Stock Add a further 100 uL of a 6.5 mg/mL solution of NHS ester Solution of monomer in ethanol (1 g in 4 mL of ethanol) and incubate for about 40 minutes at room temperature. dropwise. Degas the reaction mixture for one hour at least and Dilute with water to ~10 mL, filter, centrifuge dialyze (30kDa seal under 3 psi of Ar. Allow the reaction to proceed for 20 h cut-off). Re-dialyze from water x4. Dilute to ~10 mL, filter at RT. Brown coloration: quenching, sampling for conversion and lyophilize SEC/MALS: Mp 230.8 kDa, PDI 1.1. US 2016/01 84445 A1 Jun. 30, 2016 53

Polymer R4438 R4438 HC MPC CH3 HC O O -N

H H3C O sus N 1N1 O N-1\o N I CH3 H CH3 O O

Mc-H(MPC O

Example 27 Synthesis of Polymer R4451 0303 First, R4180 (see above) was reacted with BOC homoarg-OH.HCl to produce R4366 as shown below:

CH O 3 NH

O O B, HN l "\-o O O O NH2 HC CH H F 3 CH H-Cl O 3 O F F CH3 Boc-homo-Arg-OHHCL B-HCH, O HO NO es

R418O Compound R4180 BOC-homoarg-OHHCI

O CH3

O OX--- CH

HC HO O O CH3 CH3

R4366 Compound R4366

0304 200 mg of R4180 was dissolved in 2 ml of acetoni- solids dissolved. This reaction mixture was incubated for 40 trile. This solution was added to 80 mg of BOC-homoarg- minutes at room temperature. Another 81 mg of BOC-ho OH.HCl, which was partially soluble. 175ul of Diisopropy- E.O.C. style. it. lethylamine (DIPEA) was added and the BOC-homoarg-OH. incubated for another 50 minutes. The reaction mixture was HCl all appeared to precipitate out. However, when 2001ll of diluted with water and R4366 was purified by RV-HPLC and propylphosphonic anhydride solution (T3P) was added all confirmed by LCMS. US 2016/01 84445 A1 Jun. 30, 2016 54

0305 Next, the BOC group was removed to produce com pound R4354 as shown: O CH X-- Br O O CH3 H Br O CH N-1 no-1N1 ro O 3 TFA 1 -e- "Ni--NH O F O CH CH3 O O HC s F F

HC X." HO O Br O 3 CH CH Compound R4366 HC CH O 3

O O H.C.3 " O r N-1 no.1 nu- O N-1SN --~- NH 2 HC O NH NH

O O O d HC 3 HO O HO O CH Br 3 R4354 Compound R4354

(0306 90 mg of R4366 was dissolved in 500 ul of TFA. 30 fication by dialysis (MWCO is 3.5 kDa) before the freeze minutes at room temperature. Add waterfacetonitrile. Purify drying procedure. SEC/MALS PDI 1.1 Mp 258.4 kDa. by RV-HPLC and confirm by LCMS and NMR. 0308 Finally, an iodoacetamide functionality was added 0307 Next, R4354 was used as a substrate for MPC poly to R4386 to produce R4551 (shown below). 100 mg of R4386 mer synthesis to produce R4386. In a 25 mL Schlenktube was was dissolved in 800 ul of water. To this was added 80 ul of 0.5 loaded 4.8 mg of 2.2 Bipyridyl, then 58.3 uL of a stock M pH 7.4 sodium phosphate buffer. 100 ul of a 6.5 mg/ml solution of initiator (R4354) in DMF (7.4 mg of initiator in 74 solution of Iodoacetic acid N-Hydroxysuccinimide ester was uL of DMF). Degas the stock solution then add 2.8 mg of added. Incubate at room temperature for 5 minutes and add CuBr, degas, then add the stock Solution of monomer in another 100 ul of the NHS ester. ethanol (1 g in 4 mL of ethanol) dropwise. Degas the reaction 0309 Incubate at room temperature for 30 minutes. Dilute mixture for one hour at least and seal under 3 psi of Ar. Allow with water to ~10 mL, filter, centrifuge dialyze (30 kDa the reaction to proceed for 20 h at RT. Brown coloration: cut-off). Re-dialyze from water x4. Dilute to ~10 mL, filter quenching, sampling for conversion and MW analysis. Puri and lyophilize SEC/MALS: Mp 258.4 kDa, PDI 1.1. Compound R4451

R4451 CH H3C MPC O MPC O HC O HC O O O O HC CH3 US 2016/01 84445 A1 Jun. 30, 2016 55

Example 28 Synthesis of PEG Reagents 0310 PEG reagents are conveniently made via coupling of NHS esters to amines Branched PEGs ending in amine func tionalities are commercially available. For example, the fol lowing PEG iodoacetamide reagent may be used in accor dance with the instant invention:

PEG Reagent R500 HC-O--C-C-O H. H. pi O HC-O--C-C-O H H. H. J. N NH2 O-C-C-C -N H. H. H his O NH

where n is the number of PEG units. PEG Reagent R500 is Example 29 conveniently synthesized by the following reaction: 0311 Mammalian expression vector, cell line and expres HC-O-H-C-C-O sion optimization for rhBuChE534 (C66Y)-Fc (L234A, H. H. L235E, G237A, A330S, P331S, Q347C) HC-O--C-C-O 0312 A DNA fragment containing the rhBuChE534 H. H. pi H (C66Y)-Fc (L234A, L235E, G237A, A330S, P331S, Q347C) O-C-C-C -NH2 + H. H. construct was cloned into a mammalian expression vector O O encodingapuromycin resistance gene. The sequence that was H inserted is shown in SEQ ID NO. 9. The expression vector N NH2 with the inserted BuChE-Fc fusion is called Construct 14. N-O Construct 14 was transfected into CHO cells and a stable HN O NH transfected pool was constructed. The stable pool was sub jected to cell line development and a stably transfected clone O was isolated called C98. A shake flask study was performed to determine which media and conditions to use to maximize the I PEG Reagent R500 amount of protein produced. Seeding density for each flask was 0.4x106 cells/ml. The results are set forth in Table 2 below: TABLE 8 Shake Flask Experiment Flask Feed Feed Feed Temp Peak Titer i Medium Condition Amount Days Shift? (mg/L) 1 CD OptiCHO Glucose Up to 4 g/L. As needed No 60 Only 2 CD OptiCHO CD Efficient 10% 3, 6 No 40 Feed C CD OptiCHO CD Efficient 10% 3, 6 Yes 35 Feed C CD FortiCHO Glucose only Up to 4 g/L. As needed No 145 CD FortiCHO CD Efficient 59 3, 6 No 475 Feed C (2x) CD FortiCHO CD Efficient 59 3, 6 Yes 26S Feed C (2x) ActioP Glucose only Up to 4 g/l. As needed No 150 ActioP ActicFHO 3% + O.3% 3 No 100 Feed A+ B ActioP ActicFHO 3% + O.3% 3 Yes 8O Feed A+ B 10 BalanCD CHO Glucose only Up to 4 g/L. As needed No 35 A. US 2016/01 84445 A1 Jun. 30, 2016 56

TABLE 8-continued Shake Flask Experiment Flask Feed Feed Feed Temp Peak Titer i Medium Condition Amount Days Shift? (mg/L) 11 BalanCD CHO BalanCD 15% 3, 6 No 40 A. Feed 3 12 BalanCD CHO BalanCD 15% 3, 6 Yes 40 A. Feed 3

Example 30 control polymerization of styrene and methyl methacrylate. Low molecular weight polymers were obtained only (below 10,000 Da). Unimolecular Initiators

0313 The introduction of a basic group (e.g., guanidine or tertiary amines) covalently attached to the previous ATRP initiators resulted in potential interference during the poly merization process by ATRP due to potential complexing of ()- Y / the copper halide species (CuBr) with Such basic groups N when the ligand was added (2,2'-Bipyridine noted bpy). This TMGpy example tests whether if Such basic group claimed in the invention could also act as ligands in an ATRP process and to be able to skip the use of Such compounds when aiming to obtain tailor-made polymers with Such initiators. 0314 Unimolecular ATRP initiators containing tertiary amine group covalently attached were already reported in the polymerization of styrene (polym. Preprint 2012 43(2), p -NX=N 7-() N / 245). The goal of using such initiators in an ATRP system for vinylic monomers (styrene, methyl methacrylate) was only to Suppress the use of external agent by covalently attach the DMEGPy amine onto the initiator but not for conjugation purpose where a bioactive compound is involved. Studies on unimolecular initiator were only performed on styrene and MMA in organic 0316. This example tests whether groups newly intro Solvent (toluene) requiring heating. According to the authors, duced on the initiator do not interfere with the traditional ATRP process described above and check on their capability moderate control of the molecular weights were obtained. to be used as a ligand in an ATRP polymerization process. High molecular weights were achieved with a broader range 0317. A first block Poly (HEMA-PC) was synthesized distribution. (step 1) without any bpy introduced to check on the polymer 0315. The use of guanidine molecules as ligand in ATRP formation and the control of the molecular weight. This step process was reported as guanidine-pyridine derivatives (dim was followed by the introduction of bpy and an incremental ethylethyleneguanidine)methylenepyridine (DMEGpy) or addition of monomer (step 2) to check on a chain extension (tetramethylguanidine)methylenepyridine (TMGpy). These and the livingness of the system according to the scheme structures were not used as ligands in an ATRP process for the below. OXuk O O H O R N O O O stage 1 ul H 1N1'N-1N ~~ -C CuBr, DMF -e- O O Br HEMA-PC in ethanol

O

Br US 2016/01 84445 A1 Jun. 30, 2016 57

-continued O polymer,

O O R 1- N-1N1 S-1 Sno y O O polymer, O

polymer,

HEMA-PC in ethanol stage 2 bpy

O Xuk" polymer 2 O O O --~'N-1N1\- '^c O H O x polymer 2

polymer polymer 2 0318 Compounds R4235 was used as a reference. The structure of all the other compounds used are described below: Compound R4235 R4235 Br O CH

O CH3 H3C O O CH3 H Br O sus 1n-N-1N1\-O N NH2 CH H CH3 O O

tic- {O Br TFA Salt Compound R4315 R4315 Br HN NH

O y CH YNH HC O HC H Br O Nulls 1N1'N-1O no-1N1 N NH2 CH3 H CH3 O O He- { F O Br O A-on F 2

US 2016/01 84445 A1 Jun. 30, 2016 59

-continued Compound R4182 Compound R4182 O Br

O H3C O HC NH2 N H y Br O O N---- O N-1 no-1N-1 N N CH H V CH3 to-H.CH3 O Br O

0319. The protocol used was the same for all the initiators: HEMA-PC monomer for step 2, a brown coloration a brown 0320 Polymerization protocol without bpy as ligand. A coloration immediately appeared Suggesting a recomplex stock solution of initiator as a 100 mg/mL in DMF was freshly ation of the copper species with bpy. Polymerizations were prepared. The solution was dispensed to introduce 5x10-6 resumed in all case by a chain extensions of the polymers mol of initiator in the Schlenk tube. The solution was obtained in step 1 with the desired molecular weights and degassed before the addition of 2.48 mg of CuBr (1.7x10-5 narrow polydispersities. mol) (no change of coloration). A stock solution of HEMA 0326. The newly introduced group not only participates at PC in ethanol (1 g in 4 mL) was added dropwise. Reaction the conjugation at neutral pH but can also act as efficient mixture was seated under 3 psi and polymerization and ligand in an ATRP. For such polymerizations, the use of an allowed to proceed at RT for 20 hours at RT external ligand (such as bpy) is unnecessary. 0321 Reaction stayed colorless and a aliquots was taken 0327 All patent filings, websites, other publications, to analyze the conversion and molecular weights in each case accession numbers and the like cited above or below are (see table below on step 1). incorporated by reference in their entirety for all purposes to 0322. In step 2, bpy was added to the crude mixture of step the same extent as if each individual item were specifically 1 immediately followed by an incremental addition of mono and individually indicated to be so incorporated by reference. mer (HEMA-PC) in solution (1 g in 2 mL of ethanol) and 4.8 If different versions of a sequence are associated with an mg of bpy (3.1x10-5 mol) inside the reaction mixture (a accession number at different times, the version associated brown coloration immediately appears). Reaction was sealed, with the accession number at the effective filing date of this thoroughly degassed, homogenized and allowed to proceed at application is meant. The effective filing date means the ear room temperature until Viscous before final quenching. lier of the actual filing date or filing date of a priority appli 0323 Results are shown in table 9 below: cation referring to the accession number if applicable. Like TABLE 9 2nd block (polymerization continuation afte 1st block (no bpy introduced) introduction ofbpy as external ligand) Stepl Step 2 Initiator Min Mp PDI conversion Mn Mp PDI conversion Reference R4235 NAA NAA NA NA 335 406 1.15 92 Guanidine R4315 220 24S 1.15 81 350 415 1.15 95 Series R4334 2SS 318 1.2 96 368 436 1.2 95 R4037 210 2SS 1.2 91 3S4 410 1.15 95 R4354 260 33O 1.2 94 360 420 1.2 95 Tertiary amine R4314 21S 283 1.2 94 323 410 1.2 91 Series R4211 218 28O 1.2 90 3S4 425 1.15 90 Histidine series R4182 2OS 26O 1.2 95 335 415 1.2 97

0324. In the absence of bpy for the reference (compound wise if different versions of a publication, website or the like R4235) no polymer formation was obtained with in the pres are published at different times, the version most recently ence of bpy. Polymerization proceeded only when bipyridyl published at the effective filing date of the application is as ligand was introduced inside the reaction mixture to reach meant unless otherwise indicated. Any feature, step, element, the desired molecular weight and high conversion. embodiment, or aspect of the invention can be used in com 0325 For all the other initiators listed in the table (com bination with any other unless specifically indicated other pounds R4315, R4334, R4037, R4354, R4314, R4211 and wise. Although the present invention has been described in R4182) polymerization proceeded in all cases without any Some detail by way of illustration and example for purposes bpy ligand introduced in the reaction for step one. Controlled of clarity and understanding, it will be apparent that certain molecular weights were obtained with low PDIs (1.2 and changes and modifications may be practiced within the scope below). When adding bpy with an incremental addition of of the appended claims.

US 2016/01 84445 A1 Jun. 30, 2016 64

- Continued

Phe Gly Gly Thr Val Thr Ala Phe Leu Gly Ile Pro Tyr Ala Glin Pro SO 55 6 O Pro Lieu. Gly Arg Lieu. Arg Phe Llys Llys Pro Glin Ser Lieu. Thir Lys Trp 65 70 7s 8O Ser Asp Ile Trp Asn Ala Thr Lys Tyr Ala Asn. Ser Cys Cys Glin Asn 85 90 95 Ile Asp Glin Ser Phe Pro Gly Phe His Gly Ser Glu Met Trp Asn Pro 1OO 105 11 O Asn. Thir Asp Lieu. Ser Glu Asp Cys Lieu. Tyr Lieu. Asn Val Trp Ile Pro 115 12 O 125 Ala Pro Llys Pro Lys Asn Ala Thr Val Lieu. Ile Trp Ile Tyr Gly Gly 13 O 135 14 O Gly Phe Glin Thr Gly Thr Ser Ser Lieu. His Val Tyr Asp Gly Llys Phe 145 150 155 160 Lieu Ala Arg Val Glu Arg Val Ile Val Val Ser Met Asn Tyr Arg Val 1.65 17O 17s Gly Ala Lieu. Gly Phe Lieu Ala Lieu Pro Gly ASn Pro Glu Ala Pro Gly 18O 185 19 O Asn Met Gly Lieu. Phe Asp Glin Glin Lieu Ala Lieu Gln Trp Val Glin Lys 195 2OO 2O5 Asn Ile Ala Ala Phe Gly Gly Asn Pro Llys Ser Val Thr Lieu. Phe Gly 21 O 215 22O Glu Ser Ala Gly Ala Ala Ser Val Ser Lieu. His Lieu Lleu Ser Pro Gly 225 23 O 235 24 O Ser His Ser Leu Phe Thr Arg Ala Ile Leu Gln Ser Gly Ser Phe Asn 245 250 255 Ala Pro Trp Ala Val Thir Ser Lieu. Tyr Glu Ala Arg Asn Arg Thr Lieu 26 O 265 27 O Asn Lieu Ala Lys Lieu. Thr Gly Cys Ser Arg Glu Asn. Glu Thr Glu Ile 27s 28O 285 Ile Lys Cys Lieu. Arg Asn Lys Asp Pro Glin Glu Ile Lieu. Lieu. Asn. Glu 29 O 295 3 OO Ala Phe Val Val Pro Tyr Gly Thr Pro Leu Ser Val Asn Phe Gly Pro 3. OS 310 315 32O Thr Val Asp Gly Asp Phe Lieu. Thir Asp Met Pro Asp Ile Lieu. Lieu. Glu 3.25 330 335 Lieu. Gly Glin Phe Llys Llys Thr Glin Ile Lieu Val Gly Val Asn Lys Asp 34 O 345 35. O Glu Gly Thr Ala Phe Leu Val Tyr Gly Ala Pro Gly Phe Ser Lys Asp 355 360 365 Asn Asn. Ser Ile Ile Thr Arg Lys Glu Phe Glin Glu Gly Lieu Lys Ile 37 O 375 38O Phe Phe Pro Gly Val Ser Glu Phe Gly Lys Glu Ser Ile Leu Phe His 385 390 395 4 OO

Tyr Thr Asp Trp Val Asp Asp Glin Arg Pro Glu Asn Tyr Arg Glu Ala 4 OS 41O 415

Lieu. Gly Asp Val Val Gly Asp Tyr Asn. Phe Ile Cys Pro Ala Lieu. Glu 42O 425 43 O

Phe Thr Lys Llys Phe Ser Glu Trp Gly Asn Asn Ala Phe Phe Tyr Tyr 435 44 O 445 US 2016/01 84445 A1 Jun. 30, 2016 65

- Continued Phe Glu. His Arg Ser Ser Lys Lieu Pro Trp Pro Glu Trp Met Gly Val 450 45.5 460 Met His Gly Tyr Glu Ile Glu Phe Val Phe Gly Lieu Pro Leu Glu Arg 465 470 47s 48O Arg Asp Asn Tyr Thr Lys Ala Glu Glu Ile Lieu. Ser Arg Ser Ile Val 485 490 495 Lys Arg Trp Ala Asn. Phe Ala Lys Tyr Gly Asn. Pro Asn. Glu Thr Glin SOO 505 51O Asn Asn Ser Thr Ser Trp Pro Val Phe Llys Ser Thr Glu Gln Lys Tyr 515 52O 525 Lieu. Thir Lieu. Asn. Thr Glu Ser Thr Arg Ile Met Thr Llys Lieu. Arg Ala 53 O 535 54 O Gln Glin Cys Arg Phe Trp Thir Ser Phe Phe Pro Llys Val Lieu. Glu Met 5.45 550 555 560 Thr Gly Asn. Ile Asp Glu Ala Glu Trp Glu Trp Lys Ala Gly Phe His 565 st O sts Arg Trp Asn. Asn Tyr Met Met Asp Trp Lys Asn Glin Phe Asn Asp Tyr 58O 585 59 O Thir Ser Lys Lys Glu Ser Cys Val Gly Lieu. 595 6OO

<210s, SEQ ID NO 2 &211s LENGTH: 772 212. TYPE PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 2 Glu Asp Asp Ile Ile Ile Ala Thir Lys Asn Gly Llys Val Arg Gly Met 1. 5 1O 15 Asn Lieu. Thr Val Phe Gly Gly Thr Val Thr Ala Phe Leu Gly Ile Pro 2O 25 3O Tyr Ala Glin Pro Pro Lieu. Gly Arg Lieu. Arg Phe Llys Llys Pro Glin Ser 35 4 O 45 Lieu. Thir Lys Trp Ser Asp Ile Trp Asn Ala Thr Llys Tyr Ala Asn. Ser SO 55 6 O Cys Tyr Glin Asn Ile Asp Glin Ser Phe Pro Gly Phe His Gly Ser Glu 65 70 7s 8O Met Trp Asn Pro Asn. Thir Asp Lieu. Ser Glu Asp Cys Lieu. Tyr Lieu. Asn 85 90 95 Val Trp Ile Pro Ala Pro Llys Pro Lys Asn Ala Thr Val Lieu. Ile Trp 1OO 105 11 O Ile Tyr Gly Gly Gly Phe Glin Thr Gly. Thir Ser Ser Lieu. His Val Tyr 115 12 O 125

Asp Gly Llys Phe Lieu Ala Arg Val Glu Arg Val Ile Val Val Ser Met 3 O 135 14 O

Asn Tyr Arg Val Gly Ala Lieu. Gly Phe Lieu Ala Lieu Pro Gly Asn Pro 145 150 155 160

Glu Ala Pro Gly Asn Met Gly Lieu. Phe Asp Glin Glin Lieu Ala Lieu. Glin 1.65 17O 17s

Trp Val Glin Lys Asn. Ile Ala Ala Phe Gly Gly ASn Pro Llys Ser Val 18O 185 19 O US 2016/01 84445 A1 Jun. 30, 2016 66

- Continued

Thir Lieu. Phe Gly Glu Ser Ala Gly Ala Ala Ser Val Ser Lieu. His Lieu. 195 2OO 2O5 Lieu. Ser Pro Gly Ser His Ser Leu Phe Thr Arg Ala Ile Leu Glin Ser 21 O 215 22O Gly Ser Phe Asn Ala Pro Trp Ala Val Thir Ser Lieu. Tyr Glu Ala Arg 225 23 O 235 24 O Asn Arg Thir Lieu. Asn Lieu Ala Lys Lieu. Thr Gly Cys Ser Arg Glu Asn 245 250 255 Glu Thr Glu Ile Ile Llys Cys Lieu. Arg Asn Lys Asp Pro Glin Glu Ile 26 O 265 27 O Lieu. Lieu. Asn Glu Ala Phe Val Val Pro Tyr Gly Thr Pro Leu Ser Val 27s 28O 285 Asn Phe Gly Pro Thr Val Asp Gly Asp Phe Lieu. Thr Asp Met Pro Asp 29 O 295 3 OO Ile Lieu. Lieu. Glu Lieu. Gly Glin Phe Llys Llys Thr Glin Ile Lieu Val Gly 3. OS 310 315 32O Val Asn Lys Asp Glu Gly Thr Ala Phe Lieu Val Tyr Gly Ala Pro Gly 3.25 330 335 Phe Ser Lys Asp Asn. Asn. Ser Ile Ile Thr Arg Lys Glu Phe Glin Glu 34 O 345 35. O Gly Lieu Lys Ile Phe Phe Pro Gly Val Ser Glu Phe Gly Lys Glu Ser 355 360 365 Ile Lieu. Phe His Tyr Thr Asp Trp Val Asp Asp Glin Arg Pro Glu Asn 37 O 375 38O Tyr Arg Glu Ala Lieu. Gly Asp Val Val Gly Asp Tyr Asn. Phe Ile Cys 385 390 395 4 OO Pro Ala Lieu. Glu Phe Thr Llys Llys Phe Ser Glu Trp Gly Asn. Asn Ala 4 OS 41O 415 Phe Phe Tyr Tyr Phe Glu. His Arg Ser Ser Lys Lieu Pro Trp Pro Glu 42O 425 43 O Trp Met Gly Val Met His Gly Tyr Glu Ile Glu Phe Val Phe Gly Lieu. 435 44 O 445 Pro Lieu. Glu Arg Arg Asp Asn Tyr Thr Lys Ala Glu Glu Ile Lieu. Ser 450 45.5 460 Arg Ser Ile Val Lys Arg Trp Ala Asn. Phe Ala Lys Tyr Gly Asn Pro 465 470 47s 48O Asn Glu Thr Glin Asn Asn Ser Thr Ser Trp Pro Val Phe Llys Ser Thr 485 490 495 Glu Glin Llys Tyr Lieu. Thir Lieu. Asn Thr Glu Ser Thr Arg Ile Met Thr SOO 505 51O Lys Lieu. Arg Ala Glin Gln Cys Arg Phe Trp Thr Ser Phe Phe Pro Llys 515 52O 525 Val Lieu. Glu Met Thr Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 53 O 535 54 O Ser Asp Llys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu 5.45 550 555 560

Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Llys Pro Lys Asp Thr Lieu. 565 st O sts

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 58O 585 59 O US 2016/01 84445 A1 Jun. 30, 2016 67

- Continued His Glu Asp Pro Glu Val Llys Phe Asn Trp Tyr Val Asp Gly Val Glu 595 6OO 605 Val His Asn Ala Lys Thr Llys Pro Arg Glu Glu Glin Tyr Asn. Ser Thr 610 615 62O Tyr Arg Val Val Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn 625 630 635 64 O Gly Lys Glu Tyr Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ser Ser 645 650 655 Ile Glu Lys Thir Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Cys 660 665 67 O Val Tyr Thr Lieu Pro Pro Ser Arg Asp Glu Lieu. Thir Lys Asn Glin Val 675 68O 685 Ser Lieu. Thr Cys Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 69 O. 695 7 OO Glu Trp Glu Ser Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro 7 Os 71O 71s 72O Pro Val Lieu. Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Lieu. Thr 72 73 O 73 Val Asp Llys Ser Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val 740 74. 7 O Met His Glu Ala Lieu. His Asn His Tyr Thr Gln Lys Ser Leu Ser Lieu. 7ss 760 765 Ser Pro Gly Lys 770

<210s, SEQ ID NO 3 &211s LENGTH: 614 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 3 Met Arg Pro Pro Gln Cys Lieu. Lieu. His Thr Pro Ser Leu Ala Ser Pro 1. 5 1O 15 Lieu. Lieu. Lieu. Lieu Lleu Lleu Trp Lieu. Lieu. Gly Gly Gly Val Gly Ala Glu 2O 25 3O Gly Arg Glu Asp Ala Glu Lieu. Lieu Val Thr Val Arg Gly Gly Arg Lieu. 35 4 O 45 Arg Gly Ile Arg Lieu Lys Thr Pro Gly Gly Pro Val Ser Ala Phe Lieu SO 55 6 O Gly Ile Pro Phe Ala Glu Pro Pro Met Gly Pro Arg Arg Phe Leu Pro 65 70 7s 8O Pro Glu Pro Lys Gln Pro Trp Ser Gly Val Val Asp Ala Thr Thr Phe 85 90 95

Gln Ser Val Cys Tyr Glin Tyr Val Asp Thr Lieu. Tyr Pro Gly Phe Glu 1OO 105 11 O

Gly Thr Glu Met Trp Asn Pro Asn Arg Glu Lieu. Ser Glu Asp Cys Lieu. 115 12 O 125

Tyr Lieu. Asn Val Trp Thr Pro Tyr Pro Arg Pro Thr Ser Pro Thr Pro 13 O 135 14 O

Val Lieu Val Trp Ile Tyr Gly Gly Gly Phe Tyr Ser Gly Ala Ser Ser 145 150 155 160

Lieu. Asp Val Tyr Asp Gly Arg Phe Lieu Val Glin Ala Glu Arg Thr Val 1.65 17O 17s US 2016/01 84445 A1 Jun. 30, 2016 68

- Continued

Lieu Val Ser Met Asn Tyr Arg Val Gly Ala Phe Gly Phe Lieu Ala Lieu. 18O 185 19 O Pro Gly Ser Arg Glu Ala Pro Gly Asn Val Gly Lieu. Lieu. Asp Glin Arg 195 2OO 2O5 Lieu Ala Lieu Gln Trp Val Glin Glu Asn. Wall Ala Ala Phe Gly Gly Asp 21 O 215 22O Pro Thir Ser Val Thr Lieu Phe Gly Glu Ser Ala Gly Ala Ala Ser Val 225 23 O 235 24 O Gly Met His Lieu Lleu Ser Pro Pro Ser Arg Gly Lieu. Phe His Arg Ala 245 250 255 Val Lieu. Glin Ser Gly Ala Pro Asn Gly Pro Trp Ala Thr Val Gly Met 26 O 265 27 O Gly Glu Ala Arg Arg Arg Ala Thr Glin Lieu Ala His Lieu Val Gly Cys 27s 28O 285 Pro Pro Gly Gly Thr Gly Gly Asn Asp Thr Glu Lieu Val Ala Cys Lieu. 29 O 295 3 OO Arg Thr Arg Pro Ala Glin Val Lieu Val Asn His Glu Trp His Val Lieu 3. OS 310 315 32O Pro Glin Glu Ser Val Phe Arg Phe Ser Phe Val Pro Val Val Asp Gly 3.25 330 335 Asp Phe Lieu. Ser Asp Thr Pro Glu Ala Lieu. Ile Asn Ala Gly Asp Phe 34 O 345 35. O His Gly Lieu. Glin Val Lieu Val Gly Val Val Lys Asp Glu Gly Ser Tyr 355 360 365 Phe Lieu Val Tyr Gly Ala Pro Gly Phe Ser Lys Asp Asn. Glu Ser Lieu. 37 O 375 38O Ile Ser Arg Ala Glu Phe Lieu Ala Gly Val Arg Val Gly Val Pro Glin 385 390 395 4 OO Val Ser Asp Lieu Ala Ala Glu Ala Val Val Lieu. His Tyr Thr Asp Trip 4 OS 41O 415 Lieu. His Pro Glu Asp Pro Ala Arg Lieu. Arg Glu Ala Lieu. Ser Asp Wall 42O 425 43 O Val Gly Asp His Asn Val Val Cys Pro Val Ala Glin Lieu Ala Gly Arg 435 44 O 445 Lieu Ala Ala Glin Gly Ala Arg Val Tyr Ala Tyr Val Phe Glu. His Arg 450 45.5 460 Ala Ser Thr Lieu Ser Trp Pro Leu Trp Met Gly Val Pro His Gly Tyr 465 470 47s 48O Glu Ile Glu Phe Ile Phe Gly Ile Pro Leu Asp Pro Ser Arg Asn Tyr 485 490 495 Thir Ala Glu Glu Lys Ile Phe Ala Glin Arg Lieu Met Arg Tyr Trp Ala SOO 505 51O

Asn Phe Ala Arg Thr Gly Asp Pro Asn. Glu Pro Arg Asp Pro Lys Ala 515 52O 525

Pro Gln Trp Pro Pro Tyr Thr Ala Gly Ala Glin Glin Tyr Val Ser Lieu. 53 O 535 54 O

Asp Lieu. Arg Pro Lieu. Glu Val Arg Arg Gly Lieu. Arg Ala Glin Ala Cys 5.45 550 555 560

Ala Phe Trp Asn Arg Phe Lieu Pro Llys Lieu. Lieu. Ser Ala Thr Asp Thr 565 st O sts US 2016/01 84445 A1 Jun. 30, 2016 69

- Continued Lieu. Asp Glu Ala Glu Arg Gln Trp Lys Ala Glu Phe His Arg Trp Ser 58O 585 59 O Ser Tyr Met Val His Trp Lys Asn Glin Phe Asp His Tyr Ser Lys Glin 595 6OO 605 Asp Arg Cys Ser Asp Lieu. 610

<210s, SEQ ID NO 4 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 4 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1. 5 1O

<210s, SEQ ID NO 5 &211s LENGTH: 329 212. TYPE: PRT <213> ORGANISM: Desulfairculus baarsii

<4 OOs, SEQUENCE: 5 Met Ala Ala Lys Met Val Asn. Thr Val Ala Gly Pro Val Ser Ala Asp 1. 5 1O 15 Glu Lieu. Gly Lieu. Thir Lieu Met His Glu. His Ile Val Phe Gly Tyr Pro 2O 25 3O Gly Trp Asn Gly Asp Val Thir Lieu. Gly Ala Phe Asp Arg Pro Ala Ala 35 4 O 45 Val Lys Glin Ala Val Glu Thir Lieu. Ser Ala Lieu Lys Glin Ala Phe Gly SO 55 6 O Lieu. Gly. Thir Lieu Val Asp Ala Thr Pro Asn. Glu Thr Gly Arg Asp Pro 65 70 7s 8O Lieu. Lieu. Lieu Lys Glu Val Ser Glu Lys Ser Gly Val Asn. Ile Val Cys 85 90 95 Ser Thr Gly Tyr Tyr Ser Glin Ala Glu Gly Gly Ala Ala Tyr Phe Ala 1OO 105 11 O Phe Arg Ala Ser Lieu. Gly Asp Ala Val Ala Glu Ile Arg Glu Met Phe 115 12 O 125 Lieu. Thr Glu Lieu. Thir Lys Gly Val Ala Asp Thr Gly Val Arg Pro Gly 13 O 135 14 O Val Ile Llys Lieu Ala Ser Ser Glin Gly Glin Ile Thr Asp Tyr Glu Lys 145 150 155 160 Met Phe Phe Thr Ala Ala Val Ala Ala Glin Lys Glu Thr Gly Ala Pro 1.65 17O 17s Ile Ile Thr His Thr Glu. His Gly Thr Met Gly Pro Glu Glin Ala Lys 18O 185 19 O Phe Lieu. Lieu. Glu Lieu. Gly Ala Asp Pro Lys Arg Thr Met Ile Gly His 195 2OO 2O5

Met Cys Asp Asn Lieu. Asp Lieu. Asp Tyr Glin Glu Ala Val Lieu. Arg Glin 21 O 215 22O

Gly Val Tyr Val Ser Trp Asp Arg Met Gly Lieu. Glin Gly Lieu Ala Gly 225 23 O 235 24 O US 2016/01 84445 A1 Jun. 30, 2016 70

- Continued

Cys Pro Met Glu Ala Thr Arg Tyr Pro Val Lieu. Asn Glu Lieu. Ile Glin 245 250 255 Arg Gly Trp Ala Lys Glin Lieu Met Lieu. Ser His Asp Ser Ile Asn Thr 26 O 265 27 O Trp Lieu. Gly Arg Pro Lieu. Ser Ile Pro Glu Ala Ala Lieu Pro Met Val 27s 28O 285 Ile Asp Trp Arg Pro Asp His Ile Phe Asn Llys Val Ala Pro Ala Lieu. 29 O 295 3 OO Lieu Ala Gly Gly Ala Thr Glin Ala Asp Lieu. Asp Val Ile Lieu Lys Asp 3. OS 310 315 32O Asn Pro Arg Arg Lieu. Phe Ala Gly Val 3.25

<210s, SEQ ID NO 6 &211s LENGTH: 355 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 6 Met Ala Lys Lieu. Ile Ala Lieu. Thir Lieu. Lieu. Gly Met Gly Lieu Ala Lieu. 1. 5 1O 15 Phe Arg Asn His Glin Ser Ser Tyr Glin Thr Arg Lieu. Asn Ala Lieu. Arg 2O 25 3O Glu Val Glin Pro Val Glu Lieu Pro Asn. Cys Asn Lieu Val Lys Gly Ile 35 4 O 45 Glu Thr Gly Ser Glu Asp Lieu. Glu Ile Lieu Pro Asn Gly Lieu Ala Phe SO 55 6 O Ile Ser Ser Gly Lieu Lys Tyr Pro Gly Ile Llys Ser Phe Asin Pro Asn 65 70 7s 8O Ser Pro Gly Lys Ile Lieu. Lieu Met Asp Lieu. Asn. Glu Glu Asp Pro Thr 85 90 95 Val Lieu. Glu Lieu. Gly Ile Thr Gly Ser Llys Phe Asp Val Ser Ser Phe 1OO 105 11 O Asn Pro His Gly Ile Ser Thr Phe Thr Asp Glu Asp Asn Ala Met Tyr 115 12 O 125 Lieu. Lieu Val Val Asn His Pro Asp Ala Lys Ser Thr Val Glu Lieu. Phe 13 O 135 14 O Llys Phe Glin Glu Glu Glu Lys Ser Lieu. Lieu. His Lieu Lys Thir Ile Arg 145 150 155 160 His Llys Lieu. Lieu Pro Asn Lieu. Asn Asp Ile Val Ala Val Gly Pro Glu 1.65 17O 17s His Phe Tyr Gly Thr Asn Asp His Tyr Phe Lieu. Asp Pro Tyr Lieu. Glin 18O 185 19 O Ser Trp Glu Met Tyr Lieu. Gly Lieu Ala Trp Ser Tyr Val Val Tyr Tyr 195 2OO 2O5

Ser Pro Ser Glu Val Arg Val Val Ala Glu Gly Phe Asp Phe Ala Asn 21 O 215 22O

Gly Ile Asn. Ile Ser Pro Asp Gly Llys Tyr Val Tyr Ile Ala Glu Lieu. 225 23 O 235 24 O

Lieu Ala His Lys Ile His Val Tyr Glu Lys His Ala Asn Trp Thir Lieu. 245 250 255

Thr Pro Lieu Lys Ser Lieu. Asp Phe Asn. Thir Lieu Val Asp Asn. Ile Ser 26 O 265 27 O US 2016/01 84445 A1 Jun. 30, 2016 71

- Continued

Val Asp Pro Glu Thr Gly Asp Lieu. Trp Val Gly Cys His Pro Asn Gly 27s 28O 285 Met Lys Ile Phe Phe Tyr Asp Ser Glu Asn Pro Pro Ala Ser Glu Val 29 O 295 3 OO Lieu. Arg Ile Glin Asn. Ile Lieu. Thr Glu Glu Pro Llys Val Thr Glin Val 3. OS 310 315 32O Tyr Ala Glu Asn Gly Thr Val Lieu. Glin Gly Ser Thr Val Ala Ser Val 3.25 330 335 Tyr Lys Gly Lys Lieu. Lieu. Ile Gly Thr Val Phe His Lys Ala Lieu. Tyr 34 O 345 35. O Cys Glu Lieu. 355

<210s, SEQ ID NO 7 &211s LENGTH: 314 212. TYPE: PRT <213> ORGANISM: Loligo vulgaris

<4 OO > SEQUENCE: 7 Met Glu Ile Pro Val Ile Glu Pro Leu Phe Thr Lys Val Thr Glu Asp 1. 5 1O 15 Ile Pro Gly Ala Glu Gly Pro Val Phe Asp Lys Asn Gly Asp Phe Tyr 2O 25 3O Ile Val Ala Pro Glu Val Glu Val ASn Gly Llys Pro Ala Gly Glu Ile 35 4 O 45 Lieu. Arg Ile Asp Lieu Lys Thr Gly Lys Llys Thr Val Ile Cys Llys Pro SO 55 6 O Glu Val Asn Gly Tyr Gly Gly Ile Pro Ala Gly Cys Glin Cys Asp Arg 65 70 7s 8O Asp Ala Asn. Glin Lieu. Phe Val Ala Asp Met Arg Lieu. Gly Lieu. Lieu Val 85 90 95 Val Glin Thir Asp Gly. Thir Phe Glu Glu Ile Ala Lys Lys Asp Ser Glu 1OO 105 11 O Gly Arg Arg Met Glin Gly Cys Asn Asp Cys Ala Phe Asp Tyr Glu Gly 115 12 O 125 Asn Lieu. Trp Ile Thr Ala Pro Ala Gly Glu Val Ala Pro Ala Asp Tyr 13 O 135 14 O Thr Arg Ser Met Glin Glu Lys Phe Gly Ser Ile Tyr Cys Phe Thir Thr 145 150 155 160 Asp Gly Gln Met Ile Glin Val Asp Thr Ala Phe Glin Phe Pro Asn Gly 1.65 17O 17s Ile Ala Val Arg His Met Asn Asp Gly Arg Pro Tyr Glin Lieu. Ile Val 18O 185 19 O

Ala Glu Thr Pro Thr Lys Llys Lieu. Trp Ser Tyr Asp Ile Lys Gly Pro 195 2OO 2O5 Ala Lys Ile Glu Asn Llys Llys Val Trp Gly His Ile Pro Gly. Thir His 21 O 215 22O

Glu Gly Gly Ala Asp Gly Met Asp Phe Asp Glu Asp Asn. Asn Lieu. Lieu. 225 23 O 235 24 O Val Ala Asn Trp Gly Ser Ser His Ile Glu Val Phe Gly Pro Asp Gly 245 250 255

Gly Glin Pro Llys Met Arg Ile Arg Cys Pro Phe Glu Lys Pro Ser Asn US 2016/01 84445 A1 Jun. 30, 2016 72

- Continued

26 O 265 27 O Lieu. His Phe Llys Pro Gln Thr Lys Thr Ile Phe Val Thr Glu. His Glu 27s 28O 285 Asn Asn Ala Val Trp Llys Phe Glu Trp Glin Arg Asn Gly Lys Lys Glin 29 O 295 3 OO Tyr Cys Glu Thir Lieu Lys Phe Gly Ile Phe 3. OS 310

<210s, SEQ ID NO 8 &211s LENGTH: 227 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 8 Asp Llys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lieu. Leu Gly 1. 5 1O 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Llys Pro Lys Asp Thr Lieu Met 2O 25 3O Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 4 O 45 Glu Asp Pro Glu Val Llys Phe Asn Trp Tyr Val Asp Gly Val Glu Val SO 55 6 O His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 7s 8O Arg Val Val Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ala Pro Ile 1OO 105 11 O Glu Lys Thir Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin Val 115 12 O 125 Tyr Thr Lieu Pro Pro Ser Arg Asp Glu Lieu. Thir Lys Asn Glin Val Ser 13 O 135 14 O Lieu. Thir Cys Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp. Glu Ser Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 1.65 17O 17s Val Lieu. Asp Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Llys Lieu. Thr Val 18O 185 19 O Asp Llys Ser Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val Met 195 2OO 2O5 His Glu Ala Lieu. His Asn His Tyr Thr Glin Llys Ser Lieu. Ser Lieu. Ser 21 O 215 22O Pro Gly Lys 225

<210s, SEQ ID NO 9 &211s LENGTH: 24 6 O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polynucleotide

<4 OOs, SEQUENCE: 9 gctago.gc.cg ccaccatggc atgcct ciga tittcaaagac ataaag.ccca gttgaacct C 6 O

US 2016/01 84445 A1 Jun. 30, 2016 74

- Continued alaga.gc.cggt ggcagcaggg gaacgtgttt agctgcagog tatgcatga agctctgcat 24 OO aatcattaca citcagaaaag cct gtcqctic ticgc.ccggaa agtaataata cqtagaattic 246 O

<210s, SEQ ID NO 10 &211s LENGTH: 534 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 10 Glu Asp Asp Ile Ile Ile Ala Thir Lys Asn Gly Llys Val Arg Gly Met 1. 5 1O 15 Asn Lieu. Thr Val Phe Gly Gly Thr Val Thr Ala Phe Leu Gly Ile Pro 2O 25 3O Tyr Ala Glin Pro Pro Lieu. Gly Arg Lieu. Arg Phe Llys Llys Pro Glin Ser 35 4 O 45 Lieu. Thir Lys Trp Ser Asp Ile Trp Asn Ala Thr Llys Tyr Ala Asn. Ser SO 55 6 O Cys Tyr Glin Asn Ile Asp Glin Ser Phe Pro Gly Phe His Gly Ser Glu 65 70 7s 8O Met Trp Asn Pro Asn. Thir Asp Lieu. Ser Glu Asp Cys Lieu. Tyr Lieu. Asn 85 90 95 Val Trp Ile Pro Ala Pro Llys Pro Lys Asn Ala Thr Val Lieu. Ile Trp 1OO 105 11 O Ile Tyr Gly Gly Gly Phe Glin Thr Gly. Thir Ser Ser Lieu. His Val Tyr 115 12 O 125 Asp Gly Llys Phe Lieu Ala Arg Val Glu Arg Val Ile Val Val Ser Met 3 O 135 14 O Asn Tyr Arg Val Gly Ala Lieu. Gly Phe Lieu Ala Lieu Pro Gly Asn Pro 145 150 155 160 Glu Ala Pro Gly Asn Met Gly Lieu. Phe Asp Glin Glin Lieu Ala Lieu. Glin 1.65 17O 17s Trp Val Glin Lys Asn. Ile Ala Ala Phe Gly Gly ASn Pro Llys Ser Val 18O 185 19 O Thir Lieu. Phe Gly Glu Ser Ala Gly Ala Ala Ser Val Ser Lieu. His Lieu. 195 2OO 2O5 Lieu. Ser Pro Gly Ser His Ser Leu Phe Thr Arg Ala Ile Leu Glin Ser 21 O 215 22O Gly Ser Phe Asn Ala Pro Trp Ala Val Thir Ser Lieu. Tyr Glu Ala Arg 225 23 O 235 24 O Asn Arg Thir Lieu. Asn Lieu Ala Lys Lieu. Thr Gly Cys Ser Arg Glu Asn 245 250 255

Glu Thr Glu Ile Ile Llys Cys Lieu. Arg Asn Lys Asp Pro Glin Glu Ile 26 O 265 27 O

Lieu. Lieu. Asn Glu Ala Phe Val Val Pro Tyr Gly Thr Pro Leu Ser Val 27s 28O 285

Asn Phe Gly Pro Thr Val Asp Gly Asp Phe Lieu. Thr Asp Met Pro Asp 29 O 295 3 OO

Ile Lieu. Lieu. Glu Lieu. Gly Glin Phe Llys Llys Thr Glin Ile Lieu Val Gly 3. OS 310 315 32O US 2016/01 84445 A1 Jun. 30, 2016 75

- Continued Val Asn Lys Asp Glu Gly Thr Ala Phe Lieu Val Tyr Gly Ala Pro Gly 3.25 330 335 Phe Ser Lys Asp Asn. Asn. Ser Ile Ile Thr Arg Lys Glu Phe Glin Glu 34 O 345 35. O Gly Lieu Lys Ile Phe Phe Pro Gly Val Ser Glu Phe Gly Lys Glu Ser 355 360 365 Ile Lieu. Phe His Tyr Thr Asp Trp Val Asp Asp Glin Arg Pro Glu Asn 37 O 375 38O Tyr Arg Glu Ala Lieu. Gly Asp Val Val Gly Asp Tyr Asn. Phe Ile Cys 385 390 395 4 OO Pro Ala Lieu. Glu Phe Thr Llys Llys Phe Ser Glu Trp Gly Asn. Asn Ala 4 OS 41O 415 Phe Phe Tyr Tyr Phe Glu. His Arg Ser Ser Lys Lieu Pro Trp Pro Glu 42O 425 43 O Trp Met Gly Val Met His Gly Tyr Glu Ile Glu Phe Val Phe Gly Lieu. 435 44 O 445 Pro Lieu. Glu Arg Arg Asp Asn Tyr Thr Lys Ala Glu Glu Ile Lieu. Ser 450 45.5 460 Arg Ser Ile Val Lys Arg Trp Ala Asn. Phe Ala Lys Tyr Gly Asn Pro 465 470 47s 48O Asn Glu Thr Glin Asn Asn Ser Thr Ser Trp Pro Val Phe Llys Ser Thr 485 490 495 Glu Gln Llys Tyr Lieu. Thir Lieu. ASn Thr Glu Ser Thr Arg Ile Met Thr SOO 505 51O Lys Lieu. Arg Ala Glin Gln Cys Arg Phe Trp Thr Ser Phe Phe Pro Llys 515 52O 525 Val Lieu. Glu Met Thr Gly 53 O

<210s, SEQ ID NO 11 &211s LENGTH: 524 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 11 Glu Asp Asp Ile Ile Ile Ala Thir Lys Asn Gly Llys Val Arg Gly Met 1. 5 1O 15 Asn Lieu. Thr Val Phe Gly Gly Thr Val Thr Ala Phe Leu Gly Ile Pro 2O 25 3O Tyr Ala Glin Pro Pro Lieu. Gly Arg Lieu. Arg Phe Llys Llys Pro Glin Ser 35 4 O 45

Lieu. Thir Lys Trp Ser Asp Ile Trp Asn Ala Thr Llys Tyr Ala Asn. Ser SO 55 6 O Cys Tyr Glin Asn Ile Asp Glin Ser Phe Pro Gly Phe His Gly Ser Glu 65 70 7s 8O

Met Trp Asn Pro Asn. Thir Asp Lieu. Ser Glu Asp Cys Lieu. Tyr Lieu. Asn 85 90 95

Val Trp Ile Pro Ala Pro Llys Pro Lys Asn Ala Thr Val Lieu. Ile Trp 1OO 105 11 O Ile Tyr Gly Gly Gly Phe Glin Thr Gly. Thir Ser Ser Lieu. His Val Tyr 115 12 O 125 US 2016/01 84445 A1 Jun. 30, 2016 76

- Continued

Asp Gly Llys Phe Lieu Ala Arg Val Glu Arg Val Ile Val Val Ser Met 13 O 135 14 O Asn Tyr Arg Val Gly Ala Lieu. Gly Phe Lieu Ala Lieu Pro Gly Asn Pro 145 150 155 160 Glu Ala Pro Gly Asn Met Gly Lieu. Phe Asp Glin Glin Lieu Ala Lieu. Glin 1.65 17O 17s Trp Val Glin Lys Asn. Ile Ala Ala Phe Gly Gly ASn Pro Llys Ser Val 18O 185 19 O Thir Lieu. Phe Gly Glu Ser Ala Gly Ala Ala Ser Val Ser Lieu. His Lieu. 195 2OO 2O5 Lieu. Ser Pro Gly Ser His Ser Leu Phe Thr Arg Ala Ile Leu Glin Ser 21 O 215 22O Gly Ser Phe Asn Ala Pro Trp Ala Val Thir Ser Lieu. Tyr Glu Ala Arg 225 23 O 235 24 O Asn Arg Thir Lieu. Asn Lieu Ala Lys Lieu. Thr Gly Cys Ser Arg Glu Asn 245 250 255 Glu Thr Glu Ile Ile Llys Cys Lieu. Arg Asn Lys Asp Pro Glin Glu Ile 26 O 265 27 O Lieu. Lieu. Asn Glu Ala Phe Val Val Pro Tyr Gly Thr Pro Leu Ser Val 27s 28O 285 Asn Phe Gly Pro Thr Val Asp Gly Asp Phe Lieu. Thr Asp Met Pro Asp 29 O 295 3 OO Ile Lieu. Lieu. Glu Lieu. Gly Glin Phe Llys Llys Thr Glin Ile Lieu Val Gly 3. OS 310 315 32O Val Asn Lys Asp Glu Gly Thr Ala Phe Lieu Val Tyr Gly Ala Pro Gly 3.25 330 335 Phe Ser Lys Asp Asn. Asn. Ser Ile Ile Thr Arg Lys Glu Phe Glin Glu 34 O 345 35. O Gly Lieu Lys Ile Phe Phe Pro Gly Val Ser Glu Phe Gly Lys Glu Ser 355 360 365 Ile Lieu. Phe His Tyr Thr Asp Trp Val Asp Asp Glin Arg Pro Glu Asn 37 O 375 38O Tyr Arg Glu Ala Lieu. Gly Asp Val Val Gly Asp Tyr Asn. Phe Ile Cys 385 390 395 4 OO Pro Ala Lieu. Glu Phe Thr Llys Llys Phe Ser Glu Trp Gly Asn. Asn Ala 4 OS 41O 415 Phe Phe Tyr Tyr Phe Glu. His Arg Ser Ser Lys Lieu Pro Trp Pro Glu 42O 425 43 O Trp Met Gly Val Met His Gly Tyr Glu Ile Glu Phe Val Phe Gly Lieu. 435 44 O 445 Pro Lieu. Glu Arg Arg Asp Asn Tyr Thr Lys Ala Glu Glu Ile Lieu. Ser 450 45.5 460

Arg Ser Ile Val Lys Arg Trp Ala Asn. Phe Ala Lys Tyr Gly Asn Pro 465 470 47s 48O

Asn Glu Thr Glin Asn Asn Ser Thr Ser Trp Pro Val Phe Llys Ser Thr 485 490 495

Glu Glin Llys Tyr Lieu. Thir Lieu. Asn Thr Glu Ser Thr Arg Ile Met Thr SOO 505 51O

Llys Lieu. Arg Ala Glin Glin Cys Arg Phe Trp Thir Ser 515 52O US 2016/01 84445 A1 Jun. 30, 2016 77

- Continued

<210s, SEQ ID NO 12 &211s LENGTH: 528 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 12 Glu Asp Asp Ile Ile Ile Ala Thir Lys Asn Gly Llys Val Arg Gly Met 1. 5 1O 15 Asn Lieu. Thr Val Phe Gly Gly Thr Val Thr Ala Phe Leu Gly Ile Pro 2O 25 3O Tyr Ala Glin Pro Pro Lieu. Gly Arg Lieu. Arg Phe Llys Llys Pro Glin Ser 35 4 O 45 Lieu. Thir Lys Trp Ser Asp Ile Trp Asn Ala Thr Llys Tyr Ala Asn. Ser SO 55 6 O Cys Tyr Glin Asn Ile Asp Glin Ser Phe Pro Gly Phe His Gly Ser Glu 65 70 7s 8O Met Trp Asn Pro Asn. Thir Asp Lieu. Ser Glu Asp Cys Lieu. Tyr Lieu. Asn 85 90 95 Val Trp Ile Pro Ala Pro Llys Pro Lys Asn Ala Thr Val Lieu. Ile Trp 1OO 105 11 O Ile Tyr Gly Gly Gly Phe Glin Thr Gly. Thir Ser Ser Lieu. His Val Tyr 115 120 125 Asp Gly Llys Phe Lieu Ala Arg Val Glu Arg Val Ile Val Val Ser Met 3 O 135 14 O Asn Tyr Arg Val Gly Ala Lieu. Gly Phe Lieu Ala Lieu Pro Gly Asn Pro 145 150 155 160 Glu Ala Pro Gly Asn Met Gly Lieu. Phe Asp Glin Glin Lieu Ala Lieu. Glin 1.65 17O 17s Trp Val Glin Lys Asn. Ile Ala Ala Phe Gly Gly ASn Pro Llys Ser Val 18O 185 19 O Thir Lieu. Phe Gly Glu Ser Ala Gly Ala Ala Ser Val Ser Lieu. His Lieu. 195 2OO 2O5 Lieu. Ser Pro Gly Ser His Ser Leu Phe Thr Arg Ala Ile Leu Glin Ser 21 O 215 22O Gly Ser Phe Asn Ala Pro Trp Ala Val Thir Ser Lieu. Tyr Glu Ala Arg 225 23 O 235 24 O Asn Arg Thir Lieu. Asn Lieu Ala Lys Lieu. Thr Gly Cys Ser Arg Glu Asn 245 250 255 Glu Thr Glu Ile Ile Llys Cys Lieu. Arg Asn Lys Asp Pro Glin Glu Ile 26 O 265 27 O

Lieu. Lieu. Asn Glu Ala Phe Val Val Pro Tyr Gly Thr Pro Leu Ser Val 27s 28O 285

Asn Phe Gly Pro Thr Val Asp Gly Asp Phe Lieu. Thr Asp Met Pro Asp 29 O 295 3 OO

Ile Lieu. Lieu. Glu Lieu. Gly Glin Phe Llys Llys Thr Glin Ile Lieu Val Gly 3. OS 310 315 32O

Val Asn Lys Asp Glu Gly Thr Ala Phe Lieu Val Tyr Gly Ala Pro Gly 3.25 330 335

Phe Ser Lys Asp Asn. Asn. Ser Ile Ile Thr Arg Lys Glu Phe Glin Glu 34 O 345 35. O US 2016/01 84445 A1 Jun. 30, 2016 78

- Continued

Gly Lieu Lys Ile Phe Phe Pro Gly Val Ser Glu Phe Gly Lys Glu Ser 355 360 365 Ile Lieu. Phe His Tyr Thr Asp Trp Val Asp Asp Glin Arg Pro Glu Asn 37 O 375 38O Tyr Arg Glu Ala Lieu. Gly Asp Val Val Gly Asp Tyr Asn. Phe Ile Cys 385 390 395 4 OO Pro Ala Lieu. Glu Phe Thr Llys Llys Phe Ser Glu Trp Gly Asn. Asn Ala 4 OS 41O 415 Phe Phe Tyr Tyr Phe Glu. His Arg Ser Ser Lys Lieu Pro Trp Pro Glu 42O 425 43 O Trp Met Gly Val Met His Gly Tyr Glu Ile Glu Phe Val Phe Gly Lieu. 435 44 O 445 Pro Lieu. Glu Arg Arg Asp Asn Tyr Thr Lys Ala Glu Glu Ile Lieu. Ser 450 45.5 460 Arg Ser Ile Val Lys Arg Trp Ala Asn. Phe Ala Lys Tyr Gly Asn Pro 465 470 47s 48O Asn Glu Thr Glin Asn Asn Ser Thr Ser Trp Pro Val Phe Llys Ser Thr 485 490 495 Glu Glin Llys Tyr Lieu. Thir Lieu. Asn Thr Glu Ser Thr Arg Ile Met Thr SOO 505 51O Llys Lieu. Arg Ala Glin Glin Cys Arg Phe Trp Thir Ser Gly Gly Gly Cys 515 52O 525

<210s, SEQ ID NO 13 &211s LENGTH: 537 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 13 Glu Asp Asp Ile Ile Ile Ala Thir Lys Asn Gly Llys Val Arg Gly Met 1. 5 1O 15 Asn Lieu. Thr Val Phe Gly Gly Thr Val Thr Ala Phe Leu Gly Ile Pro 2O 25 3O Tyr Ala Glin Pro Pro Lieu. Gly Arg Lieu. Arg Phe Llys Llys Pro Glin Ser 35 4 O 45 Lieu. Thir Lys Trp Ser Asp Ile Trp Asn Ala Thr Llys Tyr Ala Asn. Ser SO 55 6 O Cys Tyr Glin Asn Ile Asp Glin Ser Phe Pro Gly Phe His Gly Ser Glu 65 70 7s 8O Met Trp Asn Pro Asn. Thir Asp Lieu. Ser Glu Asp Cys Lieu. Tyr Lieu. Asn 85 90 95

Val Trp Ile Pro Ala Pro Llys Pro Lys Asn Ala Thr Val Lieu. Ile Trp 1OO 105 11 O Ile Tyr Gly Gly Gly Phe Glin Thr Gly. Thir Ser Ser Lieu. His Val Tyr 115 12 O 125

Asp Gly Llys Phe Lieu Ala Arg Val Glu Arg Val Ile Val Val Ser Met 3 O 135 14 O

Asn Tyr Arg Val Gly Ala Lieu. Gly Phe Lieu Ala Lieu Pro Gly Asn Pro 145 150 155 160

Glu Ala Pro Gly Asn Met Gly Lieu. Phe Asp Glin Glin Lieu Ala Lieu. Glin US 2016/01 84445 A1 Jun. 30, 2016 79

- Continued

1.65 17O 17s Trp Val Glin Lys Asn. Ile Ala Ala Phe Gly Gly ASn Pro Llys Ser Val 18O 185 19 O Thir Lieu. Phe Gly Glu Ser Ala Gly Ala Ala Ser Val Ser Lieu. His Lieu. 195 2OO 2O5 Lieu. Ser Pro Gly Ser His Ser Leu Phe Thr Arg Ala Ile Leu Glin Ser 21 O 215 22O Gly Ser Phe Asn Ala Pro Trp Ala Val Thir Ser Lieu. Tyr Glu Ala Arg 225 23 O 235 24 O Asn Arg Thir Lieu. Asn Lieu Ala Lys Lieu. Thr Gly Cys Ser Arg Glu Asn 245 250 255 Glu Thr Glu Ile Ile Llys Cys Lieu. Arg Asn Lys Asp Pro Glin Glu Ile 26 O 265 27 O Lieu. Lieu. Asn Glu Ala Phe Val Val Pro Tyr Gly Thr Pro Leu Ser Val 27s 28O 285 Asn Phe Gly Pro Thr Val Asp Gly Asp Phe Lieu. Thr Asp Met Pro Asp 29 O 295 3 OO Ile Lieu. Lieu. Glu Lieu. Gly Glin Phe Llys Llys Thr Glin Ile Lieu Val Gly 3. OS 310 315 32O Val Asn Lys Asp Glu Gly Thr Ala Phe Lieu Val Tyr Gly Ala Pro Gly 3.25 330 335 Phe Ser Lys Asp Asn. Asn. Ser Ile Ile Thr Arg Lys Glu Phe Glin Glu 34 O 345 35 O Gly Lieu Lys Ile Phe Phe Pro Gly Val Ser Glu Phe Gly Lys Glu Ser 355 360 365 Ile Lieu. Phe His Tyr Thr Asp Trp Val Asp Asp Glin Arg Pro Glu Asn 37 O 375 38O Tyr Arg Glu Ala Lieu. Gly Asp Val Val Gly Asp Tyr Asn. Phe Ile Cys 385 390 395 4 OO Pro Ala Lieu. Glu Phe Thr Llys Llys Phe Ser Glu Trp Gly Asn. Asn Ala 4 OS 41O 415 Phe Phe Tyr Tyr Phe Glu. His Arg Ser Ser Lys Lieu Pro Trp Pro Glu 42O 425 43 O Trp Met Gly Val Met His Gly Tyr Glu Ile Glu Phe Val Phe Gly Lieu. 435 44 O 445 Pro Lieu. Glu Arg Arg Asp Asn Tyr Thr Lys Ala Glu Glu Ile Lieu. Ser 450 45.5 460 Arg Ser Ile Val Lys Arg Trp Ala Asn. Phe Ala Lys Tyr Gly Asn Pro 465 470 47s 48O Asn Glu Thr Glin Asn Asn Ser Thr Ser Trp Pro Val Phe Llys Ser Thr 485 490 495

Glu Glin Llys Tyr Lieu. Thir Lieu. Asn Thr Glu Ser Thr Arg Ile Met Thr SOO 505 51O

Lys Lieu. Arg Ala Glin Gln Cys Arg Phe Trp Thr Ser Phe Phe Pro Llys 515 52O 525

Val Lieu. Glu Met Thr Gly Gly Gly Cys 53 O 535

<210s, SEQ ID NO 14 &211s LENGTH: 772 212. TYPE: PRT <213> ORGANISM: Artificial Sequence US 2016/01 84445 A1 Jun. 30, 2016 80

- Continued

22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 14 Glu Asp Asp Ile Ile Ile Ala Thir Lys Asn Gly Llys Val Arg Gly Met 1. 5 1O 15 Asn Lieu. Thr Val Phe Gly Gly Thr Val Thr Ala Phe Leu Gly Ile Pro 2O 25 3O Tyr Ala Glin Pro Pro Lieu. Gly Arg Lieu. Arg Phe Llys Llys Pro Glin Ser 35 4 O 45 Lieu. Thir Lys Trp Ser Asp Ile Trp Asn Ala Thr Llys Tyr Ala Asn. Ser SO 55 6 O Cys Tyr Glin Asn Ile Asp Glin Ser Phe Pro Gly Phe His Gly Ser Glu 65 70 7s 8O Met Trp Asn Pro Asn. Thir Asp Lieu. Ser Glu Asp Cys Lieu. Tyr Lieu. Asn 85 90 95 Val Trp Ile Pro Ala Pro Llys Pro Lys Asn Ala Thr Val Lieu. Ile Trp 1OO 105 11 O Ile Tyr Gly Gly Gly Phe Glin Thr Gly. Thir Ser Ser Lieu. His Val Tyr 115 12 O 125 Asp Gly Llys Phe Lieu Ala Arg Val Glu Arg Val Ile Val Val Ser Met 3 O 135 14 O ASn Tyr Arg Val Gly Ala Lieu. Gly Phe Lieu Ala Lieu Pro Gly ASn Pro 145 150 155 160 Glu Ala Pro Gly Asn Met Gly Lieu. Phe Asp Glin Glin Lieu Ala Lieu. Glin 1.65 17O 17s Trp Val Glin Lys Asn. Ile Ala Ala Phe Gly Gly ASn Pro Llys Ser Val 18O 185 19 O Thir Lieu. Phe Gly Glu Ser Ala Gly Ala Ala Ser Val Ser Lieu. His Lieu. 195 2OO 2O5 Lieu. Ser Pro Gly Ser His Ser Leu Phe Thr Arg Ala Ile Leu Glin Ser 21 O 215 22O Gly Ser Phe Asn Ala Pro Trp Ala Val Thir Ser Lieu. Tyr Glu Ala Arg 225 23 O 235 24 O Asn Arg Thir Lieu. Asn Lieu Ala Lys Lieu. Thr Gly Cys Ser Arg Glu Asn 245 250 255 Glu Thr Glu Ile Ile Llys Cys Lieu. Arg Asn Lys Asp Pro Glin Glu Ile 26 O 265 27 O Lieu. Lieu. Asn Glu Ala Phe Val Val Pro Tyr Gly Thr Pro Leu Ser Val 27s 28O 285 Asn Phe Gly Pro Thr Val Asp Gly Asp Phe Lieu. Thr Asp Met Pro Asp 29 O 295 3 OO

Ile Lieu. Lieu. Glu Lieu. Gly Glin Phe Llys Llys Thr Glin Ile Lieu Val Gly 3. OS 310 315 32O

Val Asn Lys Asp Glu Gly Thr Ala Phe Lieu Val Tyr Gly Ala Pro Gly 3.25 330 335

Phe Ser Lys Asp Asn. Asn. Ser Ile Ile Thr Arg Lys Glu Phe Glin Glu 34 O 345 35. O

Gly Lieu Lys Ile Phe Phe Pro Gly Val Ser Glu Phe Gly Lys Glu Ser 355 360 365 Ile Lieu. Phe His Tyr Thr Asp Trp Val Asp Asp Glin Arg Pro Glu Asn US 2016/01 84445 A1 Jun. 30, 2016 81

- Continued

37 O 375 38O Tyr Arg Glu Ala Lieu. Gly Asp Val Val Gly Asp Tyr Asn. Phe Ile Cys 385 390 395 4 OO Pro Ala Lieu. Glu Phe Thr Llys Llys Phe Ser Glu Trp Gly Asn. Asn Ala 4 OS 41O 415 Phe Phe Tyr Tyr Phe Glu. His Arg Ser Ser Lys Lieu Pro Trp Pro Glu 42O 425 43 O Trp Met Gly Val Met His Gly Tyr Glu Ile Glu Phe Val Phe Gly Lieu. 435 44 O 445 Pro Lieu. Glu Arg Arg Asp Asn Tyr Thr Lys Ala Glu Glu Ile Lieu. Ser 450 45.5 460 Arg Ser Ile Val Lys Arg Trp Ala Asn. Phe Ala Lys Tyr Gly Asn Pro 465 470 47s 48O Asn Glu Thr Glin Asn Asn Ser Thr Ser Trp Pro Val Phe Llys Ser Thr 485 490 495 Glu Glin Llys Tyr Lieu. Thir Lieu. Asn Thr Glu Ser Thr Arg Ile Met Thr SOO 505 51O Lys Lieu. Arg Ala Glin Gln Cys Arg Phe Trp Thr Ser Phe Phe Pro Llys 515 52O 525 Val Lieu. Glu Met Thr Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 53 O 535 54 O Ser Asp Llys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lieu. Leu 545 550 555 560 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Llys Pro Lys Asp Thr Lieu. 565 st O sts Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 58O 585 59 O His Glu Asp Pro Glu Val Llys Phe Asn Trp Tyr Val Asp Gly Val Glu 595 6OO 605 Val His Asn Ala Lys Thr Llys Pro Arg Glu Glu Glin Tyr Asn. Ser Thr 610 615 62O Tyr Arg Val Val Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn 625 630 635 64 O Gly Lys Glu Tyr Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ala Pro 645 650 655 Ile Glu Lys Thir Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin 660 665 67 O Val Tyr Thr Lieu Pro Pro Ser Arg Asp Glu Lieu. Thir Lys Asn Glin Val 675 68O 685 Ser Lieu. Thr Cys Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 69 O. 695 7 OO

Glu Trp Glu Ser Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro 7 Os 71O 71s 72O

Pro Val Lieu. Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Lieu. Thr 72 73 O 73

Val Asp Llys Ser Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val 740 74. 7 O

Met His Glu Ala Lieu. His Asn His Tyr Thr Gln Lys Ser Leu Ser Lieu. 7ss 760 765

Ser Pro Gly Lys 770 US 2016/01 84445 A1 Jun. 30, 2016 82

- Continued

<210s, SEQ ID NO 15 &211s LENGTH: 772 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 15 Glu Asp Asp Ile Ile Ile Ala Thir Lys Asn Gly Llys Val Arg Gly Met 1. 5 1O 15 Asn Lieu. Thr Val Phe Gly Gly Thr Val Thr Ala Phe Leu Gly Ile Pro 2O 25 3O Tyr Ala Glin Pro Pro Lieu. Gly Arg Lieu. Arg Phe Llys Llys Pro Glin Ser 35 4 O 45 Lieu. Thir Lys Trp Ser Asp Ile Trp Asn Ala Thr Llys Tyr Ala Asn. Ser SO 55 6 O Cys Tyr Glin Asn Ile Asp Glin Ser Phe Pro Gly Phe His Gly Ser Glu 65 70 7s 8O Met Trp Asn Pro Asn. Thir Asp Lieu. Ser Glu Asp Cys Lieu. Tyr Lieu. Asn 85 90 95 Val Trp Ile Pro Ala Pro Llys Pro Lys Asn Ala Thr Val Lieu. Ile Trp 1OO 105 11 O Ile Tyr Gly Gly Gly Phe Glin Thr Gly. Thir Ser Ser Lieu. His Val Tyr 115 12 O 125 Asp Gly Llys Phe Lieu Ala Arg Val Glu Arg Val Ile Val Val Ser Met 3 O 135 14 O Asn Tyr Arg Val Gly Ala Lieu. Gly Phe Lieu Ala Lieu Pro Gly Asn Pro 145 150 155 160 Glu Ala Pro Gly Asn Met Gly Lieu. Phe Asp Glin Glin Lieu Ala Lieu. Glin 1.65 17O 17s Trp Val Glin Lys Asn. Ile Ala Ala Phe Gly Gly ASn Pro Llys Ser Val 18O 185 19 O Thir Lieu. Phe Gly Glu Ser Ala Gly Ala Ala Ser Val Ser Lieu. His Lieu. 195 2OO 2O5 Lieu. Ser Pro Gly Ser His Ser Leu Phe Thr Arg Ala Ile Leu Glin Ser 21 O 215 22O Gly Ser Phe Asn Ala Pro Trp Ala Val Thir Ser Lieu. Tyr Glu Ala Arg 225 23 O 235 24 O Asn Arg Thir Lieu. Asn Lieu Ala Lys Lieu. Thr Gly Cys Ser Arg Glu Asn 245 250 255 Glu Thr Glu Ile Ile Llys Cys Lieu. Arg Asn Lys Asp Pro Glin Glu Ile 26 O 265 27 O

Lieu. Lieu. Asn Glu Ala Phe Val Val Pro Tyr Gly Thr Pro Leu Ser Val 27s 28O 285

Asn Phe Gly Pro Thr Val Asp Gly Asp Phe Lieu. Thr Asp Met Pro Asp 29 O 295 3 OO

Ile Lieu. Lieu. Glu Lieu. Gly Glin Phe Llys Llys Thr Glin Ile Lieu Val Gly 3. OS 310 315 32O

Val Asn Lys Asp Glu Gly Thr Ala Phe Lieu Val Tyr Gly Ala Pro Gly 3.25 330 335

Phe Ser Lys Asp Asn. Asn. Ser Ile Ile Thr Arg Lys Glu Phe Glin Glu US 2016/01 84445 A1 Jun. 30, 2016 83

- Continued

34 O 345 35. O Gly Lieu Lys Ile Phe Phe Pro Gly Val Ser Glu Phe Gly Lys Glu Ser 355 360 365 Ile Lieu. Phe His Tyr Thr Asp Trp Val Asp Asp Glin Arg Pro Glu Asn 37 O 375 38O Tyr Arg Glu Ala Lieu. Gly Asp Val Val Gly Asp Tyr Asn. Phe Ile Cys 385 390 395 4 OO Pro Ala Lieu. Glu Phe Thr Llys Llys Phe Ser Glu Trp Gly Asn. Asn Ala 4 OS 41O 415 Phe Phe Tyr Tyr Phe Glu. His Arg Ser Ser Lys Lieu Pro Trp Pro Glu 42O 425 43 O Trp Met Gly Val Met His Gly Tyr Glu Ile Glu Phe Val Phe Gly Lieu. 435 44 O 445 Pro Lieu. Glu Arg Arg Asp Asn Tyr Thr Lys Ala Glu Glu Ile Lieu. Ser 450 45.5 460 Arg Ser Ile Val Lys Arg Trp Ala Asn. Phe Ala Lys Tyr Gly Asn Pro 465 470 47s 48O Asn Glu Thr Glin Asn Asn Ser Thr Ser Trp Pro Val Phe Llys Ser Thr 485 490 495 Glu Glin Llys Tyr Lieu. Thir Lieu. Asn Thr Glu Ser Thr Arg Ile Met Thr SOO 505 51O Lys Lieu. Arg Ala Glin Gln Cys Arg Phe Trp Thr Ser Phe Phe Pro Llys 515 52O 525 Val Lieu. Glu Met Thr Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 53 O 535 54 O Ser Asp Llys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu 5.45 550 555 560 Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Llys Pro Lys Asp Thr Lieu. 565 st O sts Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 58O 585 59 O His Glu Asp Pro Glu Val Llys Phe Asn Trp Tyr Val Asp Gly Val Glu 595 6OO 605 Val His Asn Ala Lys Thr Llys Pro Arg Glu Glu Glin Tyr Asn. Ser Thr 610 615 62O Tyr Arg Val Val Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn 625 630 635 64 O Gly Lys Glu Tyr Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ser Ser 645 650 655 Ile Glu Lys Thir Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin 660 665 67 O

Val Tyr Thr Lieu Pro Pro Ser Arg Asp Glu Lieu. Thir Lys Asn Glin Val 675 68O 685

Ser Lieu. Thr Cys Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 69 O. 695 7 OO

Glu Trp Glu Ser Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro 7 Os 71O 71s 72O

Pro Val Lieu. Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Lieu. Thr 72 73 O 73

Val Asp Llys Ser Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val 740 74. 7 O US 2016/01 84445 A1 Jun. 30, 2016 84

- Continued

Met His Glu Ala Lieu. His Asn His Tyr Thr Gln Lys Ser Leu Ser Lieu. 7ss 760 765 Ser Pro Gly Lys 770

<210s, SEQ ID NO 16 &211s LENGTH: 772 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 16 Glu Asp Asp Ile Ile Ile Ala Thir Lys Asn Gly Llys Val Arg Gly Met 1. 5 1O 15 Asn Lieu. Thr Val Phe Gly Gly Thr Val Thr Ala Phe Leu Gly Ile Pro 2O 25 3O Tyr Ala Glin Pro Pro Lieu. Gly Arg Lieu. Arg Phe Llys Llys Pro Glin Ser 35 4 O 45 Lieu. Thir Lys Trp Ser Asp Ile Trp Asn Ala Thr Llys Tyr Ala Asn. Ser SO 55 6 O Cys Cys Glin Asn Ile Asp Glin Ser Phe Pro Gly Phe His Gly Ser Glu 65 70 7s 8O Met Trp ASn Pro ASn Thr Asp Lieu Ser Glu Asp Cys Lieu. Tyr Lieu. ASn 85 90 95 Val Trp Ile Pro Ala Pro Llys Pro Lys Asn Ala Thr Val Lieu. Ile Trp 1OO 105 11 O Ile Tyr Gly Gly Gly Phe Glin Thr Gly. Thir Ser Ser Lieu. His Val Tyr 115 12 O 125 Asp Gly Llys Phe Lieu Ala Arg Val Glu Arg Val Ile Val Val Ser Met 13 O 135 14 O Asn Tyr Arg Val Gly Ala Lieu. Gly Phe Lieu Ala Lieu Pro Gly Asn Pro 145 150 155 160 Glu Ala Pro Gly Asn Met Gly Lieu. Phe Asp Glin Glin Lieu Ala Lieu. Glin 1.65 17O 17s Trp Val Glin Lys Asn. Ile Ala Ala Phe Gly Gly ASn Pro Llys Ser Val 18O 185 19 O Thir Lieu. Phe Gly Glu Ser Ala Gly Ala Ala Ser Val Ser Lieu. His Lieu. 195 2OO 2O5 Lieu. Ser Pro Gly Ser His Ser Leu Phe Thr Arg Ala Ile Leu Glin Ser 21 O 215 22O Gly Ser Phe Asn Ala Pro Trp Ala Val Thir Ser Lieu. Tyr Glu Ala Arg 225 23 O 235 24 O

Asn Arg Thir Lieu. Asn Lieu Ala Lys Lieu. Thr Gly Cys Ser Arg Glu Asn 245 250 255

Glu Thr Glu Ile Ile Llys Cys Lieu. Arg Asn Lys Asp Pro Glin Glu Ile 26 O 265 27 O

Lieu. Lieu. Asn Glu Ala Phe Val Val Pro Tyr Gly Thr Pro Leu Ser Val 27s 28O 285

Asn Phe Gly Pro Thr Val Asp Gly Asp Phe Lieu. Thr Asp Met Pro Asp 29 O 295 3 OO

Ile Lieu. Lieu. Glu Lieu. Gly Glin Phe Llys Llys Thr Glin Ile Lieu Val Gly US 2016/01 84445 A1 Jun. 30, 2016 85

- Continued

3. OS 310 315 32O Val Asn Lys Asp Glu Gly Thr Ala Phe Lieu Val Tyr Gly Ala Pro Gly 3.25 330 335 Phe Ser Lys Asp Asn. Asn. Ser Ile Ile Thr Arg Lys Glu Phe Glin Glu 34 O 345 35. O Gly Lieu Lys Ile Phe Phe Pro Gly Val Ser Glu Phe Gly Lys Glu Ser 355 360 365 Ile Lieu. Phe His Tyr Thr Asp Trp Val Asp Asp Glin Arg Pro Glu Asn 37 O 375 38O Tyr Arg Glu Ala Lieu. Gly Asp Val Val Gly Asp Tyr Asn. Phe Ile Cys 385 390 395 4 OO Pro Ala Lieu. Glu Phe Thr Llys Llys Phe Ser Glu Trp Gly Asn. Asn Ala 4 OS 41O 415 Phe Phe Tyr Tyr Phe Glu. His Arg Ser Ser Lys Lieu Pro Trp Pro Glu 42O 425 43 O Trp Met Gly Val Met His Gly Tyr Glu Ile Glu Phe Val Phe Gly Lieu. 435 44 O 445 Pro Lieu. Glu Arg Arg Asp Asn Tyr Thr Lys Ala Glu Glu Ile Lieu. Ser 450 45.5 460 Arg Ser Ile Val Lys Arg Trp Ala Asn. Phe Ala Lys Tyr Gly Asn Pro 465 470 47s 48O Asn Glu Thr Glin Asn Asn Ser Thr Ser Trp Pro Val Phe Llys Ser Thr 485 490 495 Glu Glin Llys Tyr Lieu. Thir Lieu. Asn Thr Glu Ser Thr Arg Ile Met Thr SOO 505 51O Lys Lieu. Arg Ala Glin Gln Cys Arg Phe Trp Thr Ser Phe Phe Pro Llys 515 52O 525 Val Lieu. Glu Met Thr Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 53 O 535 54 O Ser Asp Llys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lieu. Leu 5.45 550 555 560 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Llys Pro Lys Asp Thr Lieu. 565 st O sts Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 58O 585 59 O His Glu Asp Pro Glu Val Llys Phe Asn Trp Tyr Val Asp Gly Val Glu 595 6OO 605 Val His Asn Ala Lys Thr Llys Pro Arg Glu Glu Glin Tyr Asn. Ser Thr 610 615 62O Tyr Arg Val Val Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn 625 630 635 64 O Gly Lys Glu Tyr Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ala Pro 645 650 655

Ile Glu Lys Thir Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin 660 665 67 O

Val Tyr Thr Lieu Pro Pro Ser Arg Asp Glu Lieu. Thir Lys Asn Glin Val 675 68O 685

Ser Lieu. Thr Cys Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 69 O. 695 7 OO

Glu Trp Glu Ser Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro 7 Os 71O 71s 72O US 2016/01 84445 A1 Jun. 30, 2016 86

- Continued

Pro Val Lieu. Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Luell Thir 72 73 O 73

Val Asp Llys Ser Arg Trp Gln Glin Gly Asn Val Phe Ser Cys Ser Wall 740 74. 7 O

Met His Glu Ala Lieu. His Asn His Tyr Thr Gln Lys Ser Lieu. Ser Luell 7ss 760 765 Ser Pro Gly Lys 770

<210s, SEQ ID NO 17 &211s LENGTH: 772 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 17

Glu Asp Asp Ile Ile Ile Ala Thir Asn Gly Wall Arg Gly Met 1. 5 15

Luell Thir Wall Phe Gly Gly Thir Wall Thir Ala Phe Lell Gly Ile Pro 2O 25

Ala Gln Pro Pro Lell Gly Arg Luell Arg Phe Lys Pro Glin Ser 35 4 O 45

Lieu Llys Trp Ser Asp Ile Trp Asn Ala Thr Lys Ala Asn Ser 55 6 O

Cys Glin Asn. Ile Asp Glin Ser Phe Pro Gly Phe His Gly Ser Glu 65 70

Met ASn Pro ASn Thir Asp Luell Ser Glu Asp Lell Tyr Luell Asn 85 90 95

Wall Ile Pro Ala Pro Pro Lys Asn Ala Thir Wall Luell Ile Trp 1OO 105 11 O

Ile Gly Gly Gly Phe Glin Thir Gly Thir Ser Ser Lell His Wall 115 12 O 125

Asp Llys Phe Lieu. Ala Arg Wall Glu Arg Wall Ile Wall Wall Ser Met 135 14 O

Asn Arg Val Gly Ala Lell Gly Phe Luell Ala Lell Pro Gly Asn Pro 145 150 155 160

Glu Ala Pro Gly Asn Met Gly Luell Phe Asp Glin Glin Lell Ala Luell Glin 1.65 17O 17s

Trp Wall Gln Lys Asn Ile Ala Ala Phe Gly Gly Asn Pro Lys Ser Wall 18O 185 19 O

Thir Luell Phe Gly Glu Ser Ala Gly Ala Ala Ser Wall Ser Luell His Luell 195

Lell Ser Pro Gly Ser His Ser Luell Phe Thir Arg Ala Ile Luell Glin Ser 21 O 215 22O

Gly Ser Phe Asn Ala Pro Trp Ala Wall Thir Ser Lell Tyr Glu Ala Arg 225 23 O 235 24 O

Asn Arg Thir Lieu. Asn Lell Ala Luell Thir Gly Ser Arg Glu Asn 245 250 255

Glu Thir Glu Ile Ile Luell Arg Asn Lys Asp Pro Glin Glu Ile 26 O 265 27 O

Lell Luell Asn. Glu Ala Phe Wall Wall Pro Tyr Gly Thir Pro Luell Ser Wall US 2016/01 84445 A1 Jun. 30, 2016 87

- Continued

27s 28O 285 Asn Phe Gly Pro Thr Val Asp Gly Asp Phe Lieu. Thr Asp Met Pro Asp 29 O 295 3 OO Ile Lieu. Lieu. Glu Lieu. Gly Glin Phe Llys Llys Thr Glin Ile Lieu Val Gly 3. OS 310 315 32O Val Asn Lys Asp Glu Gly Thr Ala Phe Lieu Val Tyr Gly Ala Pro Gly 3.25 330 335 Phe Ser Lys Asp Asn. Asn. Ser Ile Ile Thr Arg Lys Glu Phe Glin Glu 34 O 345 35. O Gly Lieu Lys Ile Phe Phe Pro Gly Val Ser Glu Phe Gly Lys Glu Ser 355 360 365 Ile Lieu. Phe His Tyr Thr Asp Trp Val Asp Asp Glin Arg Pro Glu Asn 37 O 375 38O Tyr Arg Glu Ala Lieu. Gly Asp Val Val Gly Asp Tyr Asn. Phe Ile Cys 385 390 395 4 OO Pro Ala Lieu. Glu Phe Thr Llys Llys Phe Ser Glu Trp Gly Asn. Asn Ala 4 OS 41O 415 Phe Phe Tyr Tyr Phe Glu. His Arg Ser Ser Lys Lieu Pro Trp Pro Glu 42O 425 43 O Trp Met Gly Val Met His Gly Tyr Glu Ile Glu Phe Val Phe Gly Lieu. 435 44 O 445 Pro Lieu. Glu Arg Arg Asp Asn Tyr Thr Lys Ala Glu Glu Ile Lieu. Ser 450 45.5 460 Arg Ser Ile Val Lys Arg Trp Ala Asn. Phe Ala Lys Tyr Gly Asn Pro 465 470 47s 48O Asn Glu Thr Glin Asn Asn Ser Thr Ser Trp Pro Val Phe Llys Ser Thr 485 490 495 Glu Glin Llys Tyr Lieu. Thir Lieu. Asn Thr Glu Ser Thr Arg Ile Met Thr SOO 505 51O Lys Lieu. Arg Ala Glin Gln Cys Arg Phe Trp Thr Ser Phe Phe Pro Llys 515 52O 525 Val Lieu. Glu Met Thr Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly 53 O 535 54 O Ser Asp Llys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lieu. Leu 5.45 550 555 560 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Llys Pro Lys Asp Thr Lieu. 565 st O sts Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 58O 585 59 O His Glu Asp Pro Glu Val Llys Phe Asn Trp Tyr Val Asp Gly Val Glu 595 6OO 605 Val His Asn Ala Lys Thr Llys Pro Arg Glu Glu Glin Tyr Asn. Ser Thr 610 615 62O

Tyr Arg Val Val Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn 625 630 635 64 O

Gly Lys Glu Tyr Lys Cys Llys Val Ser Asn Lys Ala Lieu Pro Ala Pro 645 650 655

Ile Glu Lys Thir Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin 660 665 67 O

Val Tyr Thr Lieu Pro Pro Ser Arg Asp Glu Lieu. Thir Lys Asn Glin Val 675 68O 685 US 2016/01 84445 A1 Jun. 30, 2016 88

- Continued

Ser Lieu. Thr Cys Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 69 O. 695 7 OO Glu Trp Glu Ser Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro 7 Os 71O 71s 72O Pro Val Lieu. Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Lieu. Thr 72 73 O 73 Val Asp Llys Ser Arg Trp Glin Glin Gly Asn Val Phe Ser Cys Ser Val 740 74. 7 O Met His Glu Ala Lieu. His Asn His Tyr Thr Gln Lys Ser Leu Ser Lieu. 7ss 760 765 Ser Pro Gly Lys 770

<210s, SEQ ID NO 18 &211s LENGTH: 37 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 18 Met Ala Cys Lieu. Gly Phe Glin Arg His Lys Ala Glin Lieu. Asn Lieu Ala 1. 5 1O 15 Thr Arg Thr Trp Pro Cys Thr Lieu Lieu Phe Phe Lieu Lleu Phe Ile Pro 2O 25 3O Val Phe Cys Lys Ala 35

<210s, SEQ ID NO 19 &211s LENGTH: 574 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 19 Glu Asp Asp Ile Ile Ile Ala Thir Lys Asn Gly Llys Val Arg Gly Met 1. 5 1O 15 Asn Lieu. Thr Val Phe Gly Gly Thr Val Thr Ala Phe Leu Gly Ile Pro 2O 25 3O Tyr Ala Glin Pro Pro Lieu. Gly Arg Lieu. Arg Phe Llys Llys Pro Glin Ser 35 4 O 45 Lieu. Thir Lys Trp Ser Asp Ile Trp Asn Ala Thr Llys Tyr Ala Asn. Ser SO 55 6 O Cys Cys Glin Asn Ile Asp Glin Ser Phe Pro Gly Phe His Gly Ser Glu 65 70 7s 8O

Met Trp Asn Pro Asn. Thir Asp Lieu. Ser Glu Asp Cys Lieu. Tyr Lieu. Asn 85 90 95

Val Trp Ile Pro Ala Pro Llys Pro Lys Asn Ala Thr Val Lieu. Ile Trp 1OO 105 11 O Ile Tyr Gly Gly Gly Phe Glin Thr Gly. Thir Ser Ser Lieu. His Val Tyr 115 12 O 125

Asp Gly Llys Phe Lieu Ala Arg Val Glu Arg Val Ile Val Val Ser Met 13 O 135 14 O

Asn Tyr Arg Val Gly Ala Lieu. Gly Phe Lieu Ala Lieu Pro Gly Asn Pro 145 150 155 160 US 2016/01 84445 A1 Jun. 30, 2016 89

- Continued

Glu Ala Pro Gly Asn Met Gly Lieu. Phe Asp Glin Glin Lieu Ala Lieu. Glin 1.65 17O 17s Trp Val Glin Lys Asn. Ile Ala Ala Phe Gly Gly ASn Pro Llys Ser Val 18O 185 19 O Thir Lieu. Phe Gly Glu Ser Ala Gly Ala Ala Ser Val Ser Lieu. His Lieu. 195 2OO 2O5 Lieu. Ser Pro Gly Ser His Ser Leu Phe Thr Arg Ala Ile Leu Glin Ser 21 O 215 22O Gly Ser Phe Asn Ala Pro Trp Ala Val Thir Ser Lieu. Tyr Glu Ala Arg 225 23 O 235 24 O Asn Arg Thir Lieu. Asn Lieu Ala Lys Lieu. Thr Gly Cys Ser Arg Glu Asn 245 250 255 Glu Thr Glu Ile Ile Llys Cys Lieu. Arg Asn Lys Asp Pro Glin Glu Ile 26 O 265 27 O Lieu. Lieu. Asn Glu Ala Phe Val Val Pro Tyr Gly Thr Pro Leu Ser Val 27s 28O 285 Asn Phe Gly Pro Thr Val Asp Gly Asp Phe Lieu. Thr Asp Met Pro Asp 29 O 295 3 OO Ile Lieu. Lieu. Glu Lieu. Gly Glin Phe Llys Llys Thr Glin Ile Lieu Val Gly 3. OS 310 315 32O Val Asn Lys Asp Glu Gly Thr Ala Phe Lieu Val Tyr Gly Ala Pro Gly 3.25 330 335 Phe Ser Lys Asp Asn. Asn. Ser Ile Ile Thr Arg Lys Glu Phe Glin Glu 34 O 345 35. O Gly Lieu Lys Ile Phe Phe Pro Gly Val Ser Glu Phe Gly Lys Glu Ser 355 360 365 Ile Lieu. Phe His Tyr Thr Asp Trp Val Asp Asp Glin Arg Pro Glu Asn 37 O 375 38O Tyr Arg Glu Ala Lieu. Gly Asp Val Val Gly Asp Tyr Asn. Phe Ile Cys 385 390 395 4 OO Pro Ala Lieu. Glu Phe Thr Llys Llys Phe Ser Glu Trp Gly Asn. Asn Ala 4 OS 41O 415 Phe Phe Tyr Tyr Phe Glu. His Arg Ser Ser Lys Lieu Pro Trp Pro Glu 42O 425 43 O Trp Met Gly Val Met His Gly Tyr Glu Ile Glu Phe Val Phe Gly Lieu. 435 44 O 445 Pro Lieu. Glu Arg Arg Asp Asn Tyr Thr Lys Ala Glu Glu Ile Lieu. Ser 450 45.5 460 Arg Ser Ile Val Lys Arg Trp Ala Asn. Phe Ala Lys Tyr Gly Asn Pro 465 470 47s 48O Asn Glu Thr Glin Asn Asn Ser Thr Ser Trp Pro Val Phe Llys Ser Thr 485 490 495

Glu Glin Llys Tyr Lieu. Thir Lieu. Asn Thr Glu Ser Thr Arg Ile Met Thr SOO 505 51O

Lys Lieu. Arg Ala Glin Gln Cys Arg Phe Trp Thr Ser Phe Phe Pro Llys 515 52O 525

Val Lieu. Glu Met Thr Gly Asn. Ile Asp Glu Ala Glu Trp Glu Trp Llys 53 O 535 54 O Ala Gly Phe His Arg Trp Asn. Asn Tyr Met Met Asp Trp Lys Asn Glin 5.45 550 555 560 US 2016/01 84445 A1 Jun. 30, 2016 90

- Continued Phe Asn Asp Tyr Thr Ser Llys Lys Glu Ser Cys Val Gly Lieu 565 st O

<210s, SEQ ID NO 2 O &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 2O Gly Gly Gly Cys 1.

<210s, SEQ ID NO 21 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 21 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1. 5 1O

<210s, SEQ ID NO 22 & 211 LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 22 Gly Gly Gly Gly Ser 1. 5

<210s, SEQ ID NO 23 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 23 Gly Gly Gly Ser 1.

<210s, SEQ ID NO 24 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 24 Gly Gly Gly Glu Ser 1. 5

<210s, SEQ ID NO 25 &211s LENGTH: 11 US 2016/01 84445 A1 Jun. 30, 2016

- Continued

212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 25 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1. 5 1O

<210s, SEQ ID NO 26 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs, SEQUENCE: 26 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1. 5 1O 15 Gly Gly Gly Ser Gly 2O

<210s, SEQ ID NO 27 &211s LENGTH: 28 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 27 Met His Ser Llys Val Thr Ile Ile Cys Ile Arg Phe Leu Phe Trp Phe 1. 5 1O 15 Lieu. Lieu. Lieu. Cys Met Lieu. Ile Gly Lys Ser His Thr 2O 25

<210s, SEQ ID NO 28 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 28 Glu Asp Asp Ile Ile Ile 1. 5

1. A conjugate comprising an organophosphorus (OP) bio 4. A conjugate according to claim3 wherein the cholinest Scavenger selected from the group consisting of a stoichio erase is purified from blood plasma. metric OP bioscavenger and a catalytic OP bio scavenger 5. A conjugate according to claim3 wherein the cholinest covalently bound to a Zwitterionic polymer wherein the poly erase is produced by recombinant DNA technology. mer comprises one or more types of monomer units and wherein at least one type of monomer unit comprises a Zwit 6. A conjugate according to claim3 wherein the cholinest terionic group. erase is a butyrylcholinesterase. 2. A conjugate according to claim 1 wherein the OP bios 7. A conjugate according to claim 6 wherein the butyryl cavenger is a catalytic bio Scavenger selected from the group cholinesterase is fused to a non-butyrylcholinesterase protein consisting of aryldialkylphosphatases, organophosphate as fusion protein. hydrolases (OPH), carboxylesterases, triesterases, phospho 8. A conjugate according to claim 7 wherein the non triesterases, arylesterases, paraoxonases (PON), organophos butyrylcholinesterase protein comprises an immunoglobulin phate acid anhydrases and diisopropylfluorophosphatases (Ig) domain. (DFPases). 9. A conjugate according to claim 8 wherein the Ig domain 3. A conjugate according to claim 1 wherein the OP bios is selected from the group consisting of IgG-Fc, IgG-CH and cavenger is a stoichiometric OP bioscavenger comprising a IgG-CL. cholinesterase selected from the group consisting of acetyl 10. A conjugate according to claim 9 wherein the IgG is cholinesterase (AChE) and butyrylcholinesterase (BChE). IgG1. US 2016/01 84445 A1 Jun. 30, 2016 92

11. A conjugate according to claim 10 wherein the Ig 26. A conjugate according to claim 15 wherein the at least domain is IgG-Fc (SEQID NO. 8). one type of monomer unit comprises 2-(methacryloyloxy 12. A conjugate according to claim 11 wherein the Fc ethyl)-2'-(trimethylammoniumethyl) phosphate (HEMA domain has amino acid Substitutions in one or more of the PC). positions selected from the group consisting of E233, L234, 27. A conjugate according to claim 25 wherein the polymer L235, G236, G237, A327, A330 and P331. has 3 or more arms. 13. A conjugate according to claim 12 wherein the Fc 28. A conjugate according to claim 27 wherein the polymer domain has the following substitutions L234A, L235E, has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 arms. G237A, A330S and P331S. 29. A conjugate according to claim 28 wherein the polymer 14. A conjugate according to claim 12 wherein the Fc has 3, 6 or 9 arms. domain further has one of the following substitutions Q347C or L443C. 30. A conjugate according to claim 29 wherein the polymer 15. A conjugate according to claim 14 wherein the Substi has 9 arms. tution is Q347C. 31. A conjugate according to claim 28 wherein the polymer 16. A conjugate according to claim 7 wherein the fusion portion of the conjugate has a peak molecular weight of protein further comprises a linker between the butyrylcho between 300,000 and 1,750,000 Daltons. linesterase enzyme and the non-butyrylcholinesterase pro 32. A conjugate according to claim 31 wherein the polymer tein. portion of the conjugate has a peak molecular weight between 17. A conjugate according to claim 16 wherein the linker is 500,000 and 1,000,000 Daltons. selected from the group consisting of G, GG, GGGGS (SEQ 33. A conjugate according to claim32 wherein the polymer ID NO: 22), GGGS (SEQID NO. 23), and GGGES (SEQID portion of the conjugate has a peak molecular weight between NO: 24), and oligomers of the foregoing. 600,000 to 800,000 Daltons. 18. A conjugate according to claim 17 wherein the linker is 34. A conjugate according to claim 28 wherein the polymer selected from the group consisting of GGGSGGGSGGGS is covalently bonded to at least one of an amino group, a (SEQID NO: 21) and GGSGGGSGGGS (SEQID NO: 25). hydroxyl group, a Sulfhydryl group and a carboxyl group of 19. A conjugate according to claim 7 wherein the butyryl the cholinesterase. cholinesterase enzyme is a carboxyl truncate. 35. A conjugate according to claim 34 wherein the sulfhy 20. A conjugate according to claim 19 wherein the butyryl dryl group is from a cysteine residue in the cholinesterase. cholinesterase enzyme comprises or consists of amino acids 36. A conjugate according to claim 35 wherein the cysteine 29-561 of SEQID NO. 1. residue is at a position at which cysteine is not naturally 21. A conjugate according to claim 7 wherein the butyryl present in the cholinesterase. cholinesterase enzyme comprises or consists of amino acids 37. A conjugate according to claim 36 wherein the cho 29-562 of SEQID NO. 1. linesterase is abutyrylcholinesterase fusion corresponding to 22. A conjugate according to claim 20 wherein the butyryl that of SEQID NO. 2 and the cysteine residue is that at amino cholinesterase enzyme has a mutation wherein the C at resi acid 672. due 94 of SEQID NO.1 is Y. 38. Abutyrylcholinesterase enzyme comprising or consists 23. A conjugate according to claim 15 wherein the butyryl of amino acids 29 to 562 of SEQID NO. 1. cholinesterase fusion has the amino acid sequence set forth in 39-58. (canceled) SEQID NO. 2. 24. A conjugate according to claim 1 wherein the Zwitte 59. A method for treatment or prophylaxis of OP poisoning rionic group comprises phosphorylcholine. comprising administering to a patient an effective amount of 25. A conjugate according to claim 24 wherein the at least the conjugates or fusion proteins of claim 1. one type of monomer unit comprises 2-(acryloyloxyethyl)- 60-119. (canceled) 2'-(trimethylammoniumethyl) phosphate. k k k k k