(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`UMA IOAU UAATAA
`
`(10) International Publication Number
`WO 2018/170152 Al
`
`= a
`
`WIPO! PCT
`
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(43) International Publication Date
`20 September 2018 (20.09.2018)
`
`CA, CH,CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, Do,
`DZ, EC, EE, EG, ES, FL GB, GD, GE, GH, GM,GT, HN,
`HR, HU,ID,IL, IN,IR,IS, JO, JP, KE, KG, KH, KN,KP,
`KR, KW,KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME,
`MG, MK, MN, MW, MX, MY, MZ, NA, NG,NL, NO, NZ,
`OM,PA, PE, PG, PH,PL, PT, QA, RO, RS, RU, RW, SA,
`SC, SD, SE, SG, SK, SL, SM,ST, SV, SY, TH, TJ, TM, TN,
`TR,TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM,KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ,
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU,TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, HR, HU,IE, IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO,PL, PT, RO, RS, SE, SI, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`KM,ML, MR, NE, SN, TD, TG).
`
`Published:
`
`with international search report (Art. 21(3))
`with sequence listing part ofdescription (Rule 5.2(a))
`
`(51) International Patent Classification:
`A61K 38/54 (2006.01)
`CO7K 19/00 (2006.01)
`A6IK 38/43 (2006.01)
`
`(21) International Application Number:
`
`PCT/US2018/022471
`
`(22) InternationalFiling Date:
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`14 March 2018 (14.03.2018)
`
`(84)
`
`English
`
`English
`
`(30) Priority Data:
`1704071.8
`
`14 March 2017 (14.03.2017)
`
`GB
`
`(71) Applicant: GEMINI THERAPEUTICS [US/US]; 36 Irv-
`ing Street, No. 3, Cambridge, MA 02138 (US).
`
`(72)
`
`Inventors: KAVANAGH, David; Framlington Place,
`Neweastle-upon-Tyne NE2 4HH (GB). MARCHBANK,
`Kevin; Framlington Place, Newcastle-upon-Tyne NE2
`4HH (GB).
`
`(74)
`
`Agent: VARMA,Anita et al.; White & Case LLP, 75 State
`Street, Boston, MA 02109 (US).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO,AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ,
`
`(54) Title: RECOMBINANT MATURE COMPLEMENT FACTORI
`
`Complement Activation
`
`Complement Requlation
`
`aCFH Ab =e
`é
`
`=eCF Ab
`
`
`
`\
`
`Cell Lysis
`Membrane Attack Complex (C5b-9}
`<.
`inflammation
`
`
`Anaphylatoxins (C3a,C5a)a
`
`oe
`Opsonisation te
`
`(C3b)
`6
`
`
`Figure 1
`
`(57) Abstract: The disclosure provides, in part, compositions comprising mature recombinant mature Complement Factor I (CFI)
`protein and methods of making and using those compositions.
`
`
`
`©2018/170152A.IMINININMIINANTANTCATAAA
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`WO 2018/170152
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`PCT/US2018/022471
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`RECOMBINANT MATURE COMPLEMENT FACTOR I
`
`Cross-Reference to Related Apolication
`
`This application claims the benefit of priority from Great Britain Patent Application No.
`
`1704071.8, filed on March 14, 2017. The foregoing application is incorporated herein by
`
`reference in its entirety.
`
`Technical Field
`
`Aspects of the oresent
`
`invention relate to a recombinant mature Camplement Factor |
`
`protein, compositions comprising such proteins and methods cf manufacture and uses
`
`thereof. Also inclided herein are methads of treating a complerment-mediated disorder
`
`comprising administering a composition comprising a recombinant mature Complement
`
`Factor | protein to a patient in need thereaf,
`
`Background to the invention
`
`The complement systern is a part of the innate immune systern which is made up of a large
`
`number of discrete plasma proteins that react with one another to opsonize pathogens and
`
`induce a series of inflammatory responses that help te fight
`
`infection. A number of
`
`complement oroteins
`
`are proteases
`
`that
`
`are
`
`themselves
`
`activated
`
`by protealytic
`
`cleavage. There are three ways in which the complement system protects against infection.
`
`First, # generates large numbers of activated complement oroteins that bind covalently to
`
`pathogens,
`
`opsonizing thern fer
`
`enguifment
`
`by phagocytes bearing
`
`receptors
`
`for
`
`complement. Second, the smail fragments of seme complement proteins act as cherno-
`
`attractants to recruit more phagocytes to the site of complement activation, and alse to
`
`activate
`
`these
`
`phagocytes. Third,
`
`ine
`
`terminal
`
`complement
`
`camponenis damage
`
`certain bacteria by creating pores in the bacterial membrane.
`
`Complement Factor i, also known as C3b/C4b inhibitor,
`
`is a serine proteinase that
`
`is
`
`essential for requiating the complement cascade.
`
`it is axpressed in numerous tissues but
`
`principally by liver hepatocytes. The encoded prepraprotein is cleaved io produce both
`
`heavy and fight chains, which are linked by disulfide bonds to form a heterodimeric
`
`giycoprotein. This neterodimer can cleave and inactivate the complement components C4b
`
`and C3b, anc it prevents the assembly of the CS and C5 convertase enzymes. Defects in
`
`this gene cause complement factor | deficiency, an autosomal recessive disease associated
`
`with a susceptibility te pyogenic infections.
`
`15
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`NO JT
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`Mutations in this gene have been associated with a predisposition te atypical hemelytic
`
`uremic syndrome, a disease characterized by acute renal faiture, microanglapathic hemolytic
`
`anemia and thrombocytopenia. Recently iow levels of circulating CFi have been identified in
`
`individuals with very rare CFI variant genes and these individuals associated with advanced
`
`Age-Related Macular Degeneration (AMD) supporting the role of CFI
`
`in risk of AMD
`
`(Kavanagh et al (2015). AMD is the most common cause of vision loss in those aged over 50
`
`and currently there are few treatment ootions. This research suggests that enhancing CFi
`
`activity In these individuals may have some therapeutic benefit.
`
`Currently, efforts te produce compositions comprising a high percentage of recombinant
`
`mature CF] have had limited sucocess. Typically, prior art methods result
`
`in incomplete
`
`cleavage ofthe proform ic form the mature CF! protein. Thus, the prior art typically results in
`
`compositions comprising significant amounts of uncleaved proform protein. Furthermore,
`
`previous efferts have resulted in compositions which have reduced activity as compared to
`
`plasma-derived Complement Factar |.
`
`it is therefore an air of certain embodiments of the present invention to at least partially
`
`mitigate the problems associated with the prior art.
`
`it
`
`is an aim of ceriain embodiments of the present
`
`invention to provide a method for
`
`producing a composition which comprises a high concentration of recombinant mature
`
`Complement Factor |.
`
`itis an aim of certain embodiments to provide a composition comprising recombinant mature
`
`Complement Factor | for use in the treatment of complement- mediated disorders.
`
`Summary of the Disclosure
`
`Uniess defined otherwise, ail technical and scientific terms used herein have the same
`
`meaning as commonly undersicod by a person skilled in ine ari io which to this invention
`
`belongs.
`
`Ceriain aspects of
`
`the present
`
`invention provide
`
`an
`
`iselated recombinant mature
`
`Complement Factor|.
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`The term "isclaisd"” as used herein refers to @ biological camponent (Such as a nucisic acid
`
`molecule or protein) that has been substantially separated or purified away from other
`
`biological components in the cell of the organism in which the component naturally occurs,
`
`i.@., other chromasamal and extra chromasamal DNA and RNA, and oroteins. Nucleic acids
`
`and proteins that have been “isoiated” include nucleic acids and proteins purified by
`
`standard purification methods. The term also embraces nucleic acikis and oroteins prepared
`
`by recombinant expression in a hast cell as well as chemically synthesized nucleic acids,
`
`proteins and peptides.
`
`it is considered that the present inventors have devised a method of producing an isalated
`
`recombinant mature CFI protein which is
`
`substantially isolated fram other cellular
`
`components including for example a recombinant precursor CFI protein. Ht is considered that
`
`prior art methods of producing a recombinant CFI protein have resulted in incomplete
`
`processing of a precursor CFi protein such ihat a recombinant mature CFI protein has nat
`
`ee
`
`ist
`
`been substantially isolated.
`
`in a first aspect of the present invention, there is provided @ camposition camprising a
`
`recombinant mature Complement Factor | (CFi) protein, wherein the recombinant mature
`
`CF! protein comprised in the composition represents greater than about 50% by weight of a
`
`total CFI protein content of the composition.
`
`Thus, certain embodirnents of the present
`
`invention relate to a recombinant mature
`
`Complement Factor |
`
`(CFI, sampositions comprising recombinant mature Complement
`
`Factor | and rnethocis of obtaining such 4 protein.
`
`AS used herein,
`
`the term "protein" can be used interchangeably with "peptide" or
`
`“polypeptide”, andi means at least iwo covalently attached alpha amino acid resicues linked
`
`by a peptidy! bond. The term protein encompasses purified natural products, or chemical
`
`products, which may oe produced partially or whally using recombinant or synihetic
`
`techniques. The term protein may refer to a camplex of more than one polypeptide, such as
`
`a& dimer or other mullimer, a fusion protein, a protein variant, or derivative thereof. The term
`
`aiso includes modified proteins, for example, a protein modified by glycosylation, acetylation,
`
`phosphorylation, pegylation, ubiquitination, and so forth. A protein may comprise amino
`
`acids net encoded by a nucleic acid cadon.
`
`Complement Factor |
`
`is an important complement regulator.
`
`lt
`
`is expressed in numerous
`
`tissues but principally by liver hepatocyies. CFI is a heterodimer in which the two chains are
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`WO 2018/170152
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`linked togsiher by disulphide bond. The heavy chain contains ihe Factor | module, a CDS
`
`domain and two low density Hpoprotein receptor demains (DLN. The light chain comprises
`
`@ serine protease domain, the active site of which consists of a triad of His380, Asp439 and
`
`Ser525. A CF! heavy chain amino acid sequence is shown in SEQ ID. No.
`
`1 and a CFI light
`
`chain amine acid sequence is shown in SEQ ID. No. 2 (Figure 2).
`
`When CFi
`
`is synthesised,
`
`it
`
`is initially made as a single chain precursor (precursor CFI
`
`protein}, in which a four residue linker peptide (RRKR)} connects the heavy chain to the light
`
`chain. Thus, as used herein, the term “precursor CFI protein’ is used to refer to a single
`
`chain precursor Gomplement Factor | protein which comprises a four residue linker peptide
`
`{RRER). Aptly, the precursor CFi protein is substantially inactive and has essentially na C3
`
`C3b-inactivating or iC3b-degracdation activity.
`
`In ceriain embodiments,
`
`the recombinant
`
`precursor CFI protein comprises an amino acid sequence as set forth in SEG.
`
`ID. No. 3
`
`(Figure 2}.
`
`ee
`
`ist
`
`During processing,
`
`the precursor CFI protein is cleaved by a calcium-dependent serine
`
`endoprotease, furin,
`
`leaving the heavy chain and light chain of full
`
`length mature Fi held
`
`together by a single disulphide bond. This protein is referred to herein as a mature CFI
`
`protein.
`
`Thus, aS used herein, the term “mature CFI protein” refers to a CFI protein which is or has
`
`been cleaved at or adiacent
`
`to a RRKR linker sequence e.g. by furin.
`
`In certain
`
`embodiments, the mature CFI protein lacks an RRKR linker sequence as compared io a
`
`precursor CFI protein, wherein the precursor
`
`CF! protein comprises a RRKR linker sequence
`
`at positions 3718 to 321.
`
`In other embodiments, the mature CFI protein is cleaved adjacent to
`
`the RRKR linker sequence and therefore the mature CFI protein may comprise a light chain
`
`and a heavy chain, one cr both of which comprises one or more amine acid residues of the
`
`linker sequence.
`
`In certain embodiments, the recombinant precursor CF/ protein is a nan-
`
`human mammalian CFI protein.
`
`in certain embodiments, a mature CFi protein comprises a disulphide bond and wherein the
`
`recombinant mature CFI protein is cleavable into a heavy chain and a light chain upon
`
`reduction of the disulohide boned.
`
`In cerlain embociments, ihe mature CFI protein comprises
`
`a heavy chain comprising a Factor | module, a CD5 module, an LDOLr module, LDLr module
`
`and a light chain comprising a serine protease domain.
`
`In certain embodiments, the mature
`
`CFI protein is glycosylated.
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`As used herein, the term “recombinant precursor CFi protein” is used io refer ia a precursor
`
`CFI protein as described above which is obtained using recombinant methods.
`
`As used herein,
`
`the term “total CFI protein cantent” refers to a total content of the
`
`combination of recombinant mature CFI protein and a recombinant precursor CFI protein
`
`present in a single cornposition.
`
`Aptly, a “recombinant mature CF protein” is a mature CFI protein defined above which is
`
`rnade by recombinant expression, ie. # is not naturally occurring or derived from plasma.
`
`Aptly, a wild-tyoe mature CFI protein comprises two chains, each chain undergaing
`
`giycosylation which results in a total of six N-linked glycosylation sites which adds up to
`
`3kDa of carbohydrate to the predicted molecular weight of 85kDa.
`
`ee
`
`ist
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`NO Ww
`
`The recombinant mature CFi praisin may have a differenti giycosylation pattern to a
`
`naturally-derived ie. plasma-derived mature CF! protein.
`
`The terms “recombinant” and “recombinant expression” are well-knownin the art. The term
`
`“recombinant expression”, as used herein, relates to transcription and transiation of an
`
`exogenous gene in a host organism. Exogenous DNA refers to any deoxyribonucieic acid
`
`that originates outside of the host cell. The exogenous DNA may be integrated in the
`
`genome of the host or expressed from a non-integrating element.
`
`A recombinant protein includes any polypeptide expressed or capable of being expressed
`
`fram 4& recombinant nucieic acid. Thus, a recorbinant mature CF! protein is expressed by a
`
`recombinant DNA sequence. Aptly, the recombinant mature CF! protein has undergone
`
`post-expression processing to be cleaved at ar adjacent ic a RRKR linker sequence to leave
`a heteradimer as described herein.
`
`in certain ernbodiments, the recombinant mature CFi protein represenis greater than about
`
`60% by weight of the total CFI protein conient of the composition.
`
`In certain embodiments,
`
`the recombinant mature CFI protein represents greater than about 70% by weight of the total
`
`CFI protein content of the compositian.
`
`In one embeadiment, the recombinant mature CF!
`
`protein represenis greaier than about 80% by weight of the ictal CFI protein content of the
`
`composition.
`
`35
`
`in certain embodiments, the recombinant mature CFI protein represents greater than about
`
`$0% by weight of the total CFl protein content of the cornposition. Aptly, the recornbinant
`
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`WO 2018/170152
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`PCT/US2018/022471
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`maiure CFE protein represents greater inan about 95% by weight af the total CFI protein
`
`content of the composition.
`
`in
`
`certain embadiment,
`
`the campasition further comprises a
`
`recombinant precursor
`
`Compiement Factor | protein, wherein the ratio of recombinant mature CFI: recombinant
`
`precursor CFI in the composition is from greater than 50:50 to 100:0.
`
`in a second aspect of the present invention, there is provided a compasiicn comprising a
`
`recombinant mature Complernent Factor |
`
`(CFD protein and optionally a recombinant
`
`precursor Camplement Factor | protein, wherein the ratio of recombinant mature CFI:
`
`recombinant precursor CFI in the compositicn is from greater than 50:50 to 100:0.
`
`in cenain embodiments, the ratio of recombinant mature CFI: recombinant precursor CFI in
`
`the composition is fram 60:40 to 100:0.
`
`jn certain embodiments, the rato of recombinant
`
`mature CFl: recombinant orecursor CFI in the camposition is from 70:30 to 100:0.
`
`In certain
`
`embodiments,
`
`the ratio of recombinant mature CFE recombinant precursor CFI
`
`in the
`
`compasition is fram 60:20 to 100:0, for example fram about 96:10 to 160:0, for example from
`$5.05 io 100:0.
`
`in ceriain embaodimenis, the recombinant CF protein is a human CFI protein.
`
`in certain
`
`embodiments, the recombinant mature CFI protein comprises a first amino acid molecule
`
`comprising an amine acid sequence as set forth in SEG. IB. No. 1.
`
`in certain embodiments,
`
`the recombinant mature CFI pratein comprises a first amine acid molecule comprising an
`
`amino acid sequence which has at least 80% sequence ideniity to ine amino acid sequence
`
`as set forth in SEQ. IB. No. 1. Aptly, the % sequence identity is over the entire length of the
`
`amino acid sequence set forth in SEQ. ID. No. 7.
`
`in certain embedimenis, the recambinant mature CFI protein comprises a first amino acid
`
`sequence that is ai leasi 90% identical to the amino acid sequence as set farth in SEQ ID
`
`NO: 1, e.g. at least 9t%, 92%, 93% or 94%.
`
`in certain embodiments, ihe recombinant
`
`mature CFI protein comprises 4 first amino acid moleciiie comprising an amine acid
`
`sequence that is at least 95% identical to the amino acid sequence as set forth in SEQ ID
`
`NO: 1, 2.9. 96%, 97%, 98%, 39% or 100%identical.
`
`in certain ernbodiments, the recombinant mature CFI protein comprises a further amino acid
`
`molecule comprising an amino acid sequence as set forth in SEQ. 1D. No. 2, wherein the first
`
`and further amino acid sequence are linked by a disulphide bond.
`
`ee
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`ist
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`in certain embodiments, the recambinant mature CFI protein comprises a further amino acid
`
`molecule comprising an amino acid sequence which has at least 80% sequence identity to
`
`the amino acid sequence as set forth in SEQ. ID. No. 2 wherein the first and further amino
`
`acid sequence are linked by a disulphide bond.
`
`In certain ernbodiments, the recombinant
`
`mature CF! protein comprises an aming acid sequence that is at least 90% identical io the
`
`amino acid sequence as sei forth in SE@ ID NO: 1, eg. at least 91%, 92%, 93% or 34%
`identical.
`
`in certain embodiments, the recombinant mature CFI protein comprises a further amino acid
`
`molecule comprising an amino acid sequence that is at least
`
`955%identical to the amino acid
`
`sequence as set forth in SEG ID NO: 2, e.g. ai
`identical.
`
`least 98%, 97%, 98%, 99% or 100%
`
`ee
`
`ist
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`NO Ww
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`Thus, in cerlain embodiments, proteins having minor modifications in the sequence may be
`
`equaily useful, provided they are functional. The terms “sequence identity’, “percent identity”
`
`and “sequence percent identity" in the context of two or more nucleic acids or polypeptides,
`
`refer to two or more sequences or subsequences that are ihe same or have a specified
`
`percentage of nucleotides or amino acid residues that are the same, when compared and
`
`aligned (introducing gaps, if necessary) for maximum correspondence, not considering any
`
`conservative amino acid substitutions as part of the sequence identity. The percent identity
`
`can be measured using sequence comparison software or aigerithms or by visual inspection.
`
`Various algorithms and software are known in the art that can be used to obtain alignments
`
`of amino acid or nucleotide sequences.
`
`Suitable programs to determine percent sequence identity include for exampie the BLAST
`
`suite of programs available fram ine U.S. government's National Center for Biotechnciagy
` information BLAST web site (i
`ui }. Comparisons between
`two sequences can be carried using elther the BLASTN or BLASTPalgorithm. BLASTN is
`
`used to compare nucieic acid sequences, while BLASTP is used to compare amino acid
`
`sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California} or MegAlign,
`
`available from DNASTAR, are additional publicly available software programs that can be
`
`used ia align sequences. One skilled in the art can determine appropriate parameters for
`
`maximal alignment by particular alignment software.
`
`in certain embodiments, the default
`
`35
`
`parameters of the alignment software are used.
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`WO 2018/170152
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`
`in certain embodiments, the recombinant mature CFI protein may comprise an amine acid
`
`sequence comprising one or more mutations as compared te a reference sequence.
`
`certain embodiments, the reference sequence is as shown in SEQ.
`
`ID. No.
`
`7 and 2.
`
`in
`
`In
`
`certain embadiments, the mutation may be an insertion, a deletion, or a substitution.
`
`Substitutional variants of proteins are those in which at least one amino acid residue in the
`
`amino acid sequence has been removed and a different amine acid residue inserted in its
`
`place. The mature recombinant CFi protein of certain embodiments of the present invention
`can contain conservative or non-conservative substitutions.
`
`The term “conservative substitution” as used herein relates to the substitution of one or more
`
`amino acid residues for arnine acid residues having similar biochemical properties. Typically,
`
`conservative substitutions have little or no impact on the activity of a resulting protein.
`
`Screening of variants of the CFi proteins described herein can be used to identify which
`
`amino acid residues can tolerate an amino acid residue substitution.
`
`In one example, the
`
`ralevani biological aciivity of a modified protein is not decreased by more than 25%,
`
`preferably not more than 20%, especially not more than 16%, campared with CFi when ane
`or more conservative amino acid residue substitutlons are effected.
`
`in cenain embodiments, the composition is essentially free of a furin protein or fragmenis
`
`thereof, Furin is a subtilisin-like proprotein convertase which cleaves protein in vivo at a
`
`minimal cleavage site of Arg-xX-x-Arg. A human furin protein cornprises an amino acid
`
`sequence as set forth in SEQ. ID. 4,
`
`in
`
`certain embodiments,
`
`the
`
`composition is
`
`a pharmaceutical
`
`compasition.
`
`The
`
`oharmaceutical composition furlher comprises one or more pharmaceutically acceptable
`
`excipients. Further details cf pharmaceutical compasitions are provided herein.
`
`int a further aspect of the present invention, ihere is provided a method of preparing a
`
`composition comprising a recombinant mature Camplement Factor | (CFI) protein, wherein
`
`the recombinant mature CFi protein represents greater than 50% by weight of a ictal CFI
`
`protein content of the composition, the method comprising:
`
`a.
`
`contacting a recombinant precursor CFI protein with a furin protein or
`
`fragment thereof: and
`
`b.
`
`incubating the recombinant precursor CF! protein with the furin protein or
`
`ragment thereof for a predetermined periad of time, whereby the furin protein
`
`or fragment thereof cleaves the recombinant precursor CFI protein at or
`
`ee
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`ist
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`adjacent i¢ a RRKR linker sequence site to form the recombinant mature
`
`Campiement Factor | protein.
`
`in certain embodiments, the recombinant precursor CFI protein is a human precursor CF
`
`protein. the recombinant precursor CFi protein comprises an amino acid sequence as set
`
`forth in SEQ. ID. No: 3.
`described herein.
`
`In certain embodiments, the recombinant grecursar CFI protein is as
`
`In certain embodiments, the recombinant precurser CFI protein comprises a tag.
`
`In certain
`
`embodiments, the tag is a His-tad.
`
`in certain embedirnents, the method comprises expressing ihe recombinant precursor CF
`
`protein prior to step (a).
`
`in certain embscdiments, the method comprises expressing the
`
`recombinant precursor CF! praigin in a eukaryotic cell,
`
`in certain embodiments, the method comprises expressing ihe recombinant precursor CFI
`
`protein in a prokaryotic cell. Apthy, the prokaryotic cell is Escherichia coil,
`
`in certain embodiments, the eukaryotic cell is selected from an insect, a plant, a yeast or a
`mammatian cell.
`
`Suitable host cells for cloning or expressing the DNA encoding a CFI protein include
`
`orokaryote, yeast, or higher eukaryote cells. Suitable prakaryotes for this puroose include
`
`eubacteria,
`
`such
`
`as Gram-negative
`
`or Gram-positive
`
`organisms,
`
`for
`
`exarnpie,
`
`Enterobacteriaceae such as Escherichia, ¢.g., & coli, Enterobacter, Erwinia, Klebsiella,
`
`Proteus, Salmonella, e.g., Salmoneila typhimurium, Serratia, e.g., Serratia marcescans, and
`
`Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P.
`
`aeruginosa, and Streptomyces.
`
`in addition to prokaryotes, eukaryotic microbes such as flamentous fungi or yeast may be
`
`sultable cloning or expression hosts for CFl-encoding vectors. Saccharomyces cerevisiae, or
`
`common bakers yeast,
`
`is
`
`the most commonly used among lower eukaryotic host
`
`microorganisms although others may be useful.
`
`in certain ernbodiments, the host cell is a mammalian host cell e.g.
`
`rnonkey kidney CV1 line
`
`transformed by SV40 (e.g. COS-7); human embryonic kidney line (e.g. 293 or 293 cells);
`
`baby hamster kidney cells (e.g. BHK}: Chinese hamster ovary cells-DHFR (CHO), mouse
`
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`serial cells (e.g. TM4}: monkey kidney celis (e.g. CV1}; African gresn monkey kidney cells
`
`fa.g. VERO-76); human cervical carcinoma cells (e.g. HELA); canine kidney cells (e.g.
`
`MDCK}: buffalo rat liver cells (e.g. BRL 3A}; human lung cells (e.g. W438); humanliver cells
`
`(2.9. Heo G2}; mouse mammary tumor (MMT 060562); TRI cells, MRC 5 cells and FSd4
`
`celis.
`
`in certain embodiments, the mammiatiian ceil is a CHO call.
`
`Host cells are transformed with the above-described expressian or cloning vectors for
`
`antibody production and cultured in conventional nutrient media modified as appropriate for
`
`inducing promoters, selecting transformants, or amplifying the genes encoding the desired
`sequences.
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`in certain embodiments,
`
`the method comprises transforming the cell with a nucleic acid
`
`molecule encoding 4 precursor CFI protein. Aptly, the method comprises transforming the
`
`call with a vector which encodes a precursor CFi protein as described herein.
`
`"Nucleic acid molecule’ or ‘nucleic acid sequence", as used herein, refers to a polymer of
`
`nucleotides in which the 3' position of one nucleotide sugar is linked ta the 5° position of the
`
`next by a phosphodiester bridge. in a linear nucieic acid strand, one end typically has a free
`
`§' phosphate group, ihe other a free 3' hydroxy! group. Nucleic acid sequences may be used
`
`herein io refer to oligonuclectides, or polynuciectides, and fragments or portions thereof, and
`
`to DNA or RNA af genomic or synthetic origin that may be single- or double-stranded, and
`
`represent the sense or antisense strand.
`
`The term “vector” as used herein rneans a riucieic acid sequence containing an origin of
`
`replication. A vector may be a viral vector, bacteriophage, bacterial artificial chremosome or
`
`yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be a self-
`
`replicating extracnromoesomal vector, and aptly, is a DNA plasmid.
`
`Aptly, the vector may further comprise a promoter. The ierm “promoter? as used herein
`
`means a synthetic or naturally-derived malecule which is capable of conferring, activating or
`
`enhancing expression of a nucleic acid in a cell. A promoter may comprise one or more
`
`specific transcriptional regulatory sequences to further enhance expression and/or to alter
`
`the spatial expression and/or temporal expression of same. A pramoter may also camprise
`
`distal enhancer or repressor elements, which may be located as much as several thousand
`
`base pairs from the start site of transcription. A promoter may regulate the expression of a
`
`gene component constitutively, or differentially with respect to cell, the tissue or organ in
`
`which expression occurs or, with respect to the develoomental stage at which expression
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`ceocurs, or in response to external stimuli such as ohysiological siresses, pathogens, metal
`
`ions, or inducing agents.
`
`in certain embodiments,
`
`the method comprises isalating the expressed recombinant
`
`precursor CFI protein pricr to step (a).
`
`In certain embodiments, step (@) comprises adding
`
`the furin protein or fragment thereof to a solution comprising the expressed recombinani
`
`precursor CFI protein.
`
`in certain embodiments, step (o} comprises incubating the furin protein or fragment thereof
`
`with the recornbinant orecursor CFi protein at a temperature of between about 25°C to about
`42°C.
`
`in certain embodiments, step (b} comprises incubating the furin protein or fragment thereof
`
`with the recombinant precursor CFi proigin at a temperature of between about 30°C ico about
`42°C.
`
`ee
`
`ist
`
`in certain embodiments, step (6) comprises incubating the furin protein or fragment thereof
`
`with the recombinant precursor CF! protein at a ternperature of between about 35°C to about
`38°C.
`
`in certain embodiments, step (6) comprises incubating the furin protein or fragment thereof
`
`with the recornbinant srecursor CFI protein in a solution having a oH of between about 5 and
`7.
`
`in certain embodiments, step (b) comprises incubating the furin protein or fragrnent thereof
`
`with the recombinant precursor CF! protein in a solution having a pH of between about § and
`8.
`
`in certain embodimenis, ine solution comprises calcium ions.
`
`in ceriain embodiments, the
`
`solution comprises calcium tons at a concentration of between about ImM tio about S5mM.
`
`in Certain embodiments, the solution further comprises potassiumions.
`
`in certain embodiments, step (b) carmprises incubating the furin protein or fragment thereaf
`
`with the recombinant precursor CF! protein for between about 5 hours and about 48 hours.
`
`in certain embodiments, step (6) comprises incubating the furin protein or fragrnent thereof
`
`with the recombinant precursor CF! protein for between about 8 hours and about 20 hours.
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`in certain embediments, the furin protein is a hurnan furin protein or fragment thereof.
`
`in
`
`certain ernbociments, the furin protein is a fragment of a mature furin protein. Aptly, the furin
`
`protein is a truncated furin protein which is terminated before the transmembrane damain.
`
`Aptly ihe truncated furin protein comprises at least one or more amino acid residues at a
`
`pasition at or between 595-791 thal is involved in the catalytic activity of furin e.g. to cleave
`
`at a RRER linker sequence.
`
`in certain ernbodiments, the furin protein or fragment thereof is qiycosylated. Aptly, the furin
`
`protein or fragment thereof is glycosylated at one ar more amino acid residues selected from
`
`Asn3s7, Asn440 and Asn$53.
`
`in certain embodiments, the furin protein or fragment thereof has a molecular weight of 60
`
`kDa or greater. Apily,
`
`the furin protein or fragmeni thereof has a molecular weight af
`
`between about 65 to 65 kDa.
`
`In certain embediments, the furin protein or fragment thereof
`
`comprises a tag ¢.g. a His tag.
`
`in cernain embodiments,
`
`the furin protein or fragment thereof comprises the amine acid
`
`sequence as set forth in SEQ. ID. No.4 or a fragment thereof.
`
`in cedain embodiments, the
`
`furin protein fragment comprises at
`
`least amino acid residues 108 to 715 of a protein
`
`comprising the amino acid sequence as set farth in SEQ. ID. No: 4.
`
`NO JT
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`39
`
`35
`
`in certain embodiments, the furin protein is a protein having at least 86%, e.g. at least 85%,
`
`90%, 91%, 92%, 93%, 94%, 95%, GB%, 97%, BBN, 99% or 100% Sequence identity with a
`
`protein having a sequence as depicted in SER.
`
`ID. No. 4. Aptly, the % sequence identity is
`
`over the entire length of the amino acid sequence set forth in SEQ.
`
`ID. No. 4.
`
`In certain
`
`embodiments, the furin protein is a protein having at least 80% at least 85%, 90%, 91%,
`
`92%, 93%, 94%, 95%, 9E6%, Y7%, 9E%, GO% or 100% Sequence identity with the sequence
`
`consisting of amino acid residues 168 ic 715 of SEQ. IBD. No. 4.
`
`in certain embodiments, the furin protein or fragment thereaf is expressed in a mammatan
`
`cell, Aptly, the method comprises obtaining a furin protein or fragment thereof which has
`
`been expressed in a mammalian cell.
`
`in certain ernbodiments, the method further comprises isolating the recornbinant mature CFI
`
`protein.
`
`In certain embodiments,
`
`the method further comprises purifying the isolated
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`recombinant mature CFi protein.
`
`in certain embodiments,
`
`the recombinant mature CFI
`
`protein is as described herein.
`
`in a further aspect of the present invention, there is a composition obtainable from the
`method described herein.
`
`in @ further aspect of the present invention, there is provided a composition according to
`
`aspecis of the present invention for use in the treatment of a complement-mediated disorder.
`
`in certain embodiments,
`
`the composition is for use in the treatment of a C3 myopathy.
`
`In certain embo