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US 2017/0166878 A9 (12) Patent Application Publication (48) Pub US 20170166878A9 (19) United States (10) Pub. No.: US 2017/0166878 A9 (12) Patent Application Publication (48) Pub. Date: Jun. 15, 2017 Thanos et al. CORRECTED PUBLICATION (54) COMPOSITIONS OF ADENOSINE (60) Provisional application No. 62/063.936, filed on Oct. DEAMINASE-2 (ADA2), VARIANTS 14, 2014. THEREOF AND METHODS OF USING SAME (71) Applicant: Halozyme, Inc., San Diego, CA (US) Publication Classification (72) Inventors: Christopher D. Thanos, Tiburon, CA (51) Int. Cl. (US); Lin Wang, San Diego, CA (US); CI2N 9/78 (2006.01) H. Michael Shepard, San Diego, CA A638/48 (2006.01) (US) (52) U.S. Cl. (21) Appl. No.: 15/094,908 CPC ................ CI2N 9/78 (2013.01); A61K 38/48 (22) Filed1C Apr.pr. 8,5, 2016 (2013.01); C12Y 305/04004 (2013.01) Prior Publication Data (57) ABSTRACT (15) Correction of US 2016/0272960 A1 Sep. 22, 2016 See Claim 11. Provided are variant adenosine deaminase 2 (ADA2) pro teins, conjugates thereof and compositions containing the (65) US 2016/0272960 A1 Sep. 22, 2016 proteins and/or conjugates. Also provided are methods and uses of the ADA2 proteins or conjugates for treating dis Related U.S. Application Data eases and conditions, such as a tumor or cancer, and in (63) Continuation-in-part of application No. PCT/US 15/ particular any disease or condition associated with elevated 55613, filed on Oct. 14, 2015. adenosine or other associated marker. Patent Application Publication Jun. 15, 2017. Sheet 2 of 7 US 2017/O166878 A9 09 09 OZI OZT 08T 08T 0€ 08 099 O9€. NVITT ?VÄTHOEIXIT,N'HA AVIACIXIS AVIAQIXIS (ISA'I>{'IAÕNSHADAHIdI (ISATXHTAONSTOEOATRIÐI × * * ANCIÐARGÖWISHHA?CIH,BA?IVÄHITIÐSILAH,HINE,EXISMAANÕNLÄIAETHÕLATILHNHITIS ×* ?THINKIXEXTTTHTHIEGIST,SÐHÆSINGAVITIHOTw???s?ÐGAT? ANCI?ZJ?ÕWISHRAZG?JA?V?KHITIÐSILAH,HIJ,GI,HXISMAANÕNL?CIATHAHÕJATI,HÌNHTITIS }MSKIV?HXISTV7H?H?IHLLNTW'IVOITIINHCIISLÐÕMCI MS?L?AVGIHXISTIVZH?H?INILINTW'IVOITINNIGISL5)ÕMC? ILGALVACIWIEXCIMEXXIMI?WHLNXESETTLS?, eTT?Ios) eTTTIORÐ e[|LIOÐ eTT?LIOÐ Ueu?rlH UelunH. UeuInH UpunH Patent Application Publication Jun. 15, 2017. Sheet 3 of 7 US 2017/O166878 A9 09 09 OZT OZT 0€ 0$ 09© O9£ OZ?, OZŽ 08#7 08#7 : ×°* * * NYTTERIMINTAT(OSTHWAIME,ITTHVAÐLEGISTvSÐHÆSKIVAVITIHO'I?aa?saeqAIW ANCIÐAHRHÔINSHAHAACTHHA?\{XHI"15)SILE.HILEAXISMAANÕN?L?TAGI,HÕLAT?T?NHTT?S sºIIXIÐIÐ?HLEAHYÖVAHÒAIXASMEHHQHHRÐSTÍHAA (ISATIXITAÕNSIäCA?II?IIGIV?HyISTIVÝ,HÉÐHÉÐIHSVANTIJAIT?V?C?TITNIHCIISME?ÔMCI TIGXII,VIACIVII,HXHCIMBIXAXIMI?W,HÌNXIFISTITTT.SK.) TIGALVACI,I,IXOIMHºx{MIEWILNYESETILS, AV7ACIXIST?XIIXIÐIÈGaHLEAIXÕVAGÓXIXASMEHOIHH)ÐST?NAà. duTuo,AuIÓKAI uleu.Ir.H. UIEuIn?[ duTuoWurf,KAI Patent Application Publication Jun. 15, 2017. Sheet 4 of 7 US 2017/O166878 A9 09 09 OZT OZI 08T 09T O#Z OizZ OZ# OZ7 0357 037 × CHSATYITAÓNSIAOATIT(HTS?„Tae/\\?aH>{STV7HÆÐHÆÐTÆLLINTIATIVOITIINHO?IS?,5)ÕMCI OTSATTYITAÓNST+OAT?T&TSK.V?HY?STV7H?H?IHLENTIATIVÝCHTINHOITSJ,5)ÕMCI ×* * * * ×* NVTOETRYTINTAT,THWT-II,R?GITSTVS5),H.HS/NW/AWITTT,HOTTW7&YITHSCH?GIATJAI NYÖREYÐINTAT(05)THWW.DIE™ITTHVAÐLEGIISTYSOGIRISINVAV?TTIHHTv?MTHS??CIATIN * uennõupu.O Upºnnõue.TO uenn6tup?O upºnnõue.TO uerqn6ueIO UeuInH UeunH uerqnõue:IO ueunH UeumH LIeuan?? LIelunH Patent Application Publication Jun. 15, 2017. Sheet 6 of 7 US 2017/O166878 A9 09 89 OZT 8TT 0£ 86Z O87 8/† **×× ×* ×* ×* ×* ×* XIIXII?)IHEIGHHJÆLAHYA?VARHÓXÙXÌASM?R?GIHH5H5OSTI?IXA?I XIIXIT?T,HOEH?H?RAHXHÕVARIÕ???ASMEHHGHH515)STEIXAd ×* ×* ¿Tern67) ITG(ALVACI(IHYACIMÈXIXMI?WHLNXESETT?S?, 6OCYALVAGYZI,HXHCIMÈXXIMITH??HLNXIET,HTT?S? x* upurnH enbeoeJN upurnH enbeoe? Patent Application Publication Jun. 15, 2017. Sheet 7 of 7 US 2017/O166878 A9 US 2017/O166878 A9 Jun. 15, 2017 COMPOSITIONS OF ADENOSINE SUMMARY DEAMINASE-2 (ADA2), VARIANTS 0005 Provided herein are variant Adenosine Deaminase THEREOF AND METHODS OF USING SAME 2 (ADA2) proteins or catalytically active portions thereof that contain a modification(s) in the sequence of amino acids RELATED APPLICATIONS of an unmodified ADA2 protein or a catalytically active portion thereof. In some embodiments, the unmodified 0001. This application is a continuation-in-part of Inter ADA2 protein can include the sequence of amino acids set national PCT application No. PCT/US 15/55613, filed Oct. forth in SEQID NO:5, or a sequence of amino acids that can 14, 2015, to Applicant Halozyme, Inc. and inventors Chris exhibit at least 85% sequence identity to the sequence of topher D. Thanos, Lin Wang and H. Michael Shepard, amino acids set forth in SEQ ID NO:5, or is a catalytically entitled COMPOSITIONS OF ADENOSINE DEAMI active portion thereof; the amino acid modification(s) are NASE-2 (ADA2), VARIANTS THEREOF AND METH selected from among amino acid replacement(s), deletion(s) ODS OF USING SAME, which claims the benefit of priority and insertion(s); and the variant ADA2, when in dimer form, to U.S. provisional application Ser. No. 62/063.936, filed can exhibit one or more properties selected from among Oct. 14, 2014, to inventors Christopher D Thanos, Lin Wang increased adenosine deaminase activity, reduced heparin and H. Michael Shepard, entitled COMPOSITIONS OF binding, longer serum half-life, altered pH optimum, ADENOSINE DEAMINASE-2 (ADA2), VARIANTS increased thermal stability, altered receptor binding and THEREOF AND METHODS OF USING SAME. The Sub hyperglycosylation compared to the corresponding dimer ject matter of these applications is incorporated by reference form of the unmodified ADA2 protein. A variety of amino in its entirety. acid modifications, including replacements, deletions and insertions are provided. It is understood that the discreet INCORPORATION BY REFERENCE OF modifications that confer a particular activity or property can SEQUENCE LISTING PROVIDED be combined; as in proteins effects of mutation or modifi ELECTRONICALLY cations generally are additive. Any of the variant ADA2 or catalytically active portion thereof provided herein that 0002 An electronic version of the Sequence Listing is contains modifications, including replacements, deletions filed herewith, the contents of which are incorporated by and insertions, and nucleic acids encoding the variant ADA2 reference in their entirety. The electronic file was created on or catalytically active portion thereof, can be used in any of Apr. 8, 2016, is 3,177 kilobytes in size, and titled the methods, compositions, conjugates, modified forms, 3121 SEQ001.txt. vectors, cells, combinations, uses and compositions for use, and combinations for use, provided herein. FIELD OF THE INVENTION 0006. In some embodiments, the variant ADA2 protein or catalytically active portion thereof, when in dimer form, 0003 Provided are variant adenosine deaminase 2 exhibits increased adenosine deaminase activity or increased (ADA2) proteins. Also provided are ADA2 conjugates and adenosine deaminase activity and reduced heparin binding. compositions containing an ADA2 protein or ADA2 conju 0007. In some embodiments, the unmodified ADA2 pro gate. Also provided are methods and uses of the ADA2 tein is a homodimer, and the monomer form comprises the proteins or conjugates for treating diseases and conditions, sequence of amino acid residues set forth in SEQID NO:5. Such as a tumor or cancer, and in particular any disease or In some embodiments, the variant ADA2 is a catalytically condition associated with elevated adenosine or other asso active portion of the variant ADA2 protein as provided ciated marker. herein, wherein the unmodified ADA2 protein is a homodi mer of corresponding catalytically active portions of the BACKGROUND polypeptide whose sequence is set forth in of SEQID NO:5, wherein corresponding portions are determined by align 0004 Adenosine is a well-known effector of immune ment. function. In T-cells, adenosine decreases T-cell receptor 0008. In some embodiment, the ADA2 protein or cata induced activation of NF-KB, and inhibits IL-2, IL-4, and lytically active portion thereofdoes not contain a modifica IFN-y. Adenosine decreases T-cell cytotoxicity, increases tion selected from among an amino acid replacement cor T-cell anergy, and increases T-cell differentiation to Fop3+ responding to H7R, G18A, G18R, G18V. I64T, A80D, or Lag-3+ regulatory (Treg) T-cells. On NK cells, adenosine H83Q, V90A, C108G, A120V, H121R, W133G, R125C, is known to decrease IFN-Y production, and suppress NK R140O, K141R, R142W, P164L, P222L, W235S, H306R, cell cytoxicity. Adenosine is known to block neutrophil E330G. W333G, V365L, Y424C, F464S or a deletion cor adhesion and extravasation, decrease phagocytosis, and responding to R8-K14del->--, with numbering with refer attenuate levels of Superoxide and nitric oxide. Adenosine ence to amino acid residues set forth in SEQ ID NO:5. also decreases the expression of TNF-C. IL-12, and MIP-1C. 0009. In some embodiments, the unmodified ADA2 pro on macrophages, attenuates MHC Class II expression, and tein can include a sequence of amino acids that has at least increases levels of IL-10 and IL-6. In addition, adenosine 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, decreases phagocytosis and Superoxide and nitric oxide 95%, 96%, 97%, 98%, 99% or more sequence identity to the levels on macrophages. Through these immune-related sequence of amino acids set forth in SEQ ID NO:5 or is a activities, and others, aberrant or accumulated levels of catalytically active portion thereof. For example, the adenosine is associated with a number of diseases and unmodified ADA2 protein has at least 95% sequence iden conditions, including those in which the adenosine-mediated tity with the sequence of amino acids set forth in SEQ ID immunosuppressive effects play a role. Hence, there is a NO:5 or with the corresponding catalytically active portion need for treatments of Such diseases and conditions. thereof. For example, the unmodified ADA2 protein US 2017/O166878 A9 Jun. 15, 2017 includes the sequence of amino acids set forth in any of SEQ ID NOS:5, 326-334,340,375 or 380-383 or is a catalytically active portion thereof, or the unmodified ADA2 protein has a sequence of amino acids set forth in any of SEQID NOS:5, 326-334, 340, 375 and 380-383 or is a catalytically active portion thereof. In particular embodiments, the unmodified ADA2 protein includes the sequence of amino acids set forth in SEQID NO:5 or is a catalytically active portion thereof.
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