(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(ARYA TAUAE
`
`(10) International Publication Number
`WO 2019/051501 Al
`
`= =
`
`WIPO! PCT
`
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(43) International Publication Date
`14 March 2019 (14.03.2019)
`
`(6)
`
`International Patent Classification:
`CO7K 16700 (2006.01)
`CO7K 19700 (2006.01)
`C07K 16/28 (2006.01)
`C1I2N 15/13 (2006.01)
`CO7K 16/46 (2006.01)
`A61K 47/68 (2017.01)
`
`(81)
`
`(21)
`
`International Application Number:
`
`PCT/US82018/0505 11
`
`(22)
`
`International Filing Date:
`11 September 2018 (11.09.2018)
`
`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,
`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, DD, 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,NI, 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, 8Z, 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, FL 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).
`
`
`
`protein-coupled receptor (GPCR) libraries having nucleic acids encoding for
`a scaffold comprising a GPCR binding domain. Libraries described hereinin-
`clude variegated libraries comprising nucleic acids each encoding for a prede-
`termined variantof at least one predetermined reference nucleic acid sequence.
`Further described herein are protein libraries generated whenthe nucleic acid
`libraries are translated. Further described herein are cell libraries expressing
`variegated nucleic acid libraries described herein.
`
`(54) Title: GPCR BINDING PROTEINS AND SYNTHESIS THEREOF (57) Abstract: Provided herein are methods and compositions relating to G
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`[Continued on nextpage]
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`(25)
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`Filing Language:
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`Publication Language:
`
`English
`
`English
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`(84)
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`Priority Data:
`62/556,863
`
`11 September 2017 (11.09.2017) US
`
`Applicant: TWIST BIOSCIENCE CORPORATION
`[US/US]; 455 Mission Bay Boulevard South, Suite 545, San
`Francisco, CA 94158 (US).
`
`Inventor: GLANVILLE, Jacob; 414 Lake Street, Apart-
`ment 1, San Francisco, CA 94118 CUS).
`
`Agent: HARBURGER, David, S.; Wilson Sonsini
`Goodrich & Rosati, 650 Page Mill Road, Palo Alto, CA
`94304-1050 (US).
`
`(26)
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`(30)
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`(71)
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`(72)
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`(74)
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`wo2019051501AdTITNIMITIIIICRITTTTARTRATEAAA
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`

`

`WO 2019/051501 AD |IMMTTIUTTNN ITNT TE TTNTMT ALTAR ATA CAA
`
`Published:
`
`—_with international search report (Art. 21(3))
`— before the expiration of the time limit for amending the
`claims and to be republished in the event of receipt of
`amendments (Rule 48. 2(h))
`
`

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`WO 2019/051501
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`PCT/US2018/050511
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`GPCR BINDING PROTEINS AND SYNTHESIS THEREOF
`
`CROSS-REFERENCE
`
`[0001]
`
`This application claims the benefit of U.S. Provisional Patent Application No.
`
`62/556,863 filed on September 11, 2017 which is incorporated herein by referencein its entirety.
`
`BACKGROUND
`
`[0002]
`
`G protein-coupled receptors (GPCRs) are implicated in a wide variety of diseases.
`
`Raising antibodies to GPCRs has beendifficult due to problemsin obtaining suitable antigen
`
`because GPCRsare often expressed at low levels in cells and are very unstable when purified.
`
`Thus, there is a need for improved agents for therapeutic intervention which target GPCRs.
`
`INCORPORATION BY REFERENCE
`
`[0003]
`
`All publications, patents, and patent applications mentioned in this specification are
`
`herein incorporated by reference to the same extent as if each individual publication, patent, or
`
`patent application was specifically and individually indicated to be incorporated by reference.
`
`BRIEF SUMMARY
`
`[0004]
`
`Provided herein are antibodies comprising a CDR-H3 comprising a sequence of any
`
`one of SEQ ID NOS: 2420 to 2436. Provided herein are antibodies comprising a CDR-H3
`
`comprising a sequence of any one of SEQ ID NOS: 2420to 2436; and wherein the antibody 1s a
`
`monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a
`
`grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric
`
`antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab
`
`fragment, a F(ab’)2 fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an
`
`isolated complementarity determiming region (CDR), a diabody, a fragment comprised of only a
`
`single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic
`
`(anti-Id) antibody, or ab antigen-binding fragments thereof. Provided herein are antibodies
`
`wherein the VH domain is IGHV 1-18, IGHV 1-69, IGHV 1-8 IGHV3-21, IGHV3-23, IGHV3-
`
`30/33m, IGHV3-28, IGHV3-74, IGHV4-39, or IGHV4-59/61. Provided herein are antibodies,
`
`wherein the VL domain is IGKV 1-39, IGKV1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20,
`
`IGKV4-1, IGLV 1-51, or IGLV2-14. Provided herein are methods of inhibiting GLP1R activity,
`
`comprising, administering the antibodies as described herein. Provided herein are methods for
`
`treatment of a metabolic disorder, comprising administering to a subject in need thereof the
`
`antibodies as described herein. In some instances, the antibody comprises a CDR-H3 comprising
`
`a sequence of any one of SEQ ID NOS: 2420 to 2436. Provided herein are methods for
`
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`treatment of a metabolic disorder, wherein the metabolic disorder is Type I diabetes, or obesity.
`
`Provided herein are nucleic acids encoding for a protein comprising a sequence of any one of
`
`SEQ ID NOS: 2420 to 2436.
`
`[0005]
`
`Provided herein are nucleic acid libraries comprising a plurality of nucleic acids,
`
`wherein each nucleic acid encodes for a sequence that whentranslated encodes for an
`
`immunoglobulin scaffold, wherein the immunoglobulin scaffold comprises a CDR-H3 loop that
`
`comprises a GPCR binding domain, and wherein each nucleic acid comprises a sequence
`
`encoding, for a sequence variant of the GPCR binding domain. Provided herein are nucleic acid
`
`libraries, wherein a length of the CDR-H3loopis about 20 to about 80 aminoacids. Provided
`
`herein are nucleic acid libraries, wherein a length of the CDR-H3 loopis about 80 to about 230
`
`base pairs. Provided herein are nucleic acid libraries, wherein the immunoglobulin scaffold
`
`further comprises one or more domains selected from variable domain, light chain (VL), variable
`
`domain, heavy chain (VH), constant domain, light chain (CL), and constant domain, heavy chain
`
`(CH). Provided herein are nucleic acid libraries, wherein the VH domain is IGHV 1-18, IGHV1-
`
`69, IGHV 1-8 IGHV3-21, IGHV3-23, IGHV3-30/33rn, IGHV3-28, IGHV3-74, IGHV4-39, or
`
`IGHV4-59/61. Provided herein are nucleic acid libraries, wherein the VL domainis IGKV 1-39,
`
`IGKV 1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV 1-51, or IGLV2-14.
`
`Provided herein are nucleic acid libraries, wherein a length of the VH domain is about 90 to
`
`about 100 amino acids. Provided herein are nucleic acid libraries, wherein a length of the VL
`
`domain is about 90 to about 120 amino acids. Provided hercin are nucleic acid librarics, wherein
`
`a length of the VH domainis about 280 to about 300 base pairs. Provided herein are nucleic acid
`
`libraries, wherein a length of the VL domain is about 300 to about 350 base pairs. Provided
`
`herein are nucleic acid librarics, wherein the library comprises at lcast 105 non-identical nucleic
`
`acids. Provided herein are nucleic acid libraries, wherein the immunoglobulin scaffold
`
`comprises a single immunoglobulin domain. Provided herein are nucleic acid libraries, wherein
`
`the immunoglobulin scaffold comprises a peptide of at most 100 amino acids. Provided herein
`
`are vector libraries comprising nucleic acid libraries as described herein. Provided herein are
`
`cell libraries comprising nucleic acid libraries as described herein.
`
`[0006]
`
`Provided herein are nucleic acid libraries comprising, a plurality of nucleic acids,
`
`wherein each nucleic acid encodes for a sequence that whentranslated encodes a GPCR binding
`
`domain, and wherein each nucleic acid comprises sequence encoding for a different GPCR
`
`binding domain about 20 to about 80 amino acids. Provided herein are nucleic acid libraries,
`
`wherein a length of the GPCR binding domainis about 80 to about 230 base pairs. Provided
`
`herein are nucleic acid libraries, wherein the GPCR binding domainis designed based on
`
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`conformational ligand interactions, peptide ligand interactions, small molecule ligand
`
`interactions, extracellular domains of GPCRs, or antibodies that target GPCRs. Provided herein
`
`are vector libraries comprising nucleic acid libraries as described herein. Provided herein are
`
`cell libraries comprising nucleic acid libraries as described herein.
`
`[0007]
`
`Provided herein are protein libraries comprising a plurality of proteins, wherein each
`
`of the proteins of the plurality of proteins comprise an immunoglobulin scaffold, wherein the
`
`immunoglobulin scaffold comprises a CDR-H3 loop that comprises a sequence variant of a
`
`GPCRbinding domain. Provided herein are protein libraries, wherein a length of the CDR-H3
`
`loop is about 20 to about 80 amino acids. Provided herein are protein libraries, wherein the
`
`immunoglobulin scaffold further comprises one or more domains selected from variable domain,
`
`light chain (VL), variable domain, heavy chain (VH), constant domain,light chain (CL), and
`
`constant domain, heavy chain (CH). Provided herein are protein libraries, wherein the VH
`
`domainis IGHV 1-18, IGHV 1-69, IGHV1-8 IGHV3-21, IGHV3-23, IGHV3-30/33m, IGHV3-
`
`28, IGHV3-74, IGHV4-39, or IGHV4-59/61. Provided herein are protein libraries, wherein the
`
`VL domain is IGKV 1-39, IGKV 1-9, IGKV2-28, IGKV3-11, IGK'V3-15, IGKV3-20, IGKV4-1,
`
`IGLV1-51, or IGLV2-14. Provided herein are protein libraries, wherein a length of the WH
`
`domain is about 90 to about 100 amino acids. Provided herein are protein libraries, wherein a
`
`length of the VL domain is about 90 to about 120 amino acids. Provided herein are protein
`
`libraries, wherein the plurality of proteins is used to generate a peptidomimetic library. Provided
`
`herein are protein librarics, wherein the protein library comprises peptides. Provided herein are
`
`protein libraries, wherein the protein library comprises immunoglobulins. Provided herein are
`
`protein libraries, wherein the protein library comprises antibodies. Provided herein are cell
`
`librarics comprising protcin librarics as described hercin.
`
`[0008]
`
`Provided herein are protein libraries comprising a plurality of proteins, wherein the
`
`plurality of proteins comprises sequence encoding for different GPCR binding domains, and
`
`wherein the length of each GPCR binding domain is about 20 to about 80 amino acids. Provided
`
`herein are protein libraries, wherein the protein library comprises peptides. Provided herein are
`
`protein libraries, wherein the protein library comprises immunoglobulins. Provided herein are
`
`protein libraries, wherein the protein library comprises antibodies. Provided herein are protein
`
`libraries, wherein the plurality of proteins are used to generate a peptidomimetic library.
`
`Provided herein are cell libraries comprising protein libraries as described herein.
`
`[0009]
`
`Provided herein are vector libraries comprising a nucleic acid library described
`
`herein. Provided herein are cell libraries comprising a nucleic acid library described herein.
`
`Provided herein are cell libraries comprising a protein library described herein.
`
`

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`WO 2019/051501
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`PCT/US2018/050511
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`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0010]
`
`Figure 1 depicts a schematic of G protein-coupled receptor (GPCR) ligand
`
`interaction surfaces.
`
`[0011]
`
`[0012]
`
`[0013]
`
`[0014]
`
`[0015]
`
`Figure 2A depicts a first schematic of an immunoglobulin scaffold.
`
`Figure 2B depicts a second schematic of an immunoglobulin scaffold.
`
`Figure 3 depicts a schematic of a motif for placement in a scaffold.
`
`Figure 4 depicts a schematic of a GPCR.
`
`Figure 5 depicts schematics of segments for assembly of clonal fragments and non-
`
`clonal fragments.
`
`[0016]
`
`Figure 6 depicts schematics of segments for assembly of clonal fragments and non-
`
`clonal fragments.
`
`[0017]
`
`Figure 7 presents a diagram of steps demonstrating an exemplary process workflow
`
`for gene synthesis as disclosed herein.
`
`[0018]
`
`[0019]
`
`[0020]
`
`Figure 8 illustrates an example of a computer system.
`
`Figure 9 is a block diagram illustrating an architecture of a computer system.
`
`Figure 10 is a diagram demonstrating a network configured to incorporate a plurality
`
`of computer systems, a plurality of cell phones and personal data assistants, and Network
`
`Attached Storage (NAS).
`
`[0021]
`
`Figure 11 is a block diagram of a multiprocessor computer system using a shared
`
`virtual address memory spacc.
`
`[0022]
`
`[0023]
`
`[0024
`
`chains.
`
`[0025]
`
`chains.
`
`Figures 12A-12C depict sequences of immunoglobulin scaffolds.
`
`Figure 13 depicts sequences of G protein-coupled receptors scaffolds.
`
`Figure 14 is a graph of normalized readsfor a library for variable domain, hcavy
`
`Figure 15 is a graph of normalized reads for a library for variable domain, light
`
`[0026]
`
`Figure 16 is a graph of normalized reads for a library for heavy chain
`
`complementarity determining region 3.
`
`Figure 17A is a plot of light chain frameworks assayed for folding.
`
`Figure 17B is a plot of light chain frameworksassayed for thermostability.
`
`Figure 17C is a plot of light chain frameworks assayed for motif display using FLAG
`
`[0027]
`
`[0028]
`
`[0029]
`
`tag.
`
`[0030]
`
`Figure 17D is a plot of light chain frameworks assayed for motif display using His
`
`tag.
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`WO 2019/051501
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`Figure 18A is a plot of heavy chain frameworks assayed for folding.
`
`Figure 18B is a plot of heavy chain frameworksassayed for stability.
`
`Figure 18C is a plot of heavy chain frameworks assayed for motif display using
`
`[0031
`
`[0032]
`
`[0033]
`
`FLAGtag.
`
`[0034]
`
`Figure 18D is a plot of heavy chain frameworks assayed for motif display using His
`
`tag.
`
`[0035]
`
`[0036]
`
`[0037]
`
`Figure 18E is a plot of heavy chain frameworks assayed for expression.
`
`Figure 18F is a plot of heavy chain frameworks assayed for selection specificity.
`
`Figures 19A-19C depict images of G protein-coupled receptors visualized by
`
`fluorescent antibodies.
`
`[0038]
`
`Figures 20A-20C depict images of G protein-coupled receptors visualized by auto-
`
`fluorescent proteins.
`
`[0039]
`
`Figure 21A depicts a schematic of an immunoglobulin scaffold comprising a VH
`
`domain attached to a VL domain using a linker.
`
`[0040]
`
`Figure 21B depicts a schematic of a full-domain architecture of an immunoglobulin
`
`scaffold comprising a VH domainattached to a VL domainusing a linker, a leader sequence, and
`
`pIII sequence.
`
`[0041]
`
`Figure 21C depicts a schematic of four framework elements (FW1, FW2, FW3,
`
`FW4)and the variable 3 CDR (L1, L2, L3) elements for a VL or VH domain.
`
`DETAILED DESCRIPTION
`
`[0042]
`
`The present disclosure employs, unless otherwise indicated, conventional molecular
`
`biology techniques, which are within the skill of the art. Unless defined otherwise, all technical
`
`and scientific terms used herein have the same meaning as is commonly understood by one of
`
`ordinary skill in the art.
`
`[0043]
`
`[0044]
`
`Definitions
`
`Throughout this disclosure, various embodiments are presented in a range format. It
`
`should be understood that the description in range format is merely for convenience andbrevity
`
`and should not be construed as an inflexible limitation on the scope of any embodiments.
`
`Accordingly, the description of a range should be considered to have specifically disclosed all
`
`the possible subranges as well as individual numerical values within that range to the tenth of the
`
`unit of the lower limit unless the context clearly dictates otherwise. For example, description of
`
`a range such as from | to 6 should be considered to have specifically disclosed subranges such as
`
`from | to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as
`
`individual values within that range, for example, 1.1, 2, 2.3,5, and 5.9. This applies regardless
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`of the breadth of the range. The upper and lowerlimits of these intervening ranges may
`
`independently be included in the smaller ranges, and are also encompassed within the disclosure,
`
`subject to any specifically excluded limit in the stated range. Where the stated range includes
`
`one or both of the limits, ranges excluding either or both of those included limits are also
`
`included in the disclosure, unless the context clearly dictates otherwise.
`
`[0045]
`
`The terminology used herein is for the purpose of describing particular embodiments
`
`only andis not intended to be limiting of any embodiment. As used herein, the singular forms
`
`“a,” “an” and “the” are intended to include the plural formsas well, unless the context clearly
`
`indicates otherwise. It will be further understood that the terms “comprises” and/or
`
`“comprising,” when used in this specification, specify the presence of stated features, integers,
`
`steps, operations, elements, and/or components, but do not preclude the presence or addition of
`
`one or more other features, integers, steps, operations, elements, components, and/or groups
`
`thereof. As used herein, the term “and/or” includes any and all combinations of one or more of
`
`the associated listed items.
`
`[0046]
`
`Unless specifically stated or obvious from context, as used herein, the term “about” in
`
`reference to a numberor range of numbers is understood to meanthe stated number and numbers
`
`+/- 10% thereof, or 10% below the lowerlisted limit and 10% above the higher listed limit for
`
`the values listed for a range.
`
`[0047]
`
`Unless specifically stated, as used herein, the term “nucleic acid” encompasses
`
`double- or triplce-stranded nucleic acids, as well as single-stranded molcculcs. In double- or
`
`triple-stranded nucleic acids, the nucleic acid strands need not be coextensive (i.e., a double-
`
`stranded nucleic acid need not be double-stranded along the entire length of both strands).
`
`Nucleic acid scqucnecs, when provided,arc listed in the 5’ to 3’ direction, unless stated
`
`otherwise. Methods described herein provide for the generation of isolated nucleic acids.
`
`Methods described herein additionally provide for the generation of isolated and purified nucleic
`
`acids. A “nucleic acid”as referred to herein can comprise at least 5, 10, 20, 30, 40, 50, 60, 70,
`
`80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600,
`
`700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or more
`
`bases in length. Moreover, provided herein are methods for the synthesis of any number of
`
`polypeptide-segments encoding nucleotide sequences, including sequences encoding non-
`
`ribosomal peptides (NRPs), sequences encoding non-ribosomal peptide-synthetase (NRPS)
`
`modules and synthetic variants, polypeptide segments of other modular proteins, such as
`
`antibodies, polypeptide segments from other protein families, including non-coding DNA or
`
`RNA,such as regulatory sequences e.g. promoters, transcription factors, enhancers, siRNA,
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`shRNA, RNAi, miRNA, small nucleolar RNA derived from microRNA,or any functional or
`
`structural DNA or RNA unit of interest. The following are non-limiting examples of
`
`polynucleotides: coding or non-coding regions of a gene or gene fragment, intergenic DNA,loci
`
`(locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA,
`
`ribosomal RNA,short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA
`
`(miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA), which is a DNA
`
`representation of mRNA,usually obtained by reverse transcription of messenger RNA (mRNA)
`
`or by amplification; DNA molecules produced synthetically or by amplification, genomic DNA,
`
`recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any
`
`sequence, isolated RNA of any sequence, nucleic acid probes, and primers. cDNA encoding for
`
`a gene or gene fragment referred herein may comprise at least one region encoding for exon
`
`sequences without an intervening intron sequencein the genomic equivalent sequence.
`
`[0048]
`
`[0049]
`
`GPCRLibraries
`
`Provided herein are methods and compositionsrelating to G protein-coupled receptor
`
`(GPCR)binding libraries comprising nucleic acids encoding for a scaffold comprising a GPCR
`
`binding domain. Scaffolds as described herein can stably support a GPCR binding domain. The
`
`GPCRbinding domain may be designed based on surface interactions of a GPCRligand and the
`
`GPCR.Libraries as described herein may be further variegated to provide for variant libraries
`
`comprising nucleic acids each encoding for a predetermined variant of at least one predetermined
`
`reference nucleic acid sequence. Further described herein are protein librarics that may be
`
`generated whenthe nucleic acid libraries are translated. In someinstances, nucleic acid libraries
`
`as described herein are transferred into cells to generate a cell library. Also provided herein are
`
`downstream applications for the librarics synthcsized using methods described herein.
`
`Downstream applications include identification of variant nucleic acids or protein sequences
`
`with enhanced biologically relevant functions, ¢.g., improvedstability, affinity, binding,
`
`functional activity, and for the treatment or prevention of a disease state associated with GPCR
`
`signaling.
`
`[0050]
`
`[0051]
`
`Scaffold Libraries
`
`Provided herein are libraries comprising nucleic acids encoding for a scaffold,
`
`wherein sequences for GPCR binding domainsare placed in the scaffold. Scaffold described
`
`herein allow for improved stability for a range of GPCR binding domain encoding sequences
`
`wheninserted into the scaffold, as compared to an unmodified scaffold. Exemplary scaffolds
`
`include, but are not limited to, a protein, a peptide, an immunoglobulin, derivatives thereof, or
`
`combinations thereof. In someinstances, the scaffold is an immunoglobulin. Scaffolds as
`
`

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`described herein comprise improved functional activity, structural stability, expression,
`
`specificity, or a combination thereof. In someinstances, scaffolds comprise long regions for
`
`supporting a GPCR binding domain.
`
`[0052]
`
`Provided herein are libraries comprising nucleic acids encoding for a scaffold,
`
`wherein the scaffold is an immunoglobulin. In some instances, the immunoglobulin is an
`
`antibody. As used herein, the term antibody will be understood to include proteins having the
`
`characteristic two-armed, Y-shape of a typical antibody molecule as well as one or more
`
`fragments of an antibody that retain the ability to specifically bind to an antigen. Exemplary
`
`antibodies include, but are not limited to, a monoclonal antibody, a polyclonal antibody, a bi-
`
`specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized
`
`antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs
`
`(scFv) (including fragments in which the VL and VH are joined using recombinant methods by a
`
`synthetic or natural linker that enables them to be made as a single protein chain in which the VL
`
`and VHregionspair to form monovalent molecules, including single chain Fab and scFab), a
`
`single chain antibody, a Fab fragment (including monovalent fragments comprising the VL, VH,
`
`CL, and CH1 domains), a F(ab’)2 fragment (including bivalent fragments comprising two Fab
`
`fragments linked by a disulfide bridge at the hinge region), a Fd fragment (including fragments
`
`comprising, the VH and CH1 fragment), a Fv fragment (including fragments comprising the VL
`
`and VH domains of a single arm of an antibody), a single-domain antibody (dAb or sdAb)
`
`(including fragments comprising a VH domain), an isolated complementarity determining region
`
`(CDR), a diabody (including fragments comprising bivalent dimers such as two VL and VH
`
`domains bound to each other and recognizing two different antigens), a fragment comprised of
`
`only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-
`
`idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof. In some instances, the
`
`libraries disclosed herein comprise nucleic acids encoding for a scaffold, wherein the scaffold is
`
`a Fv antibody,including Fv antibodies comprised of the minimum antibody fragment which
`
`contains a complete antigen-recognition and antigen-binding site. In some embodiments, the Fv
`
`antibody consists of a dimer of one heavy chain and one light chain variable domainin tight,
`
`non-covalent association, and the three hypervariable regions of each variable domain interact to
`
`define an antigen-bindingsite on the surface of the VH-VL dimer. In some embodiments,the six
`
`hypervariable regions confer antigen-binding specificity to the antibody. In some embodiments,
`
`a single variable domain (or half of an Fv comprising only three hypervariable regions specific
`
`for an antigen, including single domain antibodies isolated from camelid animals comprising one
`
`heavy chain variable domain such as VHH antibodies or nanobodies) has the ability to recognize
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`and bind antigen. In some instances, the libraries disclosed herein comprise nucleic acids
`
`encoding for a scaffold, wherein the scaffold is a single-chain Fv or scFv, including antibody
`
`fragments comprising a VH, a VL, or both a VH and VL domain, wherein both domainsare
`
`present in a single polypeptide chain. In some embodiments, the Fv polypeptide further
`
`comprises a polypeptide linker between the VH and VL domains allowing the scFv to form the
`
`desired structure for antigen binding. In some instances, a scFv is linked to the Fc fragmentor a
`
`VHH is linked to the Fe fragment (including minibodies). In some instances, the antibody
`
`comprises immunoglobulin molecules and immunologically active fragments of immunoglobulin
`
`molecules, e.g., molecules that contain an antigen binding site. Immunoglobulin molecules are
`
`of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG 2, IgG 3, IgG 4, IgA
`
`1 and IgA 2) or subclass.
`
`[0053]
`
`Libraries described herein comprising nucleic acids encoding for a scaffold, wherein
`
`the scaffold is an immunoglobulin, comprise variations in at least one region of the
`
`immunoglobulin. Exemplary regions of the antibody for variation include, but are not limited to,
`
`a complementarity-determining region (CDR), a variable domain, or a constant domain. In some
`
`instances, the CDR is CDR1, CDR2, or CDR3.
`
`In some instances, the CDR 1s a heavy domain
`
`including, but not limited to, CDR-H1, CDR-H2, and CDR-H3. In someinstances, the CDRis a
`
`light domain including,but not limited to, CDR-L1, CDR-L2, and CDR-L3. In someinstances,
`
`the variable domainis variable domain, light chain (VL) or variable domain, heavy chain (VHA).
`
`In some instances, the VL domain comprises kappa or lambda chains. In someinstances, the
`
`constant domain is constant domain,light chain (CL) or constant domain, heavy chain (CH).
`
`[0054]
`
`Methods described herein provide for synthesis of libraries comprising nucleic acids
`
`encoding for a scaffold, wherein cach nucleic acid encodes for a predetermined variant ofat Icast
`
`one predetermined reference nucleic acid sequence. In somecases, the predetermined reference
`
`sequence is a nucleic acid sequence encoding for a protein, and the variant library comprises
`
`sequences encoding for variation of at least a single codon such that a plurality of different
`
`variants of a single residue in the subsequent protein encoded by the synthesized nucleic acid are
`
`generated by standard translation processes. In some instances, the scaffold library comprises
`
`varied nucleic acids collectively encoding variations at multiple positions. In some instances, the
`
`variant library comprises sequences encoding for variation of at least a single codon of a CDR-
`
`H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, VL, or VH domain. In someinstances,
`
`the variant library comprises sequences encoding for variation of multiple codons of a CDR-H1,
`
`CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, VL, or VH domain. In some instances, the
`
`variant library comprises sequences encoding for variation of multiple codons of framework
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`element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework
`
`element 4 (FW4). An exemplary number of codons for variation include, but are not limited to,
`
`at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,
`
`125, 150, 175, 225, 250, 275, 300, or more than 300 codons.
`
`[0055]
`
`In someinstances, the at least one region of the immunoglobulin for variation is from
`
`heavy chain V-gene family, heavy chain D-gene family, heavy chain J-gene family, light chain
`
`V-gene family, or light chain J-gene family. In someinstances, the light chain V-gene family
`
`comprises immunoglobulin kappa (IGK) gene or immunoglobulin lambda (IGL). Exemplary
`
`genes include, but are not limited to, IGHV 1-18, IGHV 1-69, IGHV 1-8, IGHV3-21, IGHV3-23,
`
`IGHV3-30/33rm, IGHV3-28, IGHV 1-69, IGHV3-74, IGHV4-39, IGHV4-59/61, IGKV 1-39,
`
`IGKV 1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGK-V3-20, IGKV4-1, IGLV1-51, and IGLV2-14.
`
`[0056]
`
`Provided herein are libraries comprising nucleic acids encoding for immunoglobulin
`
`scaffolds, wherein the libraries are synthesized with various numbers of fragments. In some
`
`instances, the fragments comprise the CDR-H|, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-
`
`L3, VL, or VH domain. In some instances, the fragments comprise framework element 1 (FW 1),
`
`framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). In
`
`some instances, the scaffold libraries are synthesized with at least or about 2 fragments, 3
`
`fragments, 4 fragments, 5 fragments, or more than 5 fragments. The length of each of the
`
`nucleic acid fragments or average length of the nucleic acids synthesized may be at least or about
`
`50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525,
`
`550, 575, 600, or more than 600 base pairs. In some instances, the length is about 50 to 600, 75
`
`to 575, 100 to 550, 125 to 525, 150 to 500, 175 to 475, 200 to 450, 225 to 425, 250 to 400, 275
`
`to 375, or 300 to 350 bascpairs.
`
`[0057]
`
`Libraries comprising nucleic acids encoding for immunoglobulin scaffolds as
`
`described herein comprise various lengths of amino acids when translated. In someinstances,
`
`the length of each of the amino acid fragments or average length of the amino acid synthesized
`
`may beat least or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,
`
`105, 110, 115, 120, 125, 130, 135, 140, 145, 150, or more than 150 amino acids. In some
`
`instances, the length of the amino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to
`
`130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105, 70 to 100, or 75 to 95
`
`amino acids. In some instances, the length of the amino acid is about 22 amino acids to about 75
`
`amino acids. In someinstances, the immunoglobulin scaffolds comprise at least or about 100,
`
`200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than 5000 amino
`
`acids.
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`10
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`[0058]
`
`A numberofvariant sequences for the at least one region of the immunoglobulin for
`
`variation are de novo synthesized using methods as described herein. In some instances, a
`
`numberof variant sequences is de novo synthesized for CDR-H1, CDR-H2, CDR-H3, CDR-LI,
`
`CDR-L2, CDR-L3, VL, VH, or combinations thereof. In some instances, a number of variant
`
`sequences 1s de novo synthesized for framework element 1 (FW1), framework element 2 (FW2),
`
`framework element 3 (FW3), or framework element 4 (FW4). The numberof variant sequences
`
`may beat least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
`
`100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more
`
`than 500 sequences. In some i