(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`19) World Intellectual P
`rt
`ie Oreanization
`UAAAAAA
`Organization
`International Burcau
`(10) International Publication Number
`(43) International Publication Date
`WO 2022/098662 A2
`12 May 2022 (12.05.2022)
`
`WW
`
`WIPO PCT
`
`(51) International Patent Classification:
`Notclassified
`(1) International Application Number:
`PCT/US2021/057759
`
`(22) InternationalFiling Date:
`.
`02 November 2021 (02.11.2021)
`English
`
`(25) Filing Language:
`
`(26) Publication Language:
`(30) Priority Data:
`63/109,280
`.
`(71) Applicant: TWIST BIOSCIENCE CORPORATION
`[US/US]; 681 Gateway Boulevard, South San Francisco,
`California 94080 (US).
`
`03 November 2020 (03.11.2020) US
`
`English
`
`(72) Inventors: SATO, Aaron, 681 Galeway Boulevard, South
`San Francisco, California 94080 (US). LIU, Qiang; 681
`Gateway Boulevard, South San Francisco, California 94080
`;
`(US). WANG,Linya; 681 Gateway Boulevard, South San
`Francisco, California 94080 (US). AXELROD, Fumiko;
`681 Gateway Boulevard, South San Francisco, California
`94080 (US).
`
`(74) Agent: DUSABAN GONZALES,Stephanie 8.; WILSON
`SONSINI GOODRICH & ROSATI, 650 Page Mill Road,
`Palo Alto, California 94304 (US).
`(81) Designated States (unless otherwise indicated, for every
`kind ofnational 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, FI, GB, GD, GE, GH, GM, GT, HN,
`UR, HU,ID, IL, IN, IR,IS, IT, 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, 8G, 5K, SL, ST, SV, SY, TH, TJ, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, WS,ZA, ZM,ZW.
`
`(84) Designated States (iless 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.TI.
`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, 2T. RO. RS. SE. SL. SK. SM.
`TR), OAPI(BF, BJ, CF, CG, Cl, CM, GA, GN, GQ, GW
`°
`DY,
`AD,
`AM
`Uh
`NB
`SE
`INS
`OR
`KM,ML, MR,NE, SN, TD, TG).
`
`:
`
`(54) Titles MELHODS AND COMPOSITIONS RELATING TO CHEMOKINE RECEPTOR VARLAN'T'S
`
`4.5x405
`
`RL1-HMFI
`
`5.0x4104
`
`ok
`
`®
`
`.
`
`x’
`f
`
`wa
`
`+ R&D MAB
`~* CXCR5-1-65
`
`“® CXCRS-1-66
`CXCR5-1-69
`» CXCR5-1-71
`~& CXCR5-1-73
`-» CXCR5-1-80
`-» CXCRS-1-81
`
`~e CXCRS5-1-83
`-® CXCR5-1-88
`= CXCR5-1-89
`
`~a CXCR5-1-96
`
`
`
`0.01
`
`04
`
`10
`1
`[igG] nM
`
`#001008). cxCRS-1-106
`-w- CXCRS-1-107
`
`FIG. 17A
`
`(57) Abstract: Provided hercin arc mcthods and compositions relating to chemokine receptorlibrarics having nucleic acids encoding
`for immunoglobulins that bind to chemokine receptors. Libraries described herein include variegated libraries comprising nucleic acids
`each encoding for a predetermined variant of at least one predetermined reference mucleic acid sequence. Further described herein are
`protcin librarics gencrated whenthe nucleic acid librarics are translated. Further described hereinareccll librarics expressing varicgated
`nucleic acid libraries described herein.
`
`[Continued on nextpage]
`
`
`
`
`
`wo2022/098662A2IITATANITATAITAATATA
`
`

`

`WO 2022/098662 A2 [IIITINIT INNINTMAMNACTNOI UME
`
`Declarations under Rule 4.17:
`— as to applicant's entitlement to apply jor and be granted a
`patent (Rule 4.17(fi))
`Published:
`
`— without international search report and to be republished
`upon receipt ofthat report (Rule 48.2(2))
`
`

`

`WO 2022/098662
`
`PCT/US2021/057759
`
`METHODS AND COMPOSITIONS RELATING TO CHEMOKINE RECEPTOR
`VARIANTS
`
`CROSS-REFERENCE
`
`[0001]
`
`This application claims the benefit of U.S. Provisional Patent Application No.
`
`63/109,280 filed on November3, 2020, which 1s incorporated by referencein its entirety.
`
`BACKGROUND
`
`[0002]
`
`G protein-coupled receptors (GPCRs) are implicated in a wide variety ofdiseases.
`
`Raising antibodies to GPCRs has been difficult due to problems in 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 wasspecifically and individually indicated to be incorporated by reference.
`
`BRIEF SUMMARY
`
`[0004]
`
`Provided herein are antibodies or antibody fragments comprising a variable domain,
`
`heavy chain region (VH)and a variable domain, light chain region (VL), wherein VH comprises
`
`complementarity determining regions CDRH1, CDRH2, and CDRH3, wherein VL comprises
`
`complementarity determining regions CDRL1, CDRL2, and CDRL3, and wherein (a) an amino
`
`acid sequence of CDRH1 is as set forth in any one of SEQ ID NOs: 526-662; (b) an amino acid
`
`sequence of CDRHz2is as set forth in any one of SEQ ID NOs: 663-977; (c) an amino acid
`
`sequence of CDRH31s asset forth in any one of SEQ ID NOs: 978-1102; (d) an aminoacid
`
`sequence of CDRL1 1s as set forth in any one of SEQ ID NOs: 1103-1267; (e) an amino acid
`
`sequence of CDRL2is as set forth in any one of SEQ ID NOs: 1268-1328; and (f) an amino acid
`
`sequence of CDRL31s asset forth in any one of SEQ ID NOs: 1329-1493. Further provided
`
`herein are antibodies or antibody fragments, wherein the antibody is 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
`
`determining region (CDR), a diabody, a fragment comprised of only a single monomeric
`
`1
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`

`WO 2022/098662
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`
`variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody,
`
`or ab antigen-binding fragments thereof. Further provided herein are antibodies or antibody
`
`fragments, wherein the antibody or antibody fragment thereof is chimeric or humanized. Further
`
`provided herein are antibodies or antibody fragments, wherein the antibody or antibody
`
`fragment has an EC50 less than about 25 nanomolar in a cAMP assay. Further provided herein
`
`are antibodies or antibody fragments, wherein the antibody or antibody fragment has an EC50
`
`less than about 20 nanomolar in a cAMPassay. Further provided herein are antibodies or
`
`antibody fragments, wherein the antibody or antibody fragment has an EC50 less than about 10
`
`nanomolar in a cAMP assay. Further provided herein are antibodies or antibody fragments,
`
`wherein the antibody or antibody fragment binds to a chemokine receptor with a Kp of less than
`
`100 nM. Further provided herein are antibodies or antibody fragments, wherein the antibody or
`
`antibody fragment binds to a chemokine receptor with a Kp of less than 75 nM. Further
`
`provided herein are antibodies or antibody fragments, wherein the antibody or antibody
`
`fragment binds to a chemokine receptor with a Kp of less than 50 nM. Further provided herein
`
`are antibodies or antibody fragments, wherein the antibody or antibody fragment bindsto a
`
`chemokine receptor with a Kp of less than 10 nM. Further provided herein are antibodies or
`
`antibody fragments, wherein the antibody or antibody fragmentis an agonist of a chemokine
`
`receptor. Further provided herein are antibodies or antibody fragments, wherein the antibody or
`
`antibody fragmentis an antagonist of a chemokine receptor. Further provided herein are
`
`antibodies or antibody fragments, wherein the antibody or antibody fragmentis an allosteric
`
`modulator of a chemokine receptor. Further provided herein are antibodies or antibody
`
`fragments, wherein the allosteric modulator of a chemokine receptoris a negative allosteric
`
`modulator. Further provided herein are antibodies or antibody fragments, wherein the
`
`chemokine receptor is CXCR4. Further provided herein are antibodies or antibody fragments,
`
`wherein the chemokinereceptor is CXCRS.,
`
`[0005]
`
`Provided herein are antibodies or antibody fragments comprising a variable domain,
`
`heavy chain region (VH) and a variable domain, light chain region (VL), wherein the VH
`
`comprises an amino acid sequenceat least about 90% identical to a sequenceas set forth in any
`
`one of SEQ ID NOs: 24-28 or 34-356, and wherein the VL comprises an aminoacid sequenceat
`
`least about 90% identical to a sequence as set forth in any one of SEQ ID NOs: 29-33 or 357-
`
`525. Further provided herein are antibodies or antibody fragments, wherein the VH comprises
`
`an amino acid sequenceasset forth in any one of SEQ ID NOs: 24-28 or 34-356. Further
`
`provided herein are antibodies or antibody fragments, wherein the VL comprises an aminoacid
`
`sequenceas set forth in any one of SEQ ID NOs: 29-33 or 357-525. Further provided herein are
`
`antibodies or antibody fragments, wherein the VH comprises an aminoacid sequenceat least
`2
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`

`WO 2022/098662
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`PCT/US2021/057739
`
`about 90% identical to a sequence as set forth in any one of SEQ ID NOs: 24-28 and the VL
`
`comprises an amino acid sequenceat least about 90% identical to a sequenceas set forth in any
`
`one of SEQ ID NOs: 29-33. Further provided herein are antibodies or antibody fragments,
`
`wherein the VH comprises an amino acid sequenceasset forth in any one of SEQ ID NOs: 24-
`
`28 and the VL comprises an amino acid sequenceasset forth in any one of SEQ ID NOs: 29-33.
`
`Further provided herein are antibodies or antibody fragments, wherein the VH comprises an
`
`amino acid sequenceat least about 90% identical to a sequenceas set forth in any one of SEQ
`
`ID NOs: 34-356 and the VL comprises an amino acid sequenceat least about 90% identical to a
`
`sequence as set forth in any one of SEQ ID NOs: 357-525. Further provided herein are
`
`antibodies or antibody fragments, wherein the VH comprises an amino acid sequenceas set forth
`
`in any one of SEQ ID NOs: 34-356 and the VL comprises an amino acid sequenceas set forth in
`
`any one of SEQ ID NOs: 357-525. Further provided herein are antibodies or antibody
`
`fragments, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a bi-specific
`
`antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanizedantibody,
`
`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 determining 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.
`
`Further provided herein are antibodies or antibody fragments, wherein the antibody or antibody
`
`fragment thereof 1s chimeric or humanized. Further provided herein are antibodies or antibody
`
`fragments, wherein the antibody or antibody fragment has an ECS0 less than about 25
`
`nanomolar in a cAMP assay. Further provided herein are antibodies or antibody fragments,
`
`wherein the antibody or antibody fragment has an ECSO less than about 20 nanomolarin a
`
`cAMP assay. Further provided herein are antibodies or antibody fragments, wherein the
`
`antibody or antibody fragment has an EC50 less than about 10 nanomolar in a cAMP assay.
`
`Further provided herein are antibodies or antibody fragments, wherein the antibody or antibody
`
`fragment binds to a chemokine receptor with a Kp of less than 100 nM. Further provided herein
`
`are antibodies or antibody fragments, wherein the antibody or antibody fragment binds to a
`
`chemokine receptor with a Kp of less than 75 nM. Further provided herein are antibodies or
`
`antibody fragments, wherein the antibody or antibody fragment binds to a chemokine receptor
`
`with a Kp of less than 50 nM. Further provided herein are antibodies or antibody fragments,
`
`wherein the antibody or antibody fragment binds to a chemokine receptor with a Kp of less than
`
`10nM. Further provided herein are antibodies or antibody fragments, wherein the antibody or
`
`antibody fragment is an agonist of a chemokine receptor. Further provided herein are antibodies
`3
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`

`

`WO 2022/098662
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`PCT/US2021/057739
`
`or antibody fragments, wherein the antibody or antibody fragmentis an antagonist of a
`
`chemokine receptor. Further provided herein are antibodies or antibody fragments, wherein the
`
`antibody or antibody fragmentis an allosteric modulator of a chemokine receptor. Further
`
`provided herein are antibodies or antibody fragments, wherein the allosteric modulator of a
`
`chemokinereceptoris a negative allosteric modulator. Further provided herein are antibodies or
`
`antibody fragments, wherein the chemokine receptor is CXCR4. Further provided herein are
`
`antibodies or antibody fragments, wherein the chemokine receptor is CXCRS.
`
`[9006]
`
`Provided herein are antibodies or antibody fragments comprising a variable domain,
`
`heavy chain region (VH) comprising an amino acid sequenceat least about 90% identical to a
`
`sequence as set forth in any one of SEQ ID NOs: 24-28 or 34-356. Further provided herein are
`
`antibodies or antibody fragments, wherein the VH comprises an aminoacid sequenceasset forth
`
`in any one of SEQ ID NOs: 24-28 or 34-356. Further provided herein are antibodies or antibody
`
`fragments, wherein the VH comprises an amino acid sequenceat least about 90% identical toa
`
`sequenceas set forth in any one of SEQ ID NOs: 24-28. Further provided herein are antibodies
`
`or antibody fragments, wherein the VH comprises an amino acid sequenceasset forth in any
`
`one of SEQ ID NOs: 34-356. Further provided herein are antibodies or antibody fragments,
`
`wherein the VH comprises an aminoacid sequenceat least about 90% identical to a sequence as
`
`set forth in any one of SEQ ID NOs: 24-28. Further provided herein are antibodies or antibody
`
`fragments, wherein the VH comprises an amino acid sequenceas set forth in any one of SEQ ID
`
`NOs: 34-356. Further provided herein are antibodies or antibody fragments, wherein the
`
`antibody is 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 determining region (CDR), a diabody, a fragment comprised of only a
`
`single monomeric variable domain, disulfide-linked Fvs (sdF'v), an intrabody, an anti-idiotypic
`
`(anti-Id) antibody, or ab antigen-binding fragments thereof. Further provided herein are
`
`antibodies or antibody fragments, wherein the antibody is a single-domain antibody. Further
`
`provided herein are antibodies or antibody fragments, wherein the antibody or antibody
`
`fragmentthereof is chimeric or humanized. Further provided herein are antibodies or antibody
`
`fragments, wherein the antibody or antibody fragment has an EC50 less than about 25
`
`nanomolar in a cAMP assay. Further provided herein are antibodies or antibody fragments,
`
`wherein the antibody or antibody fragment has an ECSO less than about 20 nanomolarin a
`
`cAMP assay. Further provided herein are antibodies or antibody fragments, wherein the
`
`antibody or antibody fragment has an ECSO less than about 10 nanomolar in a cAMP assay.
`4
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`

`

`WO 2022/098662
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`PCT/US2021/057739
`
`Further provided herein are antibodies or antibody fragments, wherein the antibody or antibody
`
`fragment binds to a chemokine receptor with a Kp of less than 100 nM. Further provided herein
`
`are antibodies or antibody fragments, wherein the antibody or antibody fragment binds to a
`
`chemokine receptor with a Kp of less than 75 nM. Further provided herein are antibodies or
`
`antibody fragments, wherein the antibody or antibody fragment binds to a chemokine receptor
`
`with a Kp of less than 50 nM. Further provided herein are antibodies or antibody fragments,
`
`wherein the antibody or antibody fragment binds to a chemokine receptor with a Kp of less than
`
`10 nM. Further provided herein are antibodies or antibody fragments, wherein the antibody or
`
`antibody fragment is an agonist of a chemokine receptor. Further provided herein are antibodies
`
`or antibody fragments, wherein the antibody or antibody fragment is an antagonist of a
`
`chemokine receptor. Further provided herein are antibodies or antibody fragments, wherein the
`
`antibody or antibody fragment is an allosteric modulator of a chemokine receptor. Further
`
`provided herein are antibodies or antibody fragments, wherein the allosteric modulator of a
`
`chemokinereceptoris a negative allosteric modulator. Further provided herein are antibodies or
`
`antibody fragments, wherein the chemokine receptor is CXCR4. Further provided herein are
`
`antibodies or antibody fragments, wherein the chemokine receptor is CXCRS.
`
`[0007]
`
`Provided herein are methodsof treating a disease or disorder comprising
`
`administering the antibody or antibody fragment described herein. Further provided herein are
`
`methodsoftreating a disease or disorder, wherein the disease or disorder affects homeostasis.
`
`Further provided herein are methodsoftreating a disease or disorder, wherein the disease or
`
`disorder characterized by hematopoietic stem cell migration. Further provided herein are
`
`methodsoftreating a disease or disorder, wherein the disease or disorder is a solid cancer or a
`
`hematologic cancer. Further provided herein are methodsoftreating a disease or disorder,
`
`wherein the disease or disorder is gastric cancer, breast cancer, colorectal cancer, lung cancer,
`
`prostate cancer, hepatocellular carcinoma, leukemia, or lymphoma. Further provided herein are
`
`methodsoftreating a disease or disorder, wherein the disease or disorder is B-cell non-Hodgkin
`
`lymphoma. Further provided herein are methodsoftreating a disease or disorder, wherein the
`
`disease or disorder is caused by a virus. Further provided herein are methodsoftreating a
`
`disease or disorder, wherein the disease or disorder is caused by human immunodeficiency virus
`
`(HIV).
`
`[0008]
`
`Provided herein are nucleic acid compositions comprising: a) a first nucleic acid
`
`encoding a variable domain, heavy chain region (VH) comprising complementarity determining
`
`regions CDRH1, CDRH2, and CDRH3, and wherein (i) an amino acid sequence of CDRH1 is as
`
`set forth in any one of SEQ ID NOs: 526-662; (ii) an amino acid sequence of CDRH2is as set
`
`forth in any one of SEQ ID NOs: 663-977, (iii) an amino acid sequence of CDRH3is asset forth
`>
`
`

`

`WO 2022/098662
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`PCT/US2021/057759
`
`in any one of SEQ ID NOs: 978-1102; b) a second nucleic acid encoding a variable domain,
`
`light chain region (VL) comprising complementarity determining regions CDRL1, CDRL2, and
`
`CDRL3, and wherein (i) an amino acid sequence of CDRLI is as set forth in any one of SEQ ID
`
`NOs: 1103-1267; (11) an amino acid sequence of CDRL2is as set forth in any one of SEQ ID
`
`NOs: 1268-1328; and (iii) an amino acid sequence of CDRL3is as set forth in any one of SEQ
`
`ID NOs: 1329-1493.
`
`[0009]
`
`Provided herein are nucleic acid compositions comprising: a) a first nucleic acid
`
`encoding a variable domain, heavy chain region (VH) comprising an amino acid sequenceat
`
`least about 90% identical to a sequenceas set forth in any one of SEQ ID NOs: 24-28 or 34-356;
`
`b) a second nucleic acid encoding a variable domain, light chain region (VL) comprising at least
`
`about 90% identical to a sequenceas set forth in any one of SEQ ID NOs: 29-33 or 357-525; and
`
`an excipient. Further provided herein are nucleic acid compositions, wherein the VH comprises
`
`an amino acid sequenceasset forth in any one of SEQ ID NOs: 24-28 or 34-356. Further
`
`provided herein are nucleic acid compositions, wherein the VL comprises an aminoacid
`
`sequenceas set forth in any one of SEQ ID NOs: 29-33 or 357-525. Further provided herein are
`
`nucleic acid compositions, wherein the VH comprises an amino acid sequenceat least about
`
`90% identical to a sequence as set forth in any one of SEQ ID NOs: 24-28 and the VL comprises
`
`an amino acid sequenceat least about 90% identical to a sequence as set forth in any one of SEQ
`
`ID NOs: 29-33. Further provided herein are nucleic acid compositions, wherein the VH
`
`comprises an amino acid sequenceasset forth in any one of SEQ ID NOs: 24-28 and the VL
`
`comprises an amino acid sequenceasset forth in any one of SEQ ID NOs: 29-33. Further
`
`provided herein are nucleic acid compositions, wherein the VH comprises an aminoacid
`
`sequence at least about 90% identical to a sequenceasset forth in any one of SEQ ID NOs: 34-
`
`356 and the VL comprises an amino acid sequenceat least about 90% identical to a sequence as
`
`set forth in any one of SEQ ID NOs: 357-525. Further provided herein are nucleic acid
`
`compositions, wherein the VH comprises an amino acid sequenceasset forth in any one of SEQ
`
`ID NOs: 34-356 and the VL comprises an amino acid sequenceasset forth in any one of SEQ
`
`ID NOs: 357-525.
`
`[0010]
`
`Provided herein are nucleic acid compositions comprising: a nucleic acid encoding a
`
`variable domain, heavy chain region (VH) comprising an amino acid sequence at least about
`
`90% identical to a sequenceasset forth in any one of SEQ ID NOs: 24-28 or 34-356, and an
`
`excipient. Further provided herein are nucleic acid compositions, wherein the VH comprises an
`
`amino acid sequenceas set forth in any one of SEQ ID NOs: 24-28 or 34-356. Further provided
`
`herein are nucleic acid compositions, wherein the VH comprises an amino acid sequenceat least
`
`about 90% identical to a sequenceas set forth in any one of SEQ ID NOs: 24-28. Further
`6
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`

`WO 2022/098662
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`PCT/US2021/057759
`
`provided herein are nucleic acid compositions, wherein the YVH comprises an amino acid
`
`sequence as set forth in any one of SEQ ID NOs: 34-356. Further provided herein are nucleic
`
`acid compositions, wherein the VH comprises an amino acid sequenceat least about 90%
`
`identical to a sequenceas set forth in any one of SEQ ID NOs: 24-28. Further provided herein
`
`are nucleic acid compositions, wherein the VH comprises an amino acid sequenceas set forth in
`
`any one of SEQ ID NOs: 34-356.
`
`[0011]
`
`Provided herein are nucleic acid libraries, comprising: a plurality of nucleic acids,
`
`wherein each of the nucleic acids encodes for a sequence that when translated encodesfor a
`
`chemokine receptor binding immunoglobulin, wherein the chemokine receptor binding
`
`immunoglobulin comprises a variant of a chemokine receptor binding domain, wherein the
`
`chemokine receptor binding domain is a ligand for the chemokine receptor, and wherein the
`
`nucleic acid library comprisesat least 10,000 variant immunoglobulin heavy chains and at least
`
`10,000 variant immunoglobulin light chains. Further provided are nucleic acid libraries,
`
`wherein the nucleic acid library comprises at least 50,000 variant immunoglobulin heavy chains
`
`and at least 50,000 variant immunoglobulin light chains. Further provided are nucleic acid
`
`libraries, wherein the nucleic acid library comprises at least 100,000 variant immunoglobulin
`
`heavy chains and at least 100,000 variant immunoglobulin light chains. Further provided are
`nucleic acid libraries, wherein the nucleic acid library comprises at least 10° non-identical
`
`nucleic acids. Further provided are nucleic acid libraries, wherein a length of the
`
`immunoglobulin heavy chain whentranslated is about 90 to about 100 amino acids. Further
`
`provided are nucleic acid libraries, wherein a length of the immunoglobulin heavy chain when
`
`translated is about 100 to about 400 amino acids. Further provided are nucleic acid libraries,
`
`wherein the variant immunoglobulin heavy chain when translated comprises at least 80%
`
`sequenceidentity to any one of SEQ ID NOs: 24-28 or 34-356. Further provided are nucleic
`
`acid libraries, wherein the variant immunoglobulin light chain when translated comprises at least
`
`80% sequence identity to any one of SEQ ID NOs: 29-33 or 357-525.
`
`[0012]
`
`Provided herein are nucleic acid libraries comprising a plurality of nucleic acids,
`
`wherein each nucleic acid of the plurality of nucleic acids encodes for a sequence that when
`
`translated encodes for an antibody or antibody fragment thereof, wherein the antibody or
`
`antibody fragment thereof comprises a variable region of a heavy chain (WH) that comprises a
`
`chemokine receptor binding domain, wherein each nucleic acid ofthe plurality of nucleic acids
`
`comprises a sequence encoding for a sequence variant of the chemokinereceptor binding
`
`domain, and wherein the antibody or antibody fragment bindsto its antigen with a Kp ofless
`
`than 100 nM. Further provided are nucleic acid libraries, wherein a length of the VH is about 90
`
`to about 100 amino acids. Further provided are nucleic acid libraries, wherein a length of the
`7
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`VH is about 100 to about 400 amino acids. Further provided are nucleic acid libraries, wherein
`
`a length of the VH is about 270 to about 300 base pairs. Further provided are nucleic acid
`
`libraries, wherein a length of the VH is about 300 to about 1200 base pairs. Further provided are
`nucleic acid libraries, wherein the library comprises at least 10° non-identical nucleic acids.
`
`[0013]
`
`Provided herein are nucleic acid libraries comprising: a plurality of nucleic acids,
`
`wherein each of the nucleic acids encodes for a sequence that whentranslated encodes for a
`
`chemokine receptor single domain antibody, wherein each sequence of the plurality of
`
`sequences comprises a variant sequence encoding for a CDR1, CDR2, or CDR3 onavariable
`
`region of a heavy chain (VH); wherein the library comprises at least 30,000 variant sequences;
`
`and wherein the chemokine receptor single domain antibody bindsto its antigen with a Kp of
`
`less than 100 nM. Further provided are nucleic acid libraries, wherein a length of the VH when
`
`translated is about 90 to about 100 amino acids. Further provided are nucleic acid libraries,
`
`wherein a length of the VH whentranslated is about 100 to about 400 aminoacids. Further
`
`provided are nucleic acid libraries, wherein a length of the VH is about 270 to about 300 base
`
`pairs. Further provided are nucleic acid libraries, wherein a length of the VH 1s about 300 to
`
`about 1200 base pairs. Further provided are nucleic acid libraries, wherein the VH when
`
`translated comprises at least 80% sequence identity to any one of SEQ ID NO: 24-28 or 34-356.
`
`[0014]
`
`Provided herein are antibodies or antibody fragments that binds chemokine receptor,
`
`comprising an immunoglobulin heavy chain and an immunoglobulin light chain: (a) wherein the
`
`immunoglobulin heavy chain comprises an amino acid sequenceat least about 90% identical to
`
`that set forth in any one of SEQ ID NO: 24-28 or 34-356; and (b) wherein the immunoglobulin
`
`light chain comprises an amino acid sequenceat least about 90% identical to that set forth in any
`
`one of SEQ ID NO: 29-33 or 357-525. Further provided herein are antibodies or antibody
`
`fragments, wherein the antibody is 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 determining 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.
`
`Further provided herein are antibodies or antibody fragments, wherein the antibody or antibody
`
`fragment thereof is chimeric or humanized. Further provided herein are antibodies or antibody
`
`fragments, wherein the antibody has an EC50 less than about 25 nanomolar in a cAMP assay.
`
`Further provided herein are antibodies or antibody fragments, wherein the antibody has an EC50
`
`less than about 20 nanomolar in a cAMP assay. Further provided herein are antibodies or
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`antibody fragments, wherein the antibody has an EC50 less than about 10 nanomolar in a cAMP
`
`assay,
`
`[0015]
`
`Provided herein are antibedies or antibody fragments, wherein the antibody or
`
`antibody fragment comprises a complementarity determining region (CDR) comprising an
`
`aminoacid sequenceat least about 90% identical to that set forth in any one of SEQ ID NOs:
`
`526-1493.
`
`[0016]
`
`Provided herein are antibodies or antibody fragments, wherein the antibody or
`
`antibody fragment comprises a sequence of any one of SEQ ID NOs: 526-1493 and wherein the
`
`antibody is 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 determining 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 fragmentsthereof.
`
`[0017]
`
`Provided herein are methods for generating a nucleic acid library encoding for a
`
`chemokine receptor antibody or antibody fragment thereof comprising: (a) providing
`
`predetermined sequences encodingfor: i. a first plurality of polynucleotides, wherein each
`
`polynucleotide of the first plurality of polynucleotides encodes for at least 1000 variant sequence
`
`encoding for CDR1 on a heavy chain; 11. a second plurality of polynucleotides, wherein each
`
`polynucleotide of the second plurality of polynucleotides encodes for at least 1000 variant
`
`sequence encoding for CDR2 on a heavy chain;iii. a third plurality of polynucleotides, wherein
`
`each polynucleotide of the third plurality of polynucleotides encodes for at least 1000 variant
`
`sequence encoding for CDR3 on a heavy chain; and (b) mixing thefirst plurality of
`
`polynucleotides, the second plurality of polynucleotides, and the third plurality of
`
`polynucleotides to form the nucleic acid library of variant nucleic acids encodingfor the
`
`chemokine receptor antibody or antibody fragment thereof, and wherein at least about 70% of
`
`the variant nucleic acids encodefor an antibody or antibody fragment that binds to its antigen
`
`with a Kp of less than 100 nM. Further provided herein are methods, wherein the chemokine
`
`receptor antibody or antibody fragmentthereof is a single domain antibody. Further provided
`
`herein are methods, wherein the single domain antibody comprises one heavy chain variable
`
`domain. Further provided herein are methods, wherein the single domain antibody is a VHH
`
`antibody. Further provided herein are methods, wherein the nucleic acid library comprisesat
`
`least 50,000 variant sequences. Further provided herein are methods, wherein the nucleic acid
`
`library comprisesat least 100,000 variant sequences. Further provided herein are methods,
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`wherein the nucleic acid library comprises at least 10° non-identical nucleic acids. Further
`
`provided herein are methods, wherein the nucleic acid library comprises at least one sequence
`
`encoding for the chemokine receptor antibody or antibody fragment that binds to chemokine
`
`receptor with a Kp of less than 75 nM. Further provided herein are methods, wherein the
`
`nucleic acid library comprises at least one sequence encoding for the chemokine receptor
`
`antibody or antibody fragment that binds to chemokine receptor with a Kp of less than 50 nM.
`
`Further provided herein are methods, wherein the nucleic acid library comprises at least one
`
`sequence encoding for the chemokine receptor antibody or antibody fragmentthat binds to
`
`chemokine receptor with a Kp of less than 10 nM. Further provided herein are methods,
`
`wherein the nucleic acid library comprises at least 500 variant sequences. Further provided
`
`herein are methods, wherein the nucleic acid library comprises at least five sequences encoding
`
`for the chemokine receptor antibody or antibody fragment that binds to chemokine receptor with
`
`a Kp of less than 75 nM