(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(43) International Publication Date
`10 November 2016 (10.11.2016)
`
`WIPOI PCT
`
`\9
`
`(10) International Publication Number
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`WO 2016/179003 A1
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`(51)
`
`International Patent Classification:
`C07K 16/28 (2006.01)
`
`(21)
`
`International Application Number:
`
`PCT/US2016/030127
`
`(22)
`
`International Filing Date:
`
`(25)
`
`(26)
`
`(30)
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`(71)
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`(72)
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`(74)
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`(81)
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`Filing Language:
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`Publication Language:
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`29 April 2016 (29.04.2016)
`
`English
`
`English
`
`Priority Data:
`62/155,723
`
`1 May 2015 (01.05.2015)
`
`US
`
`Applicant: GENENTECH, INC. [US/US];
`South San Francisco, CA 94080-4990 (US).
`
`1 Dna Way,
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`Inventor: DENNIS, Mark, S.;
`Francisco, CA 94080-4990 (US).
`
`1 Dna Way, South San
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`Agent: ELBING, Karen, L.; Clark & Elbing LLP, 101
`Federal Street, 15th Floor, Boston, MA 021 10 (US).
`
`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, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,
`
`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, TII, TJ, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(84
`
`) 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).
`Declarations under Rule 4.17 :
`
`as to applicant’s entitlement to applyfor and be granted a
`patent (Rule 4.1 7(ii))
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`as to the applicant’s entitlement to claim the priority ofthe
`earlier application (Rule 4.1 7(iii))
`Published:
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`with international search report (Art. 21 (3))
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`with sequence listing part ofdescription (Rule 5.2(a))
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`(54) Title: MASKED ANTI-CD3 ANTIBODIES AND METHODS OF USE
`
`(57) Abstract: The invention provides masked anti-cluster of differentiation 3 (CDS) antibodies and methods of using the same.
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`MASKED ANTS—CD3 ANTIBQDIES AND METHOQS OF USE
`
`SEQUENCE LiSTtNG
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`5
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`1C!
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`15
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`20
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`30
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`35
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`40
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`The instant appiication contains a Sequence Listing which has been submitted eiectronicaiiy in
`
`ASCii format and is hereby incorncrated by reference in its entirety. Said ASCii copy, created on Aprii
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`25, 2016, is named 504-74—063WO2mSeouenceministingmatmzsm’i5_ST25 and is 39,917 bytes in size.
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`The present invention reiates to masked anti-ciuster of differentiation 3 (CD3) antibodies and
`
`methods of using the same.
`
`HELD 0? THE ENVENTEGN
`
`BACKGRGUND
`
`Cefi proiiferative disorders, such as cancer, are characterized by the uncontroifed growth of ceii
`
`subpopuiatione. They are the feeding cause of death in the deveioped worid and the second ieading
`
`cause of death in deveiopind countries, with over 12 miiiion new cancer cases diagnosed and 7 rniiiion
`
`cancer deaths occurring each year. The American Cancer Society estimates that greater than haif a
`
`miiiion Americans wiii die of cancer in 2815, accounting for neariy one out of every four deaths in the
`
`country. As the eideriy popuiation has grown, the incidence of cancer has concurrentiy risen, as the
`
`probahifity of deveioping cancer is more than two—foid higher after the age of seventy. Cancer care thus
`
`represents a significant and ever—increasing societal burden,
`
`Longstanding approaches to cancer treatment include chemotherapy, radiation therapy, and
`
`surgery to remove soiid tumors. Recentiy, T ceii—targeting therapeutic antibodies have been deveioped.
`
`These therapeutic antibodies inciude bispecific antibodies that are capabie of sirnuitaneousiy binding ceii
`
`surface antigens on T ceiis and tumor ceiis, thereby enahiing the bound T ceiis to contribute to the
`
`destruction of the tumor ceiis. However, the deyeioprnent of such a T ceii—denendent bispecific (TDB)
`
`antibody can carry an inherent risk for the devefopment of adverse immune—mediated effects. Afthough
`
`certain unwanted effects, such as Fey receptor—mediated depieticn of T ceiis, can be minimized by
`
`rendering the T33 antibodies effectcriess, there is an unmet need in the fieid forthe deveiopment of
`
`afternative TDB antibodies that aiso account for the kinetics otT ceii engagement and activation.
`
`SUMMARY
`
`The present invention reiates to masked anti—ciuster of differentiation 3 (£303) antibodies and
`
`methods of using the same.
`
`in one aspect, the invention features an anti—cluster of differentiation 3 (CD3) antibody, wherein
`
`the anti-CD3 antibody comprises (a) a binding domain and (b) a poiypeptide mash, wherein the
`
`poiypeptide mask comprises a masking moiety (MM) comprising the amino acid sequence of at feast
`
`amino acid residues 1—3 of SEQ if) NO: 1 (e.g., a poiypeptide rnasir comorising a Nth/i comprising amino
`
`acid residues 1—3, 1—4, 1-5, 1-6, 1—7, 1—8, 1—3, 1—10, 1-11, 1—12, 1—13, 1—14, 1—15, 1—16, 1—17, 1—18, 1—19, 1—
`
`26, 1—21, 1—22, 1—23, 1—24, 1—25, 1—26, or1—27 of SEQ ED NO: 1‘), or an N—terrninai cyciicized giutarnine
`
`derivative thereof (eg, a poiypeptide rnaek comprising a h/iivt comprising S—oxopyrroiidine—Z—carhoxyiic
`
`acid (PC/9a)). For exampie, in some embodiments, the MM comprises at ieast the first three amino acid
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`residues of SEQ ED NO: 1, except that the residue at position ’i
`
`is an N—terminai cyciicized gtutamine
`
`(PCA) residue instead of a giutamine residue {e.g., the MM comprises the amino acid sequence PEA-D—
`
`G).
`
`in some embodiments, the binding domain comprises a heavy chain variabte (Vi-t) domain and a tight
`
`chain variabte (VL) domain and the poiypeptide mask isjoined to the VH domain or the ‘v’t. domain.
`
`in
`
`some embodiments, the MM is extended at the C—terminus by ait or a portion of the remaining sequence
`
`of SEQ iii) NO: 1. For exampie, in some embodiments, the MM comprises the amino acid sequence of at
`
`teast amino acid residues 1—5 of SEQ ED NO: 1, or an N—terminai cycticized giutamine derivative thereof
`
`(eg, a. pciypeptide mask comprising a MM comprising at ieast the first five amino acid residues of SEQ
`
`iD NO: 1, except that the residue at position 1
`
`is PCA instead ofa giutamine).
`
`in other embodiments, the
`
`MM comprises the amino acid sequence of at ieast amino acid residues 1~5 of SEQ iD NO: 1, or an N—
`
`terminai cyciicized giutamine derivative thereof (e.g., a polypeptide mask comprising a MM comprising at
`
`least the 1first six amino acid residues of SEQ it) NO: 1, except that the residue at position 1
`
`is PCA
`
`instead of gtutamine).
`
`in some embodiments, the anti~CD3 antibody and MM are positioned retative to
`
`each other in an N—terminat to C-terminai direction as (MMHanti—CD3 antibody).
`
`in some embodiments, the poiypeptide mask further comprises a cieavabie moiety (CM).
`
`in
`
`some embodiments, the CM is capabte of being cteaved by an enzyme.
`
`in some embodiments, the
`
`enzyme is seiected from the group consisting of matrix metattoprotease (MMP)-2, MMP—Q, iegumain
`
`asparaginyi endopeptidase, thrombin, fibrobiast activation protease (PAP), MMPwt, MMP—S, MMP~7,
`
`MMP—S, MMP-tZ, MMPu’t3, MMP—M, membrane type 1 matrix metaiioprotease (MTt—MMP), ptasmin,
`
`transmembrane protease, serine (TMPRSS—Blct), cathepsin A, cathepsin B, cathepsin D, cathepsin E,
`
`cathepsin F, cathepsin i-i, cathepsin K, cathepsin L, cathepsin L2, cathepsin O, cathepsin S, caspase 1,
`
`caspase 2, caspase 3, caspase 4, caspase 5, caspase 5, caspase 7, caspase 8, caspase 9, caspase to,
`
`oaspase ti, caspase 12, caspase 13, caspase 14, human neutrophii eiastase, urokinaseiurokinase—type
`
`piasminogen activator (uPA), a disintegrin and metatioprotease (ADAMHG, ADAMtZ ADAM17, ADAM
`
`with thrombospondin motifs (ADAMTS), ADAMTSE), beta secretase (BASE), granzyme A, granzyme B,
`
`guanidinobenzoatase, hepsin, matriptase, matriptase 2, meprin, nepritysin, prostate‘specitic membrane
`
`antigen (PSMA), tumor necrosis factor—converting enzyme (FACE), kaiiikrein—retated peptidase (KLK)3,
`
`KLKS, KLKY, KLKt t, NS3/4 protease of hepatitis (3 virus (HCV—NSSI’4), tissue ptasminogen activator
`
`(tPA), caipain, caipain 2, glutamate carboxypeptidase it, piasrna kaiiikrein, AMSH—iike protease, AMSH, v—
`
`secretase component, antipiasmin cieaving enzyme (APCE), deoysin t, apoptosis—reiated cysteine
`
`peptidase, and N—acetytated atphaviinked acidic dipeptidase—iike t. For exampie, in some embodiments,
`
`the enzyme is MMP—Z, MMP—Q, iegumain asparaginyi endopeptidase, thrombin, or PAP.
`
`in some
`
`embodiments, the CM comprises an acid—iabiie tinker that is capabie of being cieaved in an acidic pH
`
`environment.
`
`in some embodiments, the acid—iabiie tinker comprises a hydrazone, an imino, an ester, or
`
`an amide group.
`
`in some embodiments, the acidic pH environment is found in the iysosome of a ceii.
`
`in
`
`another embodiment, the acidic pi-t environment is found in a tumor microenvironment.
`
`in some
`
`embodiments wherein the anti-CUE antibody, MM, and CM are positioned reiative to each other in an N~
`
`terminat to C-terminai direction as (MM)-{CM)—(anti-CD3 antibody).
`
`in some embodiments, the poiypeptide mask further comprises a tinker moiety (Ltd).
`
`in some
`
`embodiments, the Ltd is between 5 to 24 amino acids in iength (8.9., the LM is 5, 6, 7, 8, 9, it), t ‘i, 12,
`
`2
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`13, i4,15,1S,17,18,19,20,21,22,23,or 24 amino acids in Eength).
`
`in some embodiments, the LEVi is
`
`between 5 to if) amino acids in iength (eg, the LIVE is 5, t5, 7, 8, 9, 10, ii, t2, 13, 14, or 15 amino acids
`
`in iength).
`
`in some embodiments, the Livi comprises giycine (G) and serine (S) residues.
`
`in some
`
`embodiments, the LM comprises GS repeats.
`
`in some embodiments, the anti—C393 antibody, MM, and LM
`
`are positioned reiative to each other in an Nnterminai to C—terminai direction as (MM)—(LM)-(anti—CD3
`
`antibody).
`
`in some embodiments, the poiypeptide mask comprises a cieavabie moiety and a iinker
`
`moiety, and wherein the anti—CD3 antibody, Mid, CM, and LEVE are positioned reiative to each other in an
`
`N—terminaE to C—terrninai direction as (MM)—(LM)~(CE\I‘E)—(antimCD3 antibody)or (MM)—(CM)-(LM)—(anti~0£)3
`
`antibody).
`
`En some embodiments, the binding domain comprises one or more (eg, one, two, three, four,
`
`five, or six) of the foiiowing six hypervariabie regions (HVRs): (a) an HVR—Ht comprising the amino acid
`
`sequence of SEQ ii) NO: 2; (b) an E-EVR—HZ comprising the amino acid sequence of SEQ ED NO: 3; to) an
`
`HVR-HS comprising the amino acid sequence of SEQ ED NO: 4; (d) an HVR-Lt comprising the amino acid
`
`sequence oft-SEQ ED NO: 5; (e) an t-EVR—LZZ comprising the amino acid sequence of SEQ it.) NC): 6; and (i)
`
`an HVR—LS comprising the amino acid sequence of SEQ ED NO: 7.
`
`in some embodiments, the binding
`
`domain comprises (a) a heavy chain variabie (Vt-i) domain comprising an amino acid sequence having at
`
`ieast 95% sequence identity to the amino acid sequence of SEQ iD NO: 8; (b) a tight chain variabte (VL)
`
`domain comprising an amino acid sequence having at ieast 995% sequence identity to the amino acid
`
`sequence of SEQ it) NO: 9; or (c) a VE—i domain as in (a) and a VL domain as in (b).
`
`in some
`
`embodiments, the Vi—i domain comprises the amino acid sequence of SEQ iD N0: 8.
`
`in some
`
`embodiments, the VL domain comprises the amino acid sequence of SEQ iii) NO: 9.
`
`in some
`
`embodiments, the Vi—i domain comprises the amino acid sequence of SEQ it) NO: 8, and the Vi. domain
`
`comprises the amino acid sequence of SEQ ED NC): 9.
`
`En some embodiments, the binding domain comprises one or more (eg, one, two, three, four,
`
`five, or six) of the foiiowing six hypervariabie regions (Eh/Rs): (a) an HVR-Ht comprising the amino acid
`
`sequence of SEQ ED NC): 10; (b) an HVR—HZ comprising the amino acid sequence of SEQ ED NC): 11; (c)
`
`an HVR—E—E3 comprising the amino acid sequence of X1X2YSX3X4X5FDY, wherein X1 is seEected from the
`
`group consisting of D, T, and 8; X2 is seiected from the group consisting otG, A, and S; X3 is R or N; X4 is
`
`Y or A; and X5 is ‘i’ orA (SEQ iii) NO: t2); (d) an HVE-Lt comprising the amino acid sequence of SEQ ED
`
`NO: 13; (e) an HVR—L2 comprising the amino acid sequence of SEQ it} NO: 14; and (t) an HVRES
`
`comprising the amino acid sequence of X1X2SX3X4LRT, wherein X1 is K or T; X2 is Q or A; X3 is F or A;
`
`and X4 is i or A (SEQ it) NO: 15). For exampie, in some embodiments, the binding domain comprises the
`
`toEiowing six HVRs:
`
`(a) an HVE-Ht comprising the amino acid sequence of SEQ ED NO: 10; (h) an HVR-
`
`H2 comprising the amino acid sequence of SEQ iD NO: 11; (c) an HVR-HB comprising the amino acid
`
`sequence of SEQ iD NO: 16; (d) an HVR~LE comprising the amino acid sequence of SEQ ED NO: 13; (e)
`
`an HVE—LZ comprising the amino acid sequence of SEQ ED NO: E4; and (E) an HVE—LB comprising the
`
`amino acid sequence of SEQ ED NO: 17.
`
`En some embodiments, the binding domain comprises (a) a Vi-i
`
`domain comprising an amino acid sequence having at Eeast 95% sequence identity to the amino acid
`
`sequence of SEQ iii) NO: 18; (b) a VL domain comprising an amino acid sequence having at ieast 95%
`
`sequence identity to the amino acid sequence at SEQ ED NO: 19; or (c) a Vi—i domain as in (a) and a VE.
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`domain as in (o).
`
`in some embodiments, the VH domain comprises the amino acid sequence of SEQ ED
`
`NO: 18.
`
`in some embodiments, the Vi. domain comprises the amino acid sequence of SEQ ED NC): 19.
`
`in some embodiments, the VH domain comprises the amino acid sequence of SEQ 1D NC): 18, and the
`
`VL domain comprises the amino acid sequence of SEQ ED NS: 19.
`
`in other embodiments, the binding domain comprises one or more (eg, one, two, three, four,
`
`five, or six) of the fotiowing six HVRs:
`
`(a) an HVR—Ht comprising the amino acid sequence of SEQ ED
`
`NO: to; (b) an EiVR—HZ comprising the amino acid sequence of SEQ ED NO: 11; (c) an HVR-HS
`
`comprising the amino acid sequence of SEQ ED NO: 16; (d) an HVRE‘E comprising the amino acid
`
`sequence of SEQ ED NO: 13; (e) an HVR~L2 comprising the amino acid sequence of SEQ it) NO: 14; and
`
`it“) an HVR—LS comprising the amino acid sequence of SEQ it) NO: 194.
`
`in some embodiments, the
`
`binding domain comprises (a) a VE-i domain comprising an amino acid sequence having at ieast 95%
`
`sequence identity to the amino acid sequence oE'SEQ it) NO: 107; (b) a VL domain comprising an amino
`
`acid sequence having at ieast 95% sequence identity to the amino acid sequence of SEQ ED NO: too; or
`
`(c) a Vii domain as in (a) and a Vt. domain as in (b).
`
`in some embodiments, the Vii domain comprises
`
`the amino acid sequence of SEQ it) NC): 10?.
`
`in some embodiments, the Vi. domain comprises the
`
`amino acid sequence of SEQ ED NO; 198.
`
`in some embodiments, the Vi-i domain comprises the amino
`
`acid sequence of SEQ ED N0: 153?, and the Vi. domain comprises the amino acid sequence of SEQ ED
`NO: 108.
`
`in some embodiments, the binding domain comprises one or more (eg, one, two, three, tour.
`
`five, or six) otthe foiiowing six i-iVRs:
`
`(a) an i-iVFt—i-M comprising the amino acid sequence of SEQ it)
`
`NO: 10: (b) an i-iVRui-iE comprising the amino acid sequence of SEQ it) MG: 11; (c) an HVR—HE
`
`comprising the amino acid sequence of SEQ it) NC): 18; (d) an HVR—LE comprising the amino acid
`
`sequence of SEQ it) NO: 13: (e) an HVR-L2 comprising the amino acid sequence of SEQ ii) NC): 14; and
`
`(i) an HVR—LS comprising the amino acid sequence of SEQ ED N0: 105.
`
`in some embodiments, the
`
`binding domain comprises (a) a Vii domain comprising an amino acid sequence having at ieast 95%
`
`sequence identity to the amino acid sequence of SEQ ED NC): tutti; (b) a Vt. domain comprising an amino
`
`acid sequence having at ieast 95% sequence identity to the amino acid sequence of SEQ it) NC): 119; or
`
`(c) a Vi-i domain as in (a) and a Vi. domain as in (b).
`
`in some embodiments, the Vi-t domain comprises
`
`the amino acid sequence of SEQ ED NO: 109.
`
`in some embodiments, the VL domain comprises the
`
`amino acid sequence of SEQ ED NO: 110.
`
`in some embodiments, the Vii domain comprises the amino
`
`acid sequence of SEQ ED NO: 109, and the Vi. domain comprises the amino acid sequence of SEQ ED
`NO: 110.
`
`in other embodiments, the binding domain comprises one or more (eg, one, two, three, four,
`
`five, or six) otthe toiiowing six HVRs:
`
`(a) an HVR~H1 comprising the amino acid sequence of SEQ ED
`
`NO: 10; (b) an HVR-Hz comprising the amino acid sequence ct SEQ ED NO: 11; (c) an HVR—HB
`
`comprising the amino acid sequence of SEQ it) NO: 16; (d) an HVE-Lt comprising the amino acid
`
`sequence ct SEQ ED NO: 13; (e) an E-iVFt-Lz comprising the amino acid sequence of SEQ ED NO: 14; and
`
`(i) an HVR-LB comprising the amino acid sequence of SEQ it) NO: 1%.
`
`in some embodiments, the
`
`binding domain comprises (a) a VE—t domain comprising an amino acid sequence having at ieast 95%
`
`sequence identity to the amino acid sequence of SEQ ED NO: 111; (b) a Vi. domain comprising an amino
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`acid sequence having at ieast 95% sequence identity to the amino acid sequence of SEQ it) NO: 112; or
`
`(c) a VH domain as in (a) and a V1. domain as in (b).
`
`in some embodiments, the VH domain comprises
`
`the amino acid sequence of SEQ it) NC): 111.
`
`in some embodiments, the Vt. domain comprises the
`
`amino acid sequenoe of SEQ it) NO: 112.
`
`in some embodiments, the VH domain comprises the amino
`
`acid sequence of SEQ ED NO: 111, and the Vi. domain comprises the amino acid sequenee of SEQ ED
`NC): 112.
`
`Additionai anti—CD3 antibodies inciude anti—CD3 binding domains disciosed in UHSSN.
`
`14/574,132 (1.5.55. Pub. No. 2915—0188881), which is incorporated herein by reference in its entirety, and a
`
`poiypeptide mask having any of the aforementioned additionai features (ed, Nth/i, GM, Ltd). For
`
`exambie, a masked anti-CD3 antibody may inciude HVRs as in any of the referenced antimCD‘S
`
`antibodies, and further inotude any of the referenced aooebtor framework regions (FRs) in addition to a
`
`poiypeptide mask ineiuding, for exambie, a Mi‘v’i, Chit, and/or Livi.
`
`in some embodiments, a masked anti—
`
`CD3 antibody may inoiude a VH domain and/or a V1. domain as in any of the referenced anti-CD3
`
`antibodies, and further inoiude a polypeptide mask inoiuding, for exampie, a Midi, CM, and/or Ltd. For
`
`instance, a masked anti—CD3 antibody may inciude anti-CD3 antibody 81334 (Pessano et at. The E4450
`
`Journal. 4: 337—344, 1985) and a poiypeptide mask having, for exampie, a Nth/i, Cit/i, and/or Liiii.
`
`in some embodiments, the poiybebtide mask inhibits the abiiity of the anti—CD3 antibody to bind to
`
`a human CD3 poiyoebtido by at ieast 18% (ed, 18%, 11%, t2%, 13%, 14%, 15%, 18%, 17%, 18%,
`
`19%, 20%, 21%, 22%, 23%, 24%, 25%, 28%, 27%, 28%, or 29%), at ieast 30% (ed, 38%, 31%, 32%,
`
`33%, 34%, 35%, 38%, 37%, 38%, 39%, 49%, 41%, 42%, 43%, 44%, 45%, 48%, 47%, 48%, 49%, 58%,
`
`51%, 52%, 53%, 54%, 55%, 58%, 57%, 58%, or 59%), at ieast 88% (e.g., 88%, 81%, 82%, 83%, 84%,
`
`85%, 88%, 87%, 88%, 89%, 79%, 71%, 72%, 73%, 74%, 75%, 78%, 77%, 78%, or 79%), at toast 80%
`
`(e.g., 80%, 81%, 82%, 83%, 84%, 85%, 88%, 87%, 88%, or 89%), or at least 90% (eg, 90%, 91%, 92%,
`
`93%, 94%, 95%, 98%, 97%, 98%, or 99%).
`
`in some embodiments, the ooh/peptide mask oompieteiy
`
`inhibits the abiiity ofthe anti—CD3 antibodyto bind to a human CD3 poiypeptide (i.e., 100% inhibition).
`
`in
`
`some embodiments, the human CD3 ooiypeptide is a human ($1335. ooh/peptide.
`
`in some embodiments, the antirCD3 antibody comprises an agiycosyiation site mutation.
`
`in some
`
`embodiments, the agiycosyiation site mutation is a substitution mutation.
`
`in some embodiments, the
`
`substitution mutation is at amino aoid residue N297, L234, L235, and/or E3285 (EU numbering).
`
`in some
`
`embodiments, the substitution mutation is seieoted from the group oonsisting of N297G, N297A, L234A,
`
`L235A, and 9285A.
`
`in some embodiments, the substitution mutation is an N29176: mutation.
`
`in some
`
`embodiments, the agiyoosyiation site mutation reduoes effector function of the anti—CD3 antibody.
`
`in some embodiments, the anti~CDS antibody is monooionai, human, humanized, or chirnerio.
`
`in
`
`some embodiments, the anti—CD3 antibody is an antibody fragment that binds CD3.
`
`in some
`
`embodiments, the antibody fragment is selected from the group consisting of Fab, Fab’, Fab-SH, Fv,
`
`son, TaFv, (Fab); diabody, bsDb, son, DART, Bi'i‘E, and VHH fragments.
`
`in some embodiments, the
`
`anti-C3133 antibody is a fuii-iength antibody.
`
`in some embodiments, the anti—CD3 antibody is an igG
`
`antibody.
`
`in some embodiments, the anti-CD3 antibody is a monospeoit’ic antibody.
`
`in other embodiments, the anti—CD3 antibody is a muitispeoific antibody.
`
`in some embodiments,
`
`the muitispecitio antibody is a bispeoifio antibody.
`
`in some embodiments, the bispecifio antibody
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`comprises a second binding domain that binds to a second hioiogicai moiecuie, wherein the second
`
`hioiogicai moiecuie is a cell surface antigen.
`
`in some embodiments, the cell surface antigen is a tumor
`
`antigen.
`
`in some embodiments, the tumor antigen is seiected from the group consisting of CD20; FcRH5
`
`(Fc Receptor—iike 5); HERE; LYPDt; LyfiGESD (iymphocyte antigen 6 compiex, iocus C561); Lye-D,
`
`MEGT’i); PMEL17(siiver homoiog; SiLV; 012353E; PMEL17; (Si); (SEL); ME29;gp100); LySE
`
`(iymphocyte antigen 6 compiex, iocus E; Ly67, Rte—E, SEA—2, TSA—‘i); CD'iE—B; CD33; CD22 (B—ceii
`
`receptor CDEZ—B isoform); CD79a (EDNA, CD79a, immunogionutin—associated aipha; BMPRt 8 {bone
`
`morphogenetic protein receptor~type EB); ODYSh (CD798, CD793, 1 Gt) (irnrnunogiohuiin-associated
`
`beta), E329); EDAR (Ectooyspiasin A Receptor); GFRA‘E (GDNF—Ra’i); MRPz‘i (Muitidrug Resistance
`
`Protein 4); RET; STEAM {six transntemhrane epitheiiai antigen of prostate); "renew {putative
`
`transmemorane proteogiycan): E16 (LAT’E, SLC7A5); 0772:” (CAtzfi, MUCte); MPF (MPF. MSLN, SMR,
`
`megakaryocyte potentiating factor, mesotheiin); Napisp (NAPi—SB, NPTiih, SLC34A2, solute carrier famiiy
`
`34 (sodium phosphate), member 2, type it sodium-dependent phosphate transporter 3b); Sema 5b; PSCA
`
`hig (2706050012Rik, CSSGGOBOioRik, RiKEN cDNA 2700050012, RiKEN cDNA 2796050012 gene);
`
`ETBR (Endotneiin type B receptor); M38783 {RNF124, hypotheticai protein FLJ20315); STEAPZ; Trini4
`
`(BR22450, FLJZZGGM, TRPit/M, TRPM4B, transient receptor potentiai cation channei, subfamiiy M,
`
`member 4); CRiPTO (CR, CR1, CRGF, CREPTO, TDGF‘E , teratecarcinorna—deriveo growth factor); CDZ'E
`
`(CR2 (Compiement receptor 2) or C3DR (CBd/Epstein Barr virus receptor) or t-ts.73792); FcRHZ (iFGPtt-,
`
`iRTAIi, SPAP1A(SH2 domain containing phosphatase anchor protein 1a), SPAPtB, SPAPiC); NCA;
`
`MDP; ELEGRo; Brevioan; Eight-32R; ASLGSSQ; PSCA; GEDA; BAFF—R (t3 ceii—activating factor receptor,
`
`BLyS receptor 3, 8R3); CXCRfi (Burkitt‘s iyrnphorna receptor 1; HLAuDOB (Beta subunit of Nit-ii) oiass ii
`
`moiecuie); PEXS (Purinergie receptor P2X iigand-gated ion channei 5; CD72 (B-ceii differentiation antigen
`
`CD72, Lyn—2); LY54 (Lymphocyte antigen 54 (RPtOS), type i membrane protein of the ieucine rich repeat
`
`(LRR) farniiy); Fth-i’i (Fc receptorniike protein 1); iRTAZ (immunogiobuiin supertamiiy receptor
`
`transiocation associated 2); TMEFF1; TMEM46 (snisa homoiog 2 (Xenopus iaevis); SHESAB); LGRfi
`
`(ieucine—rich repeat—containing G protein-coupied receptor 5; GPR49, GPR67); LYGK (iymphocyte antigen
`
`6 compiex, iocus K; LYtSK; HSJGO'i348; FLJ35226); fiPRtQ (G protein—coupied receptor 19; Mm 4787);
`
`GPR54 (KiSSt receptor; KESS1R; GPRSA; HQT7T175; AXQRtZ); ASPHDt (aspartate betarhydroxyiase
`
`domain containing 1; LOC253982); Tyrosinase (TYR; OCAEA; OCA’iA; tyrosinase; SHEP‘E); TMEM118
`
`(ring finger protein, transnternbrane 2; RNFTZ; FLJts’i-627); GPR172A (G proteinmooupied receptor 172A;
`
`GPCRM; FLJ11856; D15Ertd747e); GPCE (Giypican 3); CLLt (Cn’i‘ype Lectin—iike ntoiecuie t); B7~H4
`
`(87x; B731); RNF43 (Ring finger protein 43); CD70; CXQRFfit (Chromosome X open reading frame 61);
`
`and SL653D3.
`
`in some embodiments, the tumor antigen is seiected from the group consisting otCD20,
`
`FcRHt-E, HER2, LYPD1;LY585D,PMEL17, LYSE, CD18. CD33, CD22, CDTQA, CD798, EDAR, GFRA1,
`
`MRP4, RET,Steap1, and TenB2.
`
`in some embodiments wherein the tumor antigen is CD29, the second binding domain comprises
`
`the foiiowing six i-iVFts: (a) an HVR—H1 comprising the amino acid sequence of SEQ it) NO: 20; (h) an
`
`HVR—t-i2 comprising the amino acid sequence of SEQ iD NC): 21; (c) an i-iVR—i-i3 comprising the amino
`
`acid sequence of SEQ ED NO: 22; (oi) an HVR-Lt comprising the amino acid sequence of SEQ ED NO: 23;
`
`(e) an HVR—LZ comprising the amino acid sequence of SEQ iD NO: 24; and (1‘) an HVR—L3 comprising the
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`amino acid sequence of SEQ it) NO: 25.
`
`in some embodiments, the binding domain comprises (a) a
`
`heavy chain variabEe (Vii) domain comprising an amino acid sequence having at least 95% sequence
`
`identity to the amino acid sequence of SEQ ED NO: 26; (b) a tight chain variabie (VL) domain comprising
`
`an amino acid sequence having at ieast 95% sequence identity to the amino acid sequence of SEQ ED
`
`NO: 27; or (c) a VH domain as in (a) and a Vi. domain as in (to).
`
`in some embodiments, the VH domain
`
`comprises the amino acid sequence of SEQ it) NC): 26. En some embodiments, the Vi. domain comprises
`
`the amino acid sequence of SEQ it) NO: 27.
`
`in some embodiments, the Vii domain comprises the amino
`
`acid sequence of SEQ ED NO: 26 and the Vt. domain comprises the amino acid sequence of SEQ it)
`NC): 27.
`
`in some embodiments, the anti—(303 antibody comprises one or more heavy chain constant
`
`domains, wherein the one or more heavy chain constant domains are seiected from a first CH1 (Ci-it 1)
`
`domain, a first CH2 (CH21) domain, a first CH3 (CH31) domain, a second {Si-it (can) domain, second
`
`CH2 (Ci-i229 domain, and a second CH3 (Ci-i32) domain.
`
`in some embodiments, at ieast one of the one
`
`or more heavy chain constant domains is paired with another heavy chain constant domain.
`
`in some
`
`embodiments, the CH31 and CH32 domains each comprise a protuberance or cavity, and wherein the
`
`protuberance or cavity in the CH31 domain is positionabie in the cavity or protuberance, respectiveiy, in
`
`the CH3; domain.
`
`in some embodiments, the CH21 and CH22 domains each comprise a protuberance or
`
`cavity, and wherein the protuberance or cavity in the CH2», domain is positionahie in the cavity or
`
`protuberance, respectiveiy, in the CH22 domain.
`
`in some embodiments. the CH21 and CH22 domains
`
`meet at an interface between said protuberance and cavity.
`
`in another aspect, the invention features an isoiated nuoieic acid that encodes any of the anti"
`
`CUB antibodies disciosed herein. The nucieic acid may be comprised in a vector (e.g., an expression
`
`vector) for expressing the antibody.
`
`in another aspect, the invention features host ceiis comprising the preceding nucieic acids
`
`arid/or vectors.
`
`in some embodiments, the host ceii is a mammaiian ceii (e.g., a Chinese hamster
`
`ovary (CHO) ceii).
`
`in other embodiments, the host ceii is a prokaryotic ceEi (eg, an E. coii ceii). A
`
`method of producing any one of the preceding anti—CD3 antibodies is aiso provided, the method
`
`comprising outturing the host oeii that produces the anti—CD3 antibody.
`
`in some embodiments, the
`
`method further comprises recovering the anti—(303 antibody from the host ceii orthe cuiture medium.
`
`in some embodiments, the invention f>atures an immunoconiugate comprising any one otthe
`
`preceding anti—CD3: antibodies conjugated to a cytotoxic agent.
`
`Aiso provided is a composition comprising any one ofthe preceding anti—ODE antibodies or
`
`immunoconiugates.
`
`in some embodiments, the composition further comprises a pharmaceuticaiiy
`
`acceptacie carrier, excipient, or diiuent.
`
`in some embodiments, the composition is a pharmaceuticai
`
`composition.
`
`in some embodiments, the composition further comprises an additionai therapeutic agent.
`
`A further aspect of the invention is a method of treating or deiaying the progression of a ceii
`
`proiiferative disorder or an autoimmune disorder in a subject in need thereof, the method comprising
`
`administering to the subject an effective amount any one of the preceding anti—CUB antibodies.
`
`in
`
`another aspect, the invention features a method of enhancing immune function in a subject having a ceii
`
`proiiferative disorder or an autoimmune disorder, the method comprising administering to the subject any
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`one of the preceding anti—CD3 antibodies.
`
`in some embodiments, the anti—CD3 antibody binds to (a) a
`
`CD3 moiecuie iocated on an immune effector ceii and (b) a second bioiogicai moiecuie iocated on a
`
`target ceit otherthan the immune effector ceii.
`
`in some embodiments, the anti—CD3 antibody binds to the
`
`second bioiogicai moiecuie prior to the CD3 moiecuie.
`
`in some embodiments, the anti—CD3 antibody
`
`accumuiates at the surface of the target ceii, for exampie, because of a decreased association constant
`
`(Kg) ofthe anti—CD3 antibody towards (393 moiecuies iocated on the immune effector ceii.
`
`in some
`
`embodiments, the anti-CD3 antibody is capabie of providing a cytotoxic effect on the target ceti i:e.g., via
`
`the activated imrnune effector ceii).
`
`in some embodiments, the anti—ODE} antibody is capabie of providing
`
`an apoptotic effect on the target ceii (eg, via the activated immune effector ceii).
`
`in some embodiments,
`
`the anti—CD3 antibody is capabie of providing a cytotoxic effect and/or an apoptotic effect on the target
`
`ceii,
`
`in some embodiments. the cytotoxic effect and/or the apoptotic effect on the target ceii is
`
`independent of activation of the immune effector ceii.
`
`in other embodiments, the cytotoxic effect and/or
`
`the apoptotio effect on the target ceii is dependent of activation of the immune effector cell.
`
`in some
`
`embodiments, the anti—CUB antibody is administered to the subject in a dosage of about 0.61 mg/kg to
`
`about 30 mg/kg.
`
`in some embodiments, the anti—CBS antibody is administered to the subject in a dosage
`
`of about (it mgikg to about so mg/kg.
`
`in some embodiments, the anti—CBS antibody is administered to
`
`the subject in a dosage of about ’i rngiitg to about 30 trig/kg.
`
`in some embodiments, the anti—CD3
`
`antibody is administered subcutaneousiy, intravenousiy, intramuscuiariy, topicaiiy, oraiiy, transdermaiiy,
`
`intraperitoneaiiy, intraorbitaiiy, by impiantation, by inhaiation, intrathecaiiy, intraventricuiariy, or
`
`intranasaiiy in some embodiments, the anti-CD3 antibody is administered subcutaneousiy.
`
`in some
`
`embodiments, the antiuCDS antibody is administered intravenousiy.
`
`in some embodiments, the method further comprises administering to the subject a PD—i axis
`
`binding antagonist or an additional therapeutic agent.
`
`in some embodiments, the additionai therapeutic
`
`agent is administered prior to or subsequent to the administration of the anti—(303 antibody in some
`
`embodiments, the additionai therapeutic agent is administered concurrentiy with the anti—C393 antibody.
`
`in some embodiments, the PD—‘i axis binding antagonist is seiected from the group consisting ofa FED-i
`
`binding antagonist, a PD—Li binding antagonist, and a Fiji—L2 binding antagonist.
`
`in some embodiments,
`
`the 3'3th axis binding antagonist is a PD—t binding antagonist.
`
`in some embodiments, the 59th binding
`
`antagonist is seiected from the group consisting of MDX~1106 {nivoiumab}, Witt—3475 (iambroiizumab),
`
`CTmGtt (pidiiizumab), and AMP~224.
`
`in other embodiments, the FEM axis binding antagonist is a PD~L1
`
`binding antagonist.
`
`in some embodiments, the PD—L’i binding antagonist is seiected from the group
`
`consisting of: W2435587G, MPDLSQSOA, MDX—‘i 105, and MEDi473b.
`
`in other embodiments, the PD—t
`
`axis binding antagonist is a PD—LZ binding antagonist,
`
`in some embodiments, the PD—LZ binding
`
`antagonist is an antibody or an immunoadhesin.
`
`in some embodiments, the method further comprises administering to the subject a
`
`giuc