RU2782355C2 - Optimized antibody compositions for treatment of eye diseases - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to new conjugates of therapeutic antibodies; it can be used in medicine. The invention discloses conjugates of antibodies, which specifically bind to VEGF, to hyaluronic acid (HA) polymer. Such conjugates show increased stability and increased time of half-life from the subject’s body.

EFFECT: invention can be used in medical practice for the treatment of diseases related to pathological angiogenesis, in particular eye diseases.

160 cl, 4 ex, 10 tbl, 14 dwg

Description

SEQUENCE LIST

This application contains a sequence listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The specified ASCII copy created on March 22, 2018 is named 50474-1600WO3_Sequence_Listing_3.22.18_ST25 and has a size of 57671 bytes.

FIELD OF TECHNOLOGY

The invention generally relates to antibody conjugates, cysteine engineered antibodies, their compositions (eg, pharmaceutical compositions), and methods for their use.

BACKGROUND OF THE INVENTION

Angiogenesis is a highly regulated process by which new blood vessels are formed from pre-existing blood vessels. Although angiogenesis is essential during development to ensure adequate blood circulation, many disorders are associated with pathological angiogenesis, such as eye diseases (eg, age-related macular degeneration, AMD) and cell proliferative diseases (eg, cancer). Vascular endothelial growth factor (VEGF) is a clinically proven stimulatory factor for angiogenesis, and neutralization of VEGF (for example, using a blocking anti-VEGF antibody) can be used to treat disorders associated with pathological angiogenesis.

Current approaches to the treatment of eye diseases associated with pathological angiogenesis (eg, AMD (AMD) (eg, wet AMD), diabetic macular edema (DME), diabetic retinopathy (DR), and retinal vein occlusion (RVO)), typically include intravitreal injections of VEGF antagonists (eg, anti-VEGF Fab ranibizumab). Because the site of action of anti-VEGF Fab fragments is at the back of the eye on the retina, and because Fab fragments can have a relatively short residence time in the eye, maximum patient benefit from anti-VEGF Fab fragments is generally obtained. by relatively frequent dosing (eg, every four weeks, Q4W) by intravitreal injection. Prolonged delivery of anti-VEGF antibodies or antibody fragments (eg, Fab fragments) in ocular diseases may be desirable, at least in part, to reduce the frequency of dosing, which may improve patient comfort and compliance.

In this case, there remains a need for sustained delivery antibody compositions for the treatment of eye diseases (e.g., AMD (e.g., wet AMD), diabetic macular edema (DME), diabetic retinopathy (DR), and retinal vein occlusion (RVO)).

SUMMARY OF THE INVENTION

The invention provides antibody conjugates that contain monodisperse polymers (for example, hyaluronic acid (HA) monodisperse polymers) covalently linked to antibodies (for example, anti-VEGF antibodies), cysteine-engineered antibodies, which can be used, for example, in the preparation of conjugates antibodies, compositions that contain antibody conjugates (eg, pharmaceutical compositions), as well as methods for their preparation and use, for example, therapeutic applications.

In one aspect, the invention provides an antibody conjugate comprising (i) an antibody and (ii) a hyaluronic acid (HA) polymer covalently attached to the antibody, the HA polymer having a polydispersity index (PDI) of 1.1 or lower. In some embodiments, the HA polymer has a PDI of 1.0 to 1.1. In some embodiments, the HA polymer has a PDI of 1.0 to about 1.05. In some embodiments, the HA polymer has a PDI of about 1.0001 to about 1.05. In some embodiments, the HA polymer has a PDI of about 1.001. In some embodiments, the HA polymer has a molecular weight of about 1 megadalton (MDa) or less. In some embodiments, the HA polymer has a molecular weight in the range of about 25 kD and about 500 kD. In some embodiments, the HA polymer has a molecular weight in the range of about 100 kD and about 250 kD. In some embodiments, the HA polymer has a molecular weight in the range of about 150 kD and about 200 kD. In some embodiments, the HA polymer is a linear HA polymer. In some embodiments, the antibody conjugate has a hydrodynamic radius ranging from about 10 nm to about 60 nm. In some embodiments, the antibody conjugate has a hydrodynamic radius ranging from about 25 nm to about 35 nm. In some embodiments, the implementation of the hydrodynamic radius is from about 20 nm to about 30 nm.

In some embodiments of the preceding aspect, the antibody conjugate has an ocular half-life that is increased compared to a reference antibody that is not covalently attached to the HA polymer. In some embodiments, the ocular half-life is increased by at least about 2-fold relative to the reference antibody. In some embodiments, the ocular half-life is increased by at least about 4-fold relative to the reference antibody. In some embodiments, the ocular half-life is the vitreous elimination period. In some embodiments, the implementation of the reference antibody is identical to the antibody of the antibody conjugate.

In some embodiments of the preceding aspect, the antibody specifically binds to a biological molecule selected from the group consisting of vascular endothelial growth factor (VEGF); IL-1β; IL-6; IL-6R; IL-13; IL-13R; TsFR (platelet growth factor); angiopoietin; angiopoietin-2; Tie2; S1P; integrins αvβ3, αvβ5 and α5β1; betacellulin; apelina/APJ; erythropoietin; complement factor D; TNF-α; HTRA1; VEGF receptor; ST-2 receptor; and a protein genetically associated with the risk of AMD. In some embodiments, the VEGF receptor is VEGF1, VEGF2, VEGF3, mbVEGF, or sVEGF. In some embodiments, a protein genetically associated with AMD risk is selected from the group consisting of complement pathway components C2, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; SETP; LIPC, COL10A1; and TNFRSF10A.

In some embodiments of the preceding aspect, the antibody specifically binds to VEGF. In some embodiments, the antibody contains the following six hypervariable regions (HVRs): (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GX ₁ TPX ₂ GGX ₃ X ₄ X ₅ YX ₆ DSVX ₇ X ₈ (SEQ ID NO: 2), where X ₁ is Ile or His, X ₂ is Ala or Arg , X ₃ is Tyr or Lys, X ₄ is Thr or Glu, X ₅ is Arg, Tyr, Gln, or Glu, X ₆ is Ala or Glu, X ₇ is Lys or Glu, and X ₈ is is Gly or Glu; (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQX ₁ VSTAVA (SEQ ID NO: 4), where X ₁ represents Asp or Arg; (e) HVR-L2 containing the amino acid sequence X ₁ ASFLYS (SEQ ID NO: 5), where X ₁ represents Ser or Met; and (f) HVR-L3 containing the amino acid sequence X ₁ QGYGX ₂ PFT (SEQ ID NO: 6), where X ₁ is Gln, Asn, or Thr and X ₂ is Ala, Asn, Gln, or Arg. In some embodiments, the antibody contains the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYTRYADSVKG (SEQ ID NO: 7), GITPAGGYEYYADSVKG (SEQ ID NO: 21), or GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGAPFT (SEQ ID NO: 10) or QQGYGNPFT (SEQ ID NO: 23).

In some embodiments, the antibody contains the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYTRYADSVKG (SEQ ID NO: 7); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGAPFT (SEQ ID NO: 10). In some embodiments, the antibody further comprises the following heavy chain variable domain (VH) framework regions (FRs): (a) FR-H1 comprising the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTIS (SEQ ID NO: 13); (b) FR-H2 containing the amino acid sequence WVRQAPGKGLEWVA (SEQ ID NO: 14); (c) FR-H3 containing the amino acid sequence RFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO: 15); and (d) FR-H4 containing the amino acid sequence WGQGTLVTVSS (SEQ ID NO: 16). In some embodiments, the antibody further comprises a light chain variable domain (VL) FR: (a) FR-L1 comprising the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20).

In some embodiments, the antibody contains the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGNPFT (SEQ ID NO: 23). In some embodiments, the antibody further comprises the following VL domain FRs: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 24); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20).

In some embodiments, the antibody contains the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGAPFT (SEQ ID NO: 10). In some embodiments, the antibody further comprises the following VL domain FRs: (a) FR-L1 comprising the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17), DIQMTQSPESLSASVGDEVTITC (SEQ ID NO: 25), or DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18) or WYQQKPGEAPKLLIY (SEQ ID NO: 27); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19) or GVPSRFSGSGSGTDFTLTIESLQPEDAATYYC (SEQ ID NO: 28); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20). In some embodiments, the antibody further comprises the following VH domain FRs: (a) FR-H1 comprising the amino acid sequence EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29) or EEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 52); (b) FR-H2 containing the amino acid sequence WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) FR-H3 containing the amino acid sequence RFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) FR-H4 containing the amino acid sequence WGQGELVTVSS (SEQ ID NO: 32).

In some embodiments of the preceding aspect, the antibody comprises (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 11, 40, or 42; (b) a VL domain containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 12, 41, or 46; or (c) a VH domain as in (a) and a VL domain as in b). In some embodiments, the VH domain further comprises the following FRs: (a) FR-H1 containing the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTIS (SEQ ID NO: 13); (b) FR-H2 containing the amino acid sequence VWRQAPGKGLEWVA (SEQ ID NO: 14) or VWRQEPGKGLEWVA (SEQ ID NO: 39); (c) FR-H3 containing the amino acid sequence RFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO: 15); and (d) FR-H4 containing the amino acid sequence WGQGTLVTVSS (SEQ ID NO: 16). In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the VH domain further comprises the following FRs: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17) or DIQMTQSPSSLSASVGDRVTIDC (SEQ ID NO : 45); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19), GVPSRFSGSGSGTDFTLTISSLQPEDSATYYC (SEQ ID NO: 44), or GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO: 54); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20) or FGQGTKVEVK (SEQ ID NO: 55). In some embodiments, the VL domain contains the amino acid sequence of SEQ ID NO: 12.

In some embodiments of the preceding aspect, the antibody comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 11, and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 12.

In some embodiments of the preceding aspect, the antibody comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 40, and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 12.

In some embodiments of the preceding aspect, the antibody comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 42, and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 12.

In some embodiments of the preceding aspect, the antibody comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 42, and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 41.

In some embodiments according to the previous aspect, the antibody comprises (a) a VH domain containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 33 or 51; (b) a VL domain containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 12, 34, 35, 36, 37, or 38; or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, the antibody further comprises the following FRs: (a) FR-H1 comprising the amino acid sequence EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29) or EEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 52); (b) FR-H2 containing the amino acid sequence WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) FR-H3 containing the amino acid sequence RFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) FR-H4 containing the amino acid sequence WGQGELVTVSS (SEQ ID NO: 32). In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the antibody further comprises the following FRs: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17), DIQMTQSPESLSASVGDEVTITC (SEQ ID NO: 25), or DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18) or WYQQKPGEAPKLLIY (SEQ ID NO: 27); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19), GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 24), or GVPSRFSGSGSGTDFTLTIESLQPEDAATYYC (SEQ ID NO: 28); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20). In some embodiments, the VL domain comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the VL domain comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the VL domain comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the domain The VL domain contains the amino acid sequence of SEQ ID NO: 37. In some embodiments, the VL domain contains the amino acid sequence of SEQ ID NO: 12. In some embodiments, the VL domain contains the amino acid sequence of SEQ ID NO: 38.

In some embodiments of the preceding aspect, the antibody comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 33, and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 38.

In some embodiments of the preceding aspect, the antibody comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 33, and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 34.

In some embodiments of the preceding aspect, the antibody comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 33, and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 35.

In some embodiments of the preceding aspect, the antibody comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 33, and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 36.

In some embodiments of the preceding aspect, the antibody comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 33, and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 37.

In some embodiments of the preceding aspect, the antibody comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 33, and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 12.

In some embodiments of the preceding aspect, the antibody comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 51, and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 38.

In some embodiments of the preceding aspect, the antibody comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 51, and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 35.

In some embodiments of the preceding aspect, the antibody comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 51, and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 37.

In some embodiments of the preceding aspect, the antibody comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 51, and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 12.

In some embodiments of the preceding aspect, the antibody comprises (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 48 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.

In some embodiments of the preceding aspect, the antibody comprises (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49 and (b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.

In some embodiments of any of the preceding aspects, the antibody is capable of inhibiting VEGF binding to the VEGF receptor. In some embodiments, the VEGF receptor is VEGF receptor-1 (Flt-1). In some embodiments, the VEGF receptor is VEGF receptor-2 (KDR).

In some embodiments of any of the preceding aspects, the antibody binds human VEGF (hVEGF) with a Kd of about 2 nM or less. In some embodiments, the antibody binds hVEGF with a Kd of about 75 pM to about 2 nM. In some embodiments, the antibody binds hVEGF with a Kd of about 75 pM to about 600 pM. In some embodiments, the antibody binds hVEGF with a Kd of about 75 pM to about 500 pM. In some embodiments, the antibody binds hVEGF with a Kd of about 80 pM. In some embodiments, the antibody binds hVEGF with a Kd of about 60 pM.

In some embodiments of any of the preceding aspects, the antibody has a melting point (Tm) of greater than about 83.5°C. In some embodiments, the antibody has a Tm of about 85°C to about 91°C. In some embodiments, the antibody has a Tm of about 89°C.

In some embodiments of any of the preceding aspects, the antibody has an isoelectric point (pl) less than 8. In some embodiments, the antibody has a pi of about 5 to about 7. In some embodiments, the antibody has a pl of about 5 to about 6.

In some embodiments of any of the preceding aspects, the antibody is monoclonal, human, humanized, or chimeric.

In some embodiments of any of the preceding aspects, the antibody is an antibody fragment that binds VEGF. In some embodiments, the antibody fragment is selected from the group consisting of Fab, Fab-C, Fab'-SH, Fv, scFv, and (Fab') ₂ fragments. In some embodiments, the antibody fragment is a Fab.

In some embodiments of any of the preceding aspects, the antibody is a monospecific antibody. In other embodiments of any of the preceding aspects, the antibody is a multispecific antibody. In some embodiments, the multispecific antibody is a bispecific antibody. In some embodiments, the bispecific antibody binds VEGF and a second biological molecule selected from the group consisting of interleukin 1β (IL-1β); interleukin-6 (IL-6); interleukin-6 receptor (IL-6R); interleukin-13 (IL-13); IL-13 receptor (IL-13R); TsFR (platelet growth factor); angiopoietin; angiopoietin-2; Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin; apelina/APJ; erythropoietin; complement factor D; TNF-α; HtrA1; VEGF receptor; ST-2 receptor; and a protein genetically associated with the risk of age-related macular degeneration (AMD). In some embodiments, the VEGF receptor is VEGF1, VEGF2, VEGF3, membrane-bound VEGF receptor (mbVEGF), or soluble VEGF receptor (sVEGF). In some embodiments, a protein genetically associated with AMD risk is selected from the group consisting of complement pathway components C2, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; interleukin-8 (IL-8); CX3CR1; TLR3; TLR4; SETP; LIPC, COL10A1; and TNFRSF10A.

In some embodiments of any of the preceding aspects, the antibody is a cysteine-engineered antibody. In some embodiments, the cysteine engineered antibody contains a heavy chain cysteine mutation selected from the group consisting of HC-A118C, HC-A140C, and HC-L174C (EU numbering), or a light chain cysteine mutation selected from the group consisting of LC-K149C and LC-V205C (Kabat numbering). In some embodiments, the heavy chain cysteine mutation is HC-A118C (EU numbering). In some embodiments, the heavy chain cysteine mutation is HC-A140C (EU numbering). In some embodiments, the heavy chain cysteine mutation is HC-L174C (EU numbering). In some embodiments, the light chain cysteine mutation is LC-K149C (Kabat numbering). In some embodiments, the light chain cysteine mutation is LC-V205C (Kabat numbering). In some embodiments, the HA polymer is covalently attached to the antibody in a cysteine mutation.

In another aspect, any of the preceding antibody conjugates may be used as a drug.

In another aspect, any of the preceding antibody conjugates may be used in the manufacture of a medicament for the treatment of an eye disease in a subject.

In another aspect, any of the preceding antibody conjugates may be used to reduce or inhibit angiogenesis in a subject having an eye disease.

In another aspect, any of the preceding antibody conjugates may be used to treat an eye disease in a subject.

In some embodiments of any of the preceding aspects, the eye disease is selected from the group consisting of age-related macular degeneration (AMD), macular degeneration, macular edema, diabetic macular edema (DME) (including focal DME outside the central zone and diffuse DME involving the central zone retinopathy, diabetic retinopathy (DR) (including proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR), other ischemia-related retinopathy, retinopathy of prematurity (RP), retinal vein occlusion (RVO) ( including forms of occlusion of the central retinal vein (CRVR) and branches of the central retinal vein (RVR)), CNV (including myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal neovascularization / choroidal neovascularization, pathological myopia, Hippel-Lindau disease, eye histoplasmosis, familial exudative vitr eoretinopathy (SEVRP), Coats' disease, Norrie's disease, osteoporosis-pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, rubeosis, neovascular eye disease, neovascular glaucoma, retinitis pigmentosa (PR), hypertensive retinopathy, retinal angiomatous proliferation, macular telangiectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystic macular edema (CMO), vasculitis, papilledema, retinitis, conjunctivitis (including infectious and non-infectious (for example, allergic) conjunctivitis), Leber's congenital amaurosis, uveitis (including infectious and non-infectious uveitis), choroiditis, ocular histoplasmosis, blepharitis, dry eye syndrome, traumatic eye injury, and Sjögren's syndrome. In some embodiments, the eye disease is AMD, DME, DR, or OBC. In some embodiments, the eye disorder is AMD. In some embodiments, AMD is wet AMD. In some embodiments, the eye disease is DME.

In another aspect, the invention relates to a pharmaceutical composition containing any of the antibody conjugates described herein, a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the pharmaceutical composition further comprises a second agent, wherein the second agent is selected from the group consisting of an antibody, an anti-angiogenic agent, a cytokine, a cytokine antagonist, a corticosteroid, an analgesic, and a compound that binds to the second biological molecule. In some embodiments, the anti-angiogenic agent is a VEGF antagonist. In some embodiments, the VEGF antagonist is an anti-VEGF antibody, an anti-VEGF receptor antibody, a soluble VEGF receptor fusion protein, an aptamer, an anti-VEGF DARPin®, or an inhibitor of VEGF tyrosine kinase (VEGF receptor). In some embodiments, the anti-VEGF antibody is ranibizumab (LUCENTIS®), RTH-258, or a bispecific anti-VEGF antibody. In some embodiments, the bispecific anti-VEGF antibody is an anti-VEGF/anti-Ang2 antibody. In some embodiments, the anti-VEGF/anti-Ang2 antibody is RG-7716. In some embodiments, the soluble VEGF receptor fusion protein is aflibercept (EYLEA®). In some embodiments, the aptamer is pegaptanib (MACUGEN®). In some embodiments, the anti-VEGF DARPin® is abicipar pegol. In some embodiments, the VEGF tyrosine kinase inhibitor is selected from the group consisting of 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline (ZD6474), 4-(4- fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline (AZD2171), vatalanib (PTK787), semaxaminib (SU5416), and SUTENT® (sunitinib). In some embodiments, the implementation of the second biological molecule is selected from the group consisting of IL-1β; IL-6; IL-6R; IL-13; IL-13R; TCFR; angiopoietin; angiopoietin-2; Tie2; S1P; integrins αvβ3, αvβ5 and α5β1; betacellulin; apelina/APJ; erythropoietin; complement factor D; TNF-α; HTRA1; VEGF receptor; ST-2 receptor; and a protein genetically associated with the risk of AMD. In some embodiments, the VEGF receptor is VEGF1, VEGF2, VEGF3, mdVEGF, or sVEGF. In some embodiments, a protein genetically associated with AMD risk is selected from the group consisting of complement pathway components C2, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; SETP; LIPC, COL10A1; and TNFRSF10A. In some embodiments, the compound that binds the second biological molecule is an antibody or an antigen-binding fragment thereof. In some embodiments, the antigen-binding antibody fragment is selected from the group consisting of Fab, Fab-C, Fab'-SH, Fv, scFv, and (Fab') ₂ fragments. In some embodiments, the antigen-binding fragment of an antibody is a Fab.

In another aspect, any of the preceding pharmaceutical compositions may be used as a drug.

In another aspect, any of the preceding pharmaceutical compositions may be used in the manufacture of a medicament for the treatment of an eye disease in a subject.

In another aspect, any of the preceding pharmaceutical compositions may be used to reduce or inhibit angiogenesis in a subject having an eye disease.

In another aspect, any of the preceding pharmaceutical compositions may be used to treat an eye disease in a subject.

In some embodiments of any of the preceding aspects, the eye disease is selected from the group consisting of age-related macular degeneration (AMD), macular degeneration, macular edema, diabetic macular edema (DME) (including focal DME outside the central zone and diffuse DME involving the central zone retinopathy, diabetic retinopathy (DR) (including proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR), other ischemia-related retinopathy, retinopathy of prematurity (RP), retinal vein occlusion (RVO) ( including forms of occlusion of the central retinal vein (CRVR) and branches of the central retinal vein (RVR)), CNV (including myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal neovascularization / choroidal neovascularization, pathological myopia, Hippel-Lindau disease, eye histoplasmosis, familial exudative vitr eoretinopathy (SEVRP), Coats' disease, Norrie's disease, osteoporosis-pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, rubeosis, neovascular eye disease, neovascular glaucoma, retinitis pigmentosa (PR), hypertensive retinopathy, retinal angiomatous proliferation, macular telangiectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystic macular edema (CMO), vasculitis, papilledema, retinitis, conjunctivitis (including infectious and non-infectious (for example, allergic) conjunctivitis), Leber's congenital amaurosis, uveitis (including infectious and non-infectious uveitis), choroiditis, ocular histoplasmosis, blepharitis, dry eye syndrome, traumatic eye injury, and Sjögren's syndrome. In some embodiments, the eye disease is AMD, DME, DR, or OBC. In some embodiments, the eye disorder is AMD. In some embodiments, AMD is wet AMD. In some embodiments, the eye disease is DME.

In another aspect, the invention relates to a method of reducing or inhibiting angiogenesis in a subject having an eye disease, comprising administering to the subject an effective amount of any of the antibody conjugates described herein, or any of the pharmaceutical compositions described herein, thereby reducing or inhibiting angiogenesis subject.

In another aspect, the invention relates to a method of treating an ocular disease comprising administering an effective amount of any of the antibody conjugates described herein, or any of the pharmaceutical compositions described herein, to a subject in need of such treatment.

In some embodiments of any of the preceding aspects, the eye disease is selected from the group consisting of age-related macular degeneration (AMD), macular degeneration, macular edema, diabetic macular edema (DME) (including focal DME outside the central zone and diffuse DME involving the central zone retinopathy, diabetic retinopathy (DR) (including proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR), other ischemia-related retinopathy, retinopathy of prematurity (RP), retinal vein occlusion (RVO) ( including forms of occlusion of the central retinal vein (CRVR) and branches of the central retinal vein (RVR)), CNV (including myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal neovascularization / choroidal neovascularization, pathological myopia, Hippel-Lindau disease, eye histoplasmosis, familial exudative vitr eoretinopathy (SEVRP), Coats' disease, Norrie's disease, osteoporosis-pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, rubeosis, neovascular eye disease, neovascular glaucoma, retinitis pigmentosa (PR), hypertensive retinopathy, retinal angiomatous proliferation, macular telangiectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystic macular edema (CMO), vasculitis, papilledema, retinitis, conjunctivitis (including infectious and non-infectious (for example, allergic) conjunctivitis), Leber's congenital amaurosis, uveitis (including infectious and non-infectious uveitis), choroiditis, ocular histoplasmosis, blepharitis, dry eye syndrome, traumatic eye injury, and Sjögren's syndrome. In some embodiments, the eye disease is AMD, DME, DR, or OBC. In some embodiments, the eye disorder is AMD. In some embodiments, AMD is wet AMD. In some embodiments, the eye disease is DME.

In some embodiments of any of the preceding aspects, the method further comprises administering to the subject an effective amount of a second agent, the second agent being selected from the group consisting of an antibody, an anti-angiogenic agent, a cytokine, a cytokine antagonist, a corticosteroid, an analgesic, and a compound that binds to the second biological agent. molecule. In some embodiments, the anti-angiogenic agent is a VEGF antagonist. In some embodiments, the VEGF antagonist is an anti-VEGF antibody, an anti-VEGF receptor antibody, a soluble VEGF receptor fusion protein, an aptamer, an anti-VEGF DARPin®, or an inhibitor of VEGF tyrosine kinase (VEGF receptor). In some embodiments, the anti-VEGF antibody is ranibizumab (LUCENTIS®), RTH-258, or a bispecific anti-VEGF antibody. In some embodiments, the bispecific anti-VEGF antibody is an anti-VEGF/anti-Ang2 antibody. In some embodiments, the anti-VEGF/anti-Ang2 antibody is RG-7716. In some embodiments, the soluble VEGF receptor fusion protein is aflibercept (EYLEA®). In some embodiments, the aptamer is pegaptanib (MACUGEN®). In some embodiments, the anti-VEGF DARPin® is abicipar pegol. In some embodiments, the VEGF tyrosine kinase inhibitor is selected from the group consisting of 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline (ZD6474), 4-(4- fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline (AZD2171), vatalanib (PTK787), semaxaminib (SU5416), and SUTENT® (sunitinib). In some embodiments, the implementation of the second biological molecule is selected from the group consisting of IL-1β; IL-6; IL-6R; IL-13; IL-13R; TCFR; angiopoietin; angiopoietin-2; Tie2; S1P; integrins αvβ3, αvβ5 and α5β1; betacellulin; apelina/APJ; erythropoietin; complement factor D; TNF-α; HTRA1; VEGF receptor; ST-2 receptor; and a protein genetically associated with the risk of AMD. In some embodiments, the VEGF receptor is VEGF1, VEGF2, VEGF3, mbVEGF, or sVEGF. In some embodiments, a protein genetically associated with AMD risk is selected from the group consisting of complement pathway components C2, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; SETP; LIPC, COL10A1; and TNFRSF10A. In some embodiments, the compound that binds the second biological molecule is an antibody or an antigen-binding fragment thereof. In some embodiments, the antigen-binding antibody fragment is selected from the group consisting of Fab, Fab-C, Fab'-SH, Fv, scFv, and (Fab') ₂ fragments.

In some embodiments of any of the preceding aspects, the antibody conjugate or pharmaceutical composition is administered intravitreally, through the eye, intraocularly, in the periscleral region, in the subtenon space, in the suprachoroidal space, topically, intravenously, intramuscularly, intradermally, transdermally, intraarterially, intraperitoneally, intralesionly, intracranially , intraarticular, inside the prostate, intrapleural, intratracheal, intrathecal, intranasal, intravaginal, rectal, topical, intratumoral, peritoneal, intraventricular, subcutaneous, subconjunctival, intravesicular, mucosal, intrapericardial, intraumbilical cord, intraorbital, oral, local, transdermal, by inhalation, by injections, in the form of eye drops, by implantation, by infusion, by continuous infusion, by localized perfusion directly through target cells, by catheter, by lavage, in creams or in lipid formulations. In some embodiments, the antibody conjugate or pharmaceutical composition is administered into the vitreous, through the eye, intraocularly, into the periscleral region, into the sub-Tenon's space, into the suprachoroidal space, or topically. In some embodiments, the antibody conjugate or pharmaceutical composition is administered by intravitreal injection. In some embodiments, the antibody conjugate or pharmaceutical composition is administered topically as an eye drop or ointment. In some embodiments, the antibody conjugate or pharmaceutical composition is administered via an implantable delivery device.

In some embodiments of any of the preceding aspects, the subject is a human.

BRIEF DESCRIPTION OF GRAPHICS

On FIG. 1A is a graph showing the population distribution of molecular weights (in terms of molar mass) in a representative sample of 200 kDa HA.

On FIG. 1B is a graph showing the population distribution of maleimides on a 200 kDa HA chain as a result of Monte Carlo simulation of stochastic modification for each acid group having a 5% probability of modification.

On FIG. 1C is a graph showing the polydispersity of 40 kDa, 200 kDa and 600 kDa HA polymers. The Table below the graph shows the number average molecular weight (Mn), weight average molecular weight (Mw), polydispersity index (PDI), and molecular weight range (MW) (in terms of Mw) for the indicated samples.

On FIG. 2 is a series of graphs demonstrating that TK-G6.31.AARR conjugates have differences in physical stability under physiologically relevant stress conditions as assessed by size exclusion chromatography (SEC) with an attached refractive index (RI) detector and a multi-angle laser light scattering detector. (MALS) (SEC-RI-MALS). The label series refer to the molecular weight of the HA backbone (40 kDa (“40K”); 200 kDa (“200K”) and 600 kDa (“600K”) and Fab loading level.

On FIG. 3 is a series of graphs showing changes in EC retention profiles over time for TK40K-G6.31.AARR-4.7% (left panel), TK200K-G6.31.AARR-4.7% (center panel), and GK600K-G6.31.AARR-2.1% (right panel), demonstrating that the EC retention time is shifted to a later time (less than the hydrodynamic size), with the degree of this shift depending on the molecular weight of the HA backbone.

On FIG. 4 is a series of graphs showing SEC-RI-MALS data plotted as cumulative mass fraction for TK40K-G6.31.AARR-4.7% (left panel), TK200K-G6.31.AARR-4.7% (center panel), and GK600K-G6.31.AARR-2.1% (right panel).

On FIG. 5 is a graph showing SEC-RI-MALS characterization results for commercial polydisperse HA (black) and monodisperse HA (grey) showing the difference in mass distributions between the two production methods. The table in the right panel shows the Mn, Mw, and PDI values determined by this analysis.

On FIG. 6 is a graph showing the results of SEC-RI-MALS characterization of commercial polydisperse TK200K-G6.31.AARR (black) and monodisperse TK150K-G6.31.AARR (grey) showing the difference in mass distributions. The table in the right panel shows the Mn, Mw, and PDI values determined by this analysis.

On FIG. 7 is a schematic diagram showing that standard Fab-C format molecules are designed to contain a free cysteine residue suitable for conjugation by extending the standard Fab hinge peptide sequence to a first or second hinge disulfide cysteine. In order to minimize or prevent scrambling between the interchain disulfide and this free cysteine residue, the cysteine can instead be mutated on the surface of the Fab at a location spatially separated from the interchain disulfide (referred to herein as "thio-Fab").

On FIG. 8A-8C are a series of schematic representations demonstrating that the flexibility and spatial proximity of the hinge sequence can result in a rearrangement in the three possible disulfide states, leaving three different cysteine residues available for conjugation. On FIG. 8A shows a putative configuration in which the hinge cysteine is reduced and available for conjugation. On FIG. 8B shows a cyclized heavy chain (HC) variant in which the hinge cysteine forms a disulfide bond with the cysteine residue HC, which is normally part of the interchain disulfide bond, making the cysteine in the light chain (LC) of the interchain disulfide bond available for conjugation. On FIG. 8C shows an LC variant in which the hinge cysteine forms a disulfide bond with the LC cysteine, which normally forms part of the interchain disulfide bond, making the cysteine in the HC of the interchain disulfide bond available for conjugation.

On FIG. 9 shows a series of graphs showing the results of a series of maleimide capping and Lys-C limited digestion experiments performed on G6.31.AARR.Fab-C and analyzed by reverse-phase ultra high performance liquid chromatography/time-of-flight (RP-UPLC-TOF) mass -spectrometry. The numbers above the peaks are the total number of ions for that peak and are a measure of the area of the peak.

On FIG. 10A and 10B are a series of graphs showing the results of limited Lys-C (FIG. 10A) and hyaluronidase (GCase) (FIG. 10B) cleavage of the TK-G6.31.AARR conjugate, confirming the presence of conjugated variants associated with conjugation by both cysteines. , normally captured by interchain disulfide bonds.

On FIG. 11 is a series of graphs showing that the conjugation of thio-Fab G6.31.AARR with GK200K-maleimide proceeded normally compared to G6.31.AARR.Fab-C, although the conversion of Fab to conjugate was lower for the thio-Fab samples.

On FIG. 12 is a graph demonstrating the deconjugation of the polyethylene glycol (PEG)-maleimide polymer model from G6.31.AARR of various formats in PBS + 2 mM oxidized glutathione (GSSG) at 37°C, evaluated by RP-UPLC-TOF.

On FIG. 13A shows the Kabat numbering scheme for the 4D5 light chain.

On FIG. 13B shows a sequential numbering scheme (left column) starting at the N-terminus compared to the Kabat numbering scheme (middle column) and the EU numbering scheme (right column) for the 4D5 antibody.

On FIG. 14 is a graph demonstrating that HA-G6.31.AARR conjugates derived from monodisperse HA show improved physical stability under physiological stress at week 4 compared to similarly sized TK-G6.31.AARR conjugates derived from polydisperse HA. The table in the right panel shows Mw (kDa) at 0, 2, and 4 weeks.

DETAILED DESCRIPTION OF THE INVENTION

I. DEFINITIONS

The term "about" as used herein refers to the usual error range for the corresponding value and is well known to one of ordinary skill in the art. As used herein, the use of "about" in relation to a value or parameter includes (and describes) embodiments that refer directly to that value or parameter, per se.

As used herein, a "human acceptor framework" is a framework that contains the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework that is derived from a human immunoglobulin framework or consensus framework. human, as defined below. A human acceptor framework "derived from" a human immunoglobulin framework or human consensus framework may contain the same amino acid sequence as indicated or may contain alterations in the amino acid sequence. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the human VL acceptor framework sequence is identical to the human immunoglobulin VL framework sequence or the human consensus framework sequence.

The term "affinity" refers to the strength of the overall overall non-covalent interactions between a single binding site of a molecule (eg, antibody) and its binding partner (eg, antigen). Unless otherwise indicated, the term "binding affinity" as used herein refers to the actual binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented as a dissociation constant (Kd). Affinity can be measured using methods generally accepted in the art, including those described herein. Specific illustrative and typical embodiments of the invention relating to the measurement of binding affinity are described below.

An “affinity matured” antibody refers to an antibody with one or more changes in one or more hypervariable regions (HVR) and/or framework regions (FR) compared to the parent antibody that does not contain such changes, and these changes result in an improvement the affinity of the antibody for the antigen.

The term "vascular endothelial growth factor" or "VEGF" refers to the vascular endothelial growth factor-A protein, an example of which is SEQ ID NO: 47 (see also Swiss Prot, accession number P15692, Gene ID (NCBI): 7422). The term "VEGF" includes a protein having the amino acid sequence of SEQ ID NO: 47, as well as its homologues and isoforms. The term "VEGF" also encompasses known isoforms, such as spliced isoforms, VEGF, such as VEGF ₁₁₁ , VEGF ₁₂₁ , VEGF ₁₄₅ , VEGF ₁₆₅ , VEGF ₁₈₉ , and VEGF ₂₀₆ , together with its natural allelic and processed forms, including endothelial growth factor human cells, consisting of 110 amino acids formed by cleavage with VEGF ₁₆₅ plasmin, as described in Ferrara Mol. Biol. Cell, 21:687 (2010), Leung et al., Science, 246:1306 (1989), and Houck et al., Mol. Endocrin. 5:1806 (1991). The term "VEGF" also refers to VEGF of a non-human species, such as mice, rats, or primates. Sometimes VEGF from a particular species is referred to by terms such as hVEGF for human VEGF, mVEGF for mouse VEGF, and the like. The term "VEGF" is also used to refer to truncated forms of the polypeptide containing amino acids 8 to 109 or 1 to 109 of the 165 amino acids from human vascular endothelial growth factor. Reference to any such forms of VEF may be identified in this application, for example, by "VFRE ₁₀₉ ", "VFRE (8-109)", "VFRE (1-109)" or "VFRE ₁₆₅ ". Amino acid positions for "truncated" native VEGF are numbered the same as indicated in the native VEGF sequence. For example, amino acid position 17 (methionine) in truncated native VEGF is also position 17 (methionine) in native VEGF. Truncated native VEGF has a binding affinity for the KDR and Flt-1 receptors comparable to native VEGF. The term "VEGF variant" as used herein refers to a VEGF polypeptide that contains one or more amino acid mutations in the native VEGF sequence. Optionally, one or more amino acid mutations contain an amino acid substitution(s). In the context of the VEGF variant abbreviations described herein, it is noted that the numbers refer to the position of the amino acid residue of the amino acid sequence of the putative native VEGF (listed in Leung et al., formerly and Houck et al., formerly). Unless otherwise indicated, the term "VEGF" as used in this document means VEGF-A.

The terms "anti-VEGF antibody" and "antibody that binds to VEGF" and "antibody that specifically binds VEGF" refer to an antibody that is capable of binding VEGF with sufficient affinity such that the antibody can be used as a diagnostic and/or a therapeutic agent for targeting VEGF. In one embodiment, the degree of binding of an anti-VEGF antibody to an unrelated non-VEGF protein is less than about 10% of the binding of the antibody to VEGF, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the antibody that binds to VEGF has a dissociation constant (Kd) ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM ( eg 10 ^-8 M or less, eg 10 ^-8 M to 10 ^-13 M, eg 10 ^-9 M to 10 ^-13 M). In some embodiments, the anti-VEGF antibody binds to a VEGF epitope that is conserved among VEGF from different species.

The term "antibody" is used herein in its broadest sense and includes various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the required antigen-binding activity.

An "antibody fragment" refers to a molecule, other than an intact antibody, that contains a portion of the intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, v, Fab, Fab', Fab-C, Fab'-SH, F(ab') ₂ ; diabody; linear antibodies; single chain antibody molecules (eg scFv); and multispecific antibodies formed from antibody fragments. In some cases, examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diabody; linear antibodies; single chain antibody molecules (eg scFv); and multispecific antibodies formed from antibody fragments.

Cleavage of antibodies with papain produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, the name of which reflects its ability to easily crystallize. The Fab fragment consists of the entire light (L) chain along with the heavy (H) chain variable region domain (VH) and the first constant domain from a single heavy chain (CH1). Treatment of the antibody with pepsin allows the isolation of a single large F(ab') ₂ fragment, which approximately corresponds to two Fab fragments with bivalent antigen-binding activity, connected by a disulfide bond, and retaining the ability to cross-link the antigen. Fab' fragments differ from Fab fragments in that they have several additional residues at the carboxyl end of the CH1 domain, including one or more cysteines from the hinge region of the antibody. Fab-C molecules are Fab molecules that are expressed such that the sequence is truncated at the first hinge cysteine, resulting in a free cysteine Fab immediately upon expression (see e.g. Shatz et al. Mol, Pharmaceutics 2016; PubMed identifier (PMID) 27244474). For example, a Fab-C molecule may have a free cysteine at the Cys227 position of the heavy chain. In other cases, the Fab-C molecule may have a free cysteine at the Cys229 position of the heavy chain. As used herein, Fab'-SH is the designation for Fab' in which the cysteine(s) residue(s) of the constant domains bear a free thiol group. F(ab') ₂ antibody fragments were originally generated as pairs of Fab' fragments that are linked together by hinge cysteines. Other chemistries of antibody fragments are also known.

As used herein, the term "Fc region" is used to refer to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variants of Fc regions. In one embodiment, the Fc region of the human IgG heavy chain ranges from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the Fc region may or may not contain a C-terminal lysine (Lys447). Unless otherwise indicated, the amino acid residue numbering of the Fc region or constant region follows the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

"Fv" consists of a dimer of one heavy chain variable region domain and one light chain variable region domain tightly linked through non-covalent bonds. When these two domains are folded, six hypervariable loops (3 loops on each H- and L-chain) protrude outward, the amino acid residues of which are involved in antigen binding and give the antibody specificity for antigen binding. At the same time, even one variable domain (or half of the Fv containing only three antigen-specific HVRs) has the ability to recognize and bind an antigen, although often with a lower affinity than the entire binding site.

"Single chain Fv", also abbreviated as "sFv" or "scFv", are antibody fragments that contain the VH and VL antibody domains linked in a single polypeptide chain. Preferably, the sFv polypeptide also contains a polypeptide linker between the VH and VL domains, allowing the sFv to form the structure required for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term "diabodies" refers to small antibody fragments obtained by constructing sFv fragments (see previous paragraph) with short linkers (about 5-10 residues) between the VH and VL domains, thereby achieving interchain rather than intrachain pairing of V domains. , leading to the formation of a bivalent fragment, i.e. a fragment carrying two antigen-binding sites. Bispecific antibodies are heterodimers of two "crossover" sFv fragments, in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in more detail, for example, in EP 404097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. sci. USA 90:6444-6448 (1993).

A "blocking" antibody or "antagonistic" antibody is an antibody that inhibits or reduces the biological activity of the antigen it binds. Some blocking antibodies or antagonistic antibodies largely or completely inhibit the biological activity of the antigen.

"An antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the reference antibody from binding to its antigen in a competitive assay by 50% or more, and conversely, a reference antibody blocks binding of an antibody to its antigen in a competitive assay by 50% or more. This paper provides an illustrative competitive analysis.

The term "chimeric" antibody refers to an antibody in which the heavy and/or light chain portion is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The "class" of an antibody refers to the type of constant domain or constant region contained in its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further subdivided into subclasses (isotypes), such as IgG ₁ , IgG2, IgG ₃ , IgG ₄ , IgA ₁ , and IgA ₂ . The heavy chain constant domains that correspond to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

A "cysteine-engineered antibody" or "cysteine-engineered antibody variant" is an antibody in which one or more antibody residues are replaced with cysteine residues. In some cases, cysteine-engineered antibodies may be referred to as THIOMAB™ antibodies or thio-Fab antibodies. The thiol(s) group(s) from cysteine-engineered antibodies may be conjugated to other components, such as polymers (eg, HA polymers, including monodisperse HA polymers). In specific embodiments, the implementation of the substituted residues are located in accessible areas of the antibody. Thus, by replacing such residues with cysteine, thiol-reactive groups are located at accessible sites on the antibody and can be used to conjugate the antibody to other components such as polymers (eg, HA polymers). For example, a cysteine-engineered antibody may be an antibody with a single non-cysteine native mutation for a cysteine in the light chain (e.g., LC-G64C, LC-I106C, LC-R108C, LC-R142C, or LC-K149C as numbered Kabat) or heavy chain (e.g. HC-D101C, HC-V184C, or HC-T205C according to Kabat numbering, or HC-T114C, HC-A140C, HC-L174C, HC-L179C, HC-T187C, HC- T209C, HC-V262C, HC-G371C, HC-Y373C, HC-E382C, HC-S424C, HC-N434C, and HC-Q438C according to EU numbering (for example, HC-A136C according to Kabat numbering is HC-A140C according to EU numbering EU)) (see Fig. 13A and 13B). In special cases, a cysteine-engineered antibody may contain a heavy chain cysteine mutation selected from the group consisting of HC-A118C, HC-A140C, and HC-L174C (EU numbering), or a light chain cysteine mutation selected from the group consisting of LC-V205C and LC-K149C (Kabat numbering). In some cases, a cysteine-engineered antibody has a single cysteine mutation in either the heavy or light chain, such that each full-length antibody (i.e., an antibody with two heavy chains and two light chains) has two engineered cysteine residues, and each Fab -fragment has one modified cysteine residue. In other instances, the cysteine-engineered antibody has more than one cysteine mutation (eg, 2, 3, 4, or 5 cysteine mutations).

"Free cysteine amino acid" refers to a cysteine amino acid residue that is introduced into the parent antibody during construction, has a thiol functional group (-SH), and is not paired as an intramolecular or intermolecular disulfide bridge.

The term "thiol reactivity value" is a quantitative characteristic of the reactivity of free cysteine amino acids. The thiol reactivity value is the percentage of free cysteine amino acid in a cysteine-engineered antibody that reacts with a thiol-reactive reagent and is converted to a maximum value of 1. For example, the free cysteine amino acid in a cysteine-engineered antibody that reacts with a reaction in 100% yield with a thiol-reactive reagent, such as a biotin-maleimide reagent, to form a biotin-labelled antibody has a thiol reactivity value of 1.0. Another amino acid, cysteine, incorporated in the same or a different parent antibody during construction that reacts in 90% yield with a thiol-reactive reagent has a thiol reactivity value of 0.9. Another amino acid cysteine, introduced during construction in the same or a different parent antibody, that reacts in 80% yield with the reagent thiol reactivity has a thiol reactivity value of 0.8. Another amino acid, cysteine, incorporated in the same or a different parent antibody during construction that reacts in 70% yield with a thiol-reactive reagent has a thiol reactivity value of 0.7. Another amino acid, cysteine, introduced in the same or a different parent antibody during construction that reacts in 60% yield with a thiol-reactive reagent, has a thiol reactivity value of 0.6. Another amino acid, cysteine, introduced during construction into the same or a different parent antibody that reacts in 50% yield with a thiol-reactive reagent, has a thiol reactivity value of 0.5. Another amino acid, cysteine, introduced in the same or a different parent antibody during construction that reacts in 40% yield with a thiol-reactive reagent, has a thiol reactivity value of 0.4. Another amino acid cysteine, introduced during construction in the same or a different parent antibody, which reacts in 30% yield with a thiol-reactive reagent, has a thiol reactivity value of 0.3. Another amino acid, cysteine, introduced in the same or a different parent antibody during construction that reacts in 20% yield with a thiol-reactive reagent, has a thiol reactivity value of 0.2. Another amino acid, cysteine, introduced in the design into the same or a different parent antibody that reacts in 10% yield with a thiol-reactive reagent has a thiol reactivity value of 0.1. Another amino acid cysteine introduced during construction into the same or a different parent antibody that does not react at all with a thiol-reactive reagent has a thiol reactivity value of 0. Determining the thiol reactivity value of a particular cysteine can be done using an ELISA assay ( for example, PHESELECTOR assay as described herein), mass spectroscopy, liquid chromatography, autoradiography, or other quantitative analytical tests.

A "parent antibody" is an antibody containing an amino acid sequence in which one or more amino acid residues are replaced with one or more cysteine residues. The parent antibody may contain a native sequence or a wild type sequence. The parent antibody may have pre-existing amino acid sequence modifications (such as additions, deletions, and/or substitutions) compared to other natural, wild type, or modified forms of the antibody. The parent antibody may be directed against a target antigen of interest, such as a biologically important polypeptide such as VEGF. Any of the antibodies described herein (eg, an anti-VEGF antibody) can be the parent antibody.

"Effector functions" refer to those biological activities inherent in the Fc region of an antibody that vary depending on the isotype of the antibody. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); binding to the Fc receptor; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; decreased expression of cell surface receptors (eg B cell receptor) and B cell activation.

"Frame" or "framework region" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4.

The terms "full antibody", "intact antibody", and "complete antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody, or containing heavy chains that contain an Fc region, as defined herein. document.

A "human antibody" is an antibody that has an amino acid sequence that matches that of an antibody produced by a human body or a human cell, or derived from a non-human source that uses a set of human antibodies or other human antibody-coding sequences. Specifically excluded from this definition of a human antibody is a humanized antibody containing antigen-binding residues of non-human origin.

A "human consensus framework" is a framework that contains the most frequently occurring amino acid residues when selecting human immunoglobulin VL or VH framework sequences. Typically, the VL or VH sequences of a human immunoglobulin are selected from a subgroup of variable domain sequences. Typically, a subgroup of sequences is a subgroup as classified by Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I, according to Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III, according to Kabat et al., supra.

"Humanized" forms of non-human (eg, rodent) antibodies are chimeric antibodies that contain a minimal sequence derived from a non-human antibody. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which the hypervariable region residues of the recipient are replaced by hypervariable region residues of a non-human species (donor antibody) such as a mouse, rat, rabbit, or non-human primate having the desired specificity, affinity and activity of the antibody. In some cases, human immunoglobulin FR residues are replaced by corresponding non-human residues. In addition, humanized antibodies may contain residues not found in the recipient antibody or the donor antibody. These modifications were made to further improve the performance of the antibodies. In general, a humanized antibody will contain substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to such non-human immunoglobulin regions and all or substantially all of the FRs correspond to such human immunoglobulin sequence regions. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically from a human immunoglobulin. For more details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

The term "variable" refers to the fact that among antibodies, some segments of the variable domains vary greatly in sequence. The variable or "V" domain mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. At the same time, variability is unevenly distributed throughout the variable domain. In fact, V regions are composed of relatively invariant fragments called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" that are 9-12 amino acids long. The term "hypervariable region" or "HVR" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region typically contains amino acid residues from, for example, about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in VL, and about residues 26-35 (H1) , 49-65 (H2) and 95-102 (H3) in VH (in one embodiment, H1 consists of about 31-35 residues); Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or these residues from the "hypervariable loop" (for example, 26-32 (L1), 50-52 (L2) and 91-96 (L3) residues in VL, and 26-32 (H1), 53 -55 (H2) and 96-101 (H3) in VH Chothia and Lesk, J Mol Biol 196:901-917 (1987) Each of the native heavy and light chain variable domains contains four FRs, predominantly assuming a beta-sheet configuration connected by three hypervariable regions that form loops connecting the beta-sheet structure, and in some cases forming part of the beta-sheet structure. with hypervariable regions of the other chain, are involved in the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). the HVR and FR sequences generally appear in the following order in the VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. The constant domains are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity (ADCC).

The term "Kabat numbering of amino acid residues in a variable domain" or "Kabat numbering of an amino acid position" and variations thereof refers to the numbering system used for heavy chain variable domains or light chain variable domains when compiling antibodies according to Kabat et al., previously. Using this numbering system, the actual linear amino acid sequence may contain fewer or more amino acids, corresponding to shortening or insertions in the FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insertion (residue 52a according to Kabat) after residue 52 in H2 and inserted amino acid residues (e.g., residues 82a, 82b and 82c, etc. according to Kabat) after the FR amino acid residue 82 of the heavy chain . Kabat residue numbering can be determined for a given antibody by aligning homologous regions of the antibody sequence with a "standard" Kabat numbered sequence.

The Kabat numbering system is generally used when considering the amino acid residue of a variable domain (approximately amino acid residues 1-107 of the light chain and amino acid residues 1-113 of the heavy chain) (for example, see Kabat et al., Sequences of Immunological Interest. 5th Ed .Public Health Service, National Institutes of Health, Bethesda, Md. (1991, which is incorporated herein by reference in its entirety). The "EU numbering system" or "EU index" is generally used when referring to an amino acid residue in an immunoglobulin heavy chain constant region (eg, the EU index presented in Kabat et al., previously). "Kabat EU index" refers to the numbering of amino acid residues of the human UgG1 EU antibody. Unless otherwise indicated, references to residue numbers in the variable domain of antibodies refer to the numbering of residues according to the Kabat numbering system. Unless otherwise indicated, references to residue numbers in the constant region antibodies means the numbering of residues according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

The term "isolated antibody", when used to describe the various antibodies described herein, refers to an antibody that has been identified and isolated and/or isolated from a cell or cell culture in which it is expressed. Environmental contaminants are substances that can typically interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse chromatography). -phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J, Chromatogr, B 848:79-87 (2007). In preferred embodiments, the antibody will be purified (1) to a degree sufficient to obtain at least 15 residues on the N-terminal or internal amino acid sequence by using a rotating cup sequencer or (2) to homogeneity obtained by non-reducing SDS-PAGE. or under reducing conditions using Coomassie blue or preferably silver staining. An isolated antibody includes antibodies produced in situ within recombinant cells, since at least one component from the polypeptide's natural environment will be missing. At the same time, the isolated polypeptide will typically be obtained using at least one purification step.

As used herein, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical and/or bind the same epitope, with the exception of possible variant antibodies, for example, containing mutations of natural origin or arising during the preparation of the preparation of monoclonal antibodies, while such variants are generally present in a small amount. Unlike polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single antigen determinant. Thus, the term "monoclonal" indicates the characterization of an antibody derived from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced in any particular manner. For example, monoclonal antibodies for use in the present invention may be prepared by a variety of methods including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods using transgenic animals containing wholly or partly human immunoglobulin loci, such methods and other illustrative methods for obtaining monoclonal antibodies are described in this document.

The term "multispecific antibody" is used in its broadest sense and specifically encompasses an antibody containing a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH-VL unit has polyepitope specificity (i.e., is able to bind to two different epitopes on the same biological molecule or each epitope on different biological molecules). Such multispecific antibodies include, but are not limited to, full length antibodies, antibodies having two or more VL and VH domains, antibody fragments such as Fab, Fab', Fab-C. Fv, dsFv, scFv, diabodies, bispecific diabodies and tribodies, antibody fragments that are covalently or non-covalently linked. "Polyepitope specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). "Double specificity" or "bispecificity" refers to the ability to specifically bind to two different epitopes on the same or different target(s). However, unlike bispecific antibodies, dual specific antibodies have two antigen-binding arms that are identical in amino acid sequence and each Fab arm is capable of recognizing two antigens. Dual specificity allows antibodies to interact with high affinity with two different antigens as a single Fab or IgG molecule. In one embodiment, the multispecific IgG1 antibody binds to each epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM, or 0 .1 µM to 0.001 pM. The term "monospecific" refers to the ability to bind only one epitope.

The term "native antibodies" refers to natural immunoglobulin molecules having different structures. For example, native IgG antibodies are heterotetrameric glycoproteins with a molecular weight of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains connected by disulfide bonds. In the N- to C-terminal direction, each heavy chain has a variable region (VH), also referred to as a heavy chain variable or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, in the N- to C-terminal direction, each light chain has a variable region (VL), which is also called a variable light domain or a light chain variable domain, followed by a light chain constant domain (CL). Depending on the amino acid sequence of its constant domain, an antibody light chain can be classified into one of two types, called kappa (κ) and lambda (λ).

In relation to the binding of an antibody to a target molecule, the term "specific binding" or "specifically bind to" or "specific to" a particular polypeptide or epitope on a particular target polypeptide means a binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule. For example, specific binding may be determined by competition with a control molecule that is similar to the target, such as in an excess of unlabeled target. In this case, specific binding demonstrates whether binding of the labeled target to the probe can be completely inhibited by an excess of unlabeled target. The term "specific binding" or "specifically binds to" or is "specific to" a particular polypeptide or epitope on a particular polypeptide target, as used herein, can be illustrated, for example, by a molecule having a Kd for the target of 10 ^-4 M alternatively 10 ^-5 M or less alternatively 10 ^-6 M or less alternatively 10 ^-7 M or less alternatively 10 ^-8 M or less alternatively 10 ^-3 M or less alternatively 10 ^-10 M or less , alternatively 10 ^-11 M or less, alternatively 10 ^-12 M or less, or Kd in the range of 10 ^-4 M to 10 ^-6 M, or 10 ^-6 M to 10 ^-10 M, or 10 ^-7 M up to 10 ^-9 M. As will be clear to a person skilled in the art, the values of affinity and Kd are inversely related. High affinity for an antigen is measured by a low Kd value. In one embodiment of the invention, the term "specific binding" refers to binding in which the molecule binds to a particular polypeptide or epitope on a particular polypeptide without significant binding to any other polypeptide or polypeptide epitope.

The expression "nucleic acid encoding an anti-VEGF antibody" refers to one or more nucleic acid molecules encoding heavy and light chains of an antibody (or fragments thereof), including nucleic acid molecules(s) in a single vector or separate vectors and molecules nucleic acid(s) present at one or more locations in the host cell.

The term "vector" in the context of this document refers to a nucleic acid molecule capable of reproducing another nucleic acid associated with it. The term includes a vector as a self-replicating nucleic acid structure, as well as a vector that is incorporated into the genome of the host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and their progeny, regardless of the number of passages. The progeny may not be completely identical to the original cell in terms of nucleic acid content, and may contain mutations. This document includes mutant progeny that have the same function or biological activity that is the subject of research or selection in the originally transformed cell.

"Percentage (%) Amino Acid Sequence Identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference polypeptide sequence, after sequence alignment and spacing, if necessary, to achieve maximum sequence identity. , but without taking into account any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be performed in a variety of ways that are known to those skilled in the art, for example using publicly available computer programs such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine parameters suitable for sequence alignment, including any algorithms necessary to achieve maximum full length alignment of compared sequences. However, for the purposes of this invention, % amino acid sequence identity values are generated using the ALIGN-2 sequence comparison computer program. The ALIGN-2 sequence alignment computer program was developed by Genentech, Inc., and the source program was filed along with user documentation with the US Copyright Office, Washington, DC 20559, where it is registered under US copyright registration number TXU510087. The ALIGN-2 program is freely available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. For use on a UNIX operating system that includes UNIX Digital Version V4.0D, the ALIGN-2 program must be compiled. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.

Where ALIGN-2 is used to compare amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A to, c, or with respect to a given amino acid sequence B (which may alternatively be phrased as a given amino acid sequence A that has or contains a specific % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 times the X/Y ratio, where X is the number of amino acid residues evaluated by the ALIGN-2 sequence alignment program as identical matches when programmatic alignment of A and B, and where Y represents the total number of amino acid residues in B. It should be understood that where the length of the amino acid sequence of A does not equal the length of the amino acid sequence of B, the % amino acid sequence identity The ratio of A to B will not equal % Amino Acid Sequence Identity B to A. Unless specifically stated otherwise, all % Amino Acid Sequence Identity values used herein are generated as described in the previous paragraph using the ALIGN-2 computer program.

As used herein, the term "administration" refers to a method of administering to a subject a dose of a compound (e.g., an antibody (e.g., an anti-VEGF antibody engineered by cysteine injection) or an antibody conjugate (e.g., a monodisperse HA conjugate) of the present invention) or a composition (e.g., pharmaceutical composition, for example, a pharmaceutical composition containing an antibody or an antibody conjugate of the invention). Compositions used in the methods described herein can be administered, for example, intravitreally (for example, by injection into the vitreous body), as eye drops, intramuscularly, intravenously, intradermally, transdermally, intraarterially, intraperitoneally, intralesionly, intracranially, intraarticularly, orally prostate, intrapleural, intratracheal, intrathecal, intranasal, intravaginal, rectal, topical, intratumoral, peritoneal, subcutaneous, subconjunctival, intravesicular, mucosal, intrapericardial, intraumbilical, intraocular, intraorbital, oral, topical, transdermal, by inhalation, by injection, by implantation , by infusion, by continuous infusion, by localized perfusion directly through target cells, by catheter, by lavage, in creams or in lipid formulations. The compositions used in the methods described herein can also be administered systemically or locally. The route of administration may vary depending on various factors (eg, the compound or composition to be administered, the severity of the condition, disease, or disorder being treated).

"Angiogenesis" refers to the process by which new blood vessels are formed from pre-existing blood vessels. Angiogenesis is distinct from vasculogenesis, which is the de novo formation of endothelial cells from mesodermal precursor cells. Diseases associated with abnormal angiogenesis can be treated with the compositions and methods of the invention. Examples of disorders associated with abnormal angiogenesis include, but are not limited to, eye diseases (non-limiting eye diseases include, for example, retinopathies, including proliferative diabetic retinopathy, choroidal neovascularization (CNV), age-related macular degeneration (AMD), diabetic and other retinopathies, associated with ischemia, diabetic macular edema (DMA), pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, retinal vein occlusion (including forms of central retinal vein occlusion (RCVO) and branches of the central retinal vein (RVO)), corneal neovascularization, retinal neovascularization, retinopathy of prematurity (RP), familial exudative vitreoretinopathy (FEVR), Coats' disease, Norrie's disease, osteoporosis-pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, rubeosis, neovascular eye disease, neovascular glaucoma, hypertensive retinopathy). Additional eye diseases are described below.

The term "eye disease" as used herein includes any eye disease (also referred to herein interchangeably as a "pathological eye condition") associated with pathological angiogenesis. Eye disease may be characterized by altered or unregulated proliferation and/or invasion of new blood vessels into ocular tissue structures such as the retina or cornea. Non-limiting eye diseases include, for example, AMD (eg, wet AMD, dry AMD, intermediate AMD, advanced AMD, and geographic retinal atrophy (GA)), macular degeneration, macular edema, DME (eg, focal DMA). extracentral and diffuse DME involving the central retina), retinopathy, diabetic retinopathy (DR) (eg, proliferative DR (PDR), nonproliferative DR (NPDR), and high altitude DR), other ischemia-related retinopathies, ROP, occlusion retinal vein occlusion (RVO) (e.g., forms of central retinal vein occlusion (CRVO) and branches of the central retinal vein (RVO))), CNV (e.g., myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases, associated with retinal neovascularization/choroidal neovascularization, pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, EVRP, Coats disease, Norrie disease, OPPG, subconjunctival e hemorrhage, rubeosis, neovascular eye disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, macular telangiectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystic macular edema (CM), vasculitis, papilledema, retinitis, conjunctivitis (eg, infectious and non-infectious (eg, allergic) conjunctivitis), Leber's congenital amaurosis (also known as Leber's amaurosis or VAL), uveitis (including infectious and non-infectious uveitis), choroiditis (eg, multifocal choroiditis), histoplasmosis eye, blepharitis, dry eye syndrome, traumatic eye injury, Sjogren's syndrome, and other diseases of the eye, and the disease or disorder is associated with neovascularization, extravasation and/or edema of the retina. Additional exemplary eye diseases include those associated with rubeosis (neovascularization of the anterior chamber angle) and diseases caused by abnormal proliferation of fibrovascular or fibrous tissue, including all forms of proliferative vitreoretinopathy.

Examples of diseases associated with corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, vitamin A deficiency, contact lens wear syndrome, atopic keratitis, superior limbal keratitis, pterygium, keratitis sicca, Sjögren's syndrome, rosacea, phlyctenular conjunctivitis, syphilis, Mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcer, fungal ulcer, herpes simplex infections, shingles, protozoal infections, Kaposi's sarcoma, Moray's ulcer, Terjen's marginal degeneration, marginal keratolysis, rheumatoid arthritis, systemic lupus erythematosus, polyarthritis , trauma, Wegener's sarcoidosis, scleritis, Stevens-Johnson syndrome, radial keratotomy for scarring pemphigoid, and corneal transplant rejection.

Examples of diseases associated with retinal neovascularization/choroidal neovascularization include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoidosis, syphilis, pseudoxanthoma elastica, Paget's disease, vein occlusions, arterial occlusion, carotid atherosclerosis, chronic uveitis/vitreitis, mycobacterial infections, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, retinitis pigmentosa, retinal edema (including macular edema), Eales' disease, Behcet's disease, infection causing retinitis or choroiditis (eg, multifocal choroiditis), presumed histoplasmosis syndrome eye, Best's disease (congenital macular degeneration), myopia, congenital optic pits, Stargardt's disease, parsplanitis, retinal detachment (eg, chronic retinal detachment), hyperviscosity syndromes, toxoplasmosis, trauma, and post-laser complications.

An "angiogenic factor or agent" is a growth factor that stimulates the development of blood vessels, for example, stimulates angiogenesis, endothelial cell growth, blood vessel stability and/or vasculogenesis, etc. For example, angiogenic factors include, but are not limited to, for example, VEGF and members of the VEGF family, PIGF (placental growth factor), TcFR family, fibroblast growth factor (FGF) family, TIE ligands (angiopoietins), ephrins, Del-1, fibroblast growth factors: acidic (cFGF) and basic (bFGF), follistatin, granulocyte colony stimulating factor (G-CSF), hepatocyte growth factor (HGF)/scattering factor (RF), interleukin-8 (IL-8), leptin, midkine , placental growth factor, platelet-derived endothelial cell growth factor (PD-ECGF), platelet-derived growth factor, especially PDGF-BB or PDGFR-beta, pleiotropin (PTN), progranulin, proliferin, transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF), etc. They also include factors that promote wound healing such as growth hormone, insulin-like growth factor I (IGF-1), VIGF, epidermal growth factor (EGF), CTGF (connective tissue growth factor) and its family members, and TGF-alpha. and TGF-beta. See, for example, Klagsbrun and D'Amore, Annu. Rev. Physiol., 53:217-39 (1991); Streit and Detmar, Oncogene, 22:3172-3179 (2003); Ferrara & Alitalo, Nature Medicine 5(12):1359-1364 (1999); Tonini et al., Oncogene, 22:6549-6556 (2003) (eg Table 1 lists known angiogenic factors); and Sato, Int. J.Clin. Oncol. 8:200-206 (2003).

"Anti-angiogenic agent" or "angiogenesis inhibitor" refers to a small molecular weight substance, polynucleotide, polypeptide, isolated protein, recombinant protein, antibody, or conjugates or fusion proteins thereof, which inhibit angiogenesis, vasculogenesis, or unwanted vascular permeability, either directly or indirectly. An anti-angiogenic agent should be understood to include those agents that bind to and block the angiogenic activity of an angiogenic factor or its receptor. For example, an anti-angiogenic agent is an antibody or other angiogenic agent antagonist as defined above, e.g. TsFR (eg, anti-TsFR inhibitors such as GLEEVEC™ (imatinib mesylate)). Anti-angiogenic agents also include native inhibitors of angiogenesis, eg angiostatin, endostatin, etc. See, for example, Klagsbrun and D'Amore, Annu, Rev, Physiol., 53:217-39 (1991); Streit and Detmar, Oncogene, 22:3172-3179 (2003) (eg, Table 3 lists antiangiogenic therapies for malignant melanoma); Ferrara & Alitalo, Nature Medicine 5(12):1359-1364 (1999); Tonini et al., Oncogene, 22:6549-6556 (2003) (eg Table 2 lists known anti-angiogenic factors); and, Sato Int. J. Clin. Oncol., 8:200-206 (2003) (eg Table 1 lists anti-angiogenic agents used in clinical trials).

The term "VEGF antagonist" as used herein refers to a molecule capable of binding to VEGF, reducing expression levels of VEGF or neutralizing, blocking, inhibiting, suppressing, reducing, or interfering with the biological activity of VEGF, including, without limitation, the binding of VEGF to one or more VEGF receptors, VEGF signaling and VEGF-mediated angiogenesis and endothelial cell survival or proliferation. For example, a molecule that is capable of neutralizing, blocking, inhibiting, suppressing, reducing, or interfering with the biological activity of VEGF may exert its effects by binding to one or more VEGF receptors (VEGF) (e.g., VEGF1, VEGF2, VEGF3, membrane-bound VEGF receptor (mbVEGF ) or soluble VEGF receptor (eFESCR)). As VEGF antagonists useful in the methods of this invention, there may be mentioned polypeptides that specifically bind to VEGF, anti-VEGF antibodies and their antigen-binding fragments, receptor molecules and their derivatives that specifically bind to VEGF, thereby preventing its binding to one or more receptor fusion proteins (eg, VEGF-Trap (Regeneron)), and VEGF- ₁₂₁ -gelonin (Peregrine). VEGF antagonists also include antagonistic modifications of VEGF polypeptides, antisense nucleotide oligomers complementary to at least one fragment of a nucleic acid molecule that encodes a VEGF polypeptide; small RNAs complementary to at least one fragment of the nucleic acid molecule that encodes the VEGF polypeptide; ribozymes that target VEGF; peptitela (peptibodies) to VEGF; and VEGF aptamers. VEGF antagonists also include polypeptides that bind to VEGF, anti-VEGF antibodies and antigen-binding fragments and derivatives thereof, which bind to VEGF in a manner that blocks, inhibits, suppresses, reduces, or interferes with the biological activity of VEGF (e.g., VEGF signaling), fused proteins. VEGF antagonists also include non-peptide small molecules that bind to VEGF or VEGF and are capable of blocking, inhibiting, suppressing, reducing or interfering with the biological activity of VEGF. Thus, the term "VEGF activity" specifically includes VEGF-mediated biological activity of VEGF. In some embodiments, the VEGF antagonist reduces or inhibits at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of VEGF expression or biological activity. In some embodiments, VEGF is inhibited by a VEGF-specific antagonist, which is VEGF(8-109), VEGF(1-109), or VEGF- ₁₆₅ .

As used herein, VEGF antagonists may include, but are not limited to, anti-VEGF2 antibodies and related molecules (e.g., ramucirumab, tanibirumab, aflibercept), anti-VEGF1 antibodies and related molecules (e.g., icrucumab, aflibercept (VEGF Trap-Eye; EYLEA®), and ziv-aflibercept (VEGF Trap; ZALTRAP®)) bispecific antibodies to VEGF (e.g. MP-0250, vanucizumab (VEGF-ANG2), and bispecific antibodies described in US 2001/0236388), bispecific antibodies, which include combinations of two anti-VEGF, anti-VEGF1, and anti-VEGF2 arms, anti-VEGF antibodies (eg, bevacizumab, sevacizumab, and ranibizumab), and non-peptide small molecule VEGF antagonists (eg, pazopanib, axitinib, vandetanib, stivarga, cabozaninib, lenvatinib, nindedanib, orantinib, telatinib, dovitinig, cediranib, motosanib, sulfatinib, apatinib, foratinib, famitinib, and tivozanib). Additional VEGF antagonists are described below.

An "effective amount" of an agent, such as a pharmaceutical, refers to an amount effective, at doses and for the required time periods, to achieve the desired therapeutic or prophylactic result.

"Individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human. The "subject" may be a "patient".

A "disorder" is any pathological condition that can be treated with antibodies. For example, mammals that suffer from or need prophylaxis against abnormal angiogenesis (excessive, inappropriate or uncontrolled angiogenesis). The term encompasses chronic and acute disorders or diseases, including those pathological conditions that increase the susceptibility of a mammal to the disorder in question. As used herein, non-limiting examples of disorders to be treated include those associated with abnormal angiogenesis (eg, eye disease).

The term "package leaflet" is used to refer to the instructions that are usually included in commercial packages of therapeutic products for sale, containing information about the indications, uses, doses, intake, combination therapy, contraindications and / or warnings that relate to the use of such therapeutic products. .

The term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, other than the active ingredient, that is non-toxic to the subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term "pharmaceutical formulation" refers to a formulation that is in such a form as to provide the biological activity of the active ingredient (e.g., antibody conjugate) contained therein to be effective, and which does not contain any additional components that are unacceptably toxic. for the subject to which the composition will be introduced.

As used herein, the term "treatment" (and its grammatical variants, e.g., "treat") refers to clinical intervention in an attempt to alter the natural course of a disease in an individual being treated, and may be carried out for prophylaxis or in the course of the clinical manifestation of pathology. Desired effects of treatment include, without limitation, prevention of the onset or recurrence of the disease, partial relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, reduction in the rate of disease progression, improvement or temporary alleviation of the disease state, and remission or improvement in prognosis. In some embodiments, antibody conjugates of the invention or other compositions that contain an antibody conjugate of the invention (eg, a pharmaceutical formulation) are used to delay the development of a disease or slow the progression of a disease.

An "isolated" nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule with which it is normally associated in the natural source of the nucleic acid. An isolated nucleic acid molecule is different from the form or state in which it occurs in nature. Therefore, isolated nucleic acid molecules differ from the nucleic acid molecule found in natural cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that normally express the antibody, where, for example, the nucleic acid molecule is present in a region of the chromosome that differs from its position in natural cells.

The expression "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Promoters, polyadenylation signals, and enhancers are known to be used in eukaryotic cells.

A nucleic acid is "operably linked" when it is placed in a functional dependency on another nucleic acid sequence. For example, a presequence or secretory leader DNA is operably linked to the DNA of a polypeptide if it is expressed as a precursor protein involved in the secretion of that polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of that sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. In general, "operably linked" means that the DNA sequences, when linked, are contiguous and, in the case of a secretory leader, contiguous in the reading phase. However, enhancers do not need to be continuous. Linking is followed by ligation at suitable restriction sites. If such sites are not present, then synthetic oligonucleotide adapters or linkers are used in accordance with a suitable technique.

As used herein, the terms "cell", "cell line", and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary cell of the subject and cultures derived from it, without regard to the number of passages. It should also be understood that all progeny may not be completely identical in DNA content due to intentional or accidental mutations. Included are mutant progeny that have the same function or biological activity that is the subject of research in the originally transformed cell. It will be clear from the context where the various designations are included.

A "variant" or "mutant" from a parent or reference polypeptide (e.g., a reference antibody or its variable domain(s)/HVR) is a polypeptide that (1) has an amino acid sequence different from the parent or reference polypeptide and (2) was derived from a parent or reference polypeptide by either natural or artificial (human-made) mutagenesis. Such variants include, for example, deletions from and/or insertions into and/or substitutions of residues in the amino acid sequence of the polypeptide of interest, referred to herein as "amino acid residue changes". Thus, a variant HVR refers to an HVR containing the sequence of the variant relative to the parent or reference polypeptide sequence (eg, as in the parent antibody or antigen-binding fragment thereof). An amino acid residue change, as used herein, refers to an amino acid other than the amino acid at the corresponding position in the parent or reference polypeptide sequence (eg, as in a reference antibody or fragment thereof). Any combination of deletion, insertion, and substitution may be made to arrive at the final variant or mutant construct, provided that the final construct has the desired functional characteristics. Amino acid changes can also change post-translational processes of the polypeptide, such as changing the number or position of glycosylation sites.

A "wild-type (WT)" or "reference" sequence, or a sequence from a wild-type protein/polypeptide or "reference" protein/polypeptide, such as an HVR or a reference antibody variable domain, may be a reference sequence from which by introducing a mutation, variant polypeptides are obtained. In general, the "wild-type" sequence for a given protein is the sequence that is most abundant in nature. Similarly, the sequence of a "wild-type" gene is the sequence for that gene, which is most commonly found in nature. Mutations can be introduced into a "wild-type" gene (and thus code for a protein) either by natural processes or by technical means. The products of such processes are "variant" or "mutant" forms of the original protein or "wild-type" gene.

The term "isoelectric point (pl)" means the pH at which a molecule (e.g., a protein such as an antibody) carries no net electrical charge, also referred to in the art as "pH(I)" or "IEP.

As used herein, the term "antibody conjugate" is an antibody covalently attached to one or more polymers. Any suitable polymer may be conjugated to the antibody, such as a hydrophilic polymer (eg, hyaluronic acid (HA) or polyethylene glycol (PEG)) or a hydrophobic polymer (eg, polylactide-coglycolide (PLGA)). In specific embodiments, the polymer is HA (also referred to herein as "HA conjugates").

As used herein, the term "polymer" means a molecule that contains repeating structural units (ie, monomers) connected by chemical bonds in a linear, circular, branched, cross-linked or dendrimeric manner, or a combination thereof. The polymer may be synthetic or natural, or a combination thereof. It should be understood that the term "polymer" includes copolymers, which are polymers that contain two or more different monomers. The polymer may also be a homopolymer, which is a polymer that contains only one type of monomer.

The term "polydispersity index (PDI)" refers to a measurement of the breadth of the molecular weight distribution of a polymer. PDI is also referred to in the art as "dispersity index", "polydispersity index", or "degree of dispersion (D)". The PDI of a polymer sample can be calculated using equation (I): D _M =M _w /M _n , where M _w is the weight average molecular weight and M _n is the number average molecular weight. Unless otherwise stated, PDI is calculated according to Equation (I).

A polymer sample can be considered "monodisperse" (also known in the art as homogeneous) or "polydisperse" (also known in the art as heterogeneous). As used herein, the term "monodisperse" with respect to a sample of HA polymer or HA conjugate means that the HA polymer sample or HA conjugate of the sample has a PDI of less than or equal to about 1.1, e.g., about 1.001, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, or about 1.1. For example, a sample of a monodisperse HA polymer or HA conjugate may have a PDI of 1.0 to about 1.1 (e.g., 1 to about 1.1, 1 to about 1.09, 1 to about 1.08, 1 to about 1.07, 1 to about 1.06, 1 to about 1.05, 1 to about 1.04, 1 to about 1.03, 1 to about 1.02, 1 to about 1.01, 1 to about 1.005, about 1.001 to about 1.1, about 1.001 to about 1.1, about 1.001 to about 1.09, about 1.001 to about 1.08, about 1.001 to about 1.07, about 1.001 to about 1.06, about 1.001 to about 1.05, about 1.001 to about 1.04, about 1.001 to about 1.03, about 1.001 to about 1.02, about 1.001 to about 1.01, about 1.001 to about 1.005, about 1.001 to about 1.004, about 1.001 to about 1.003, about 1.001 to about 1.002, about 1.0001 to about 1.1, from about 1.0001 to about 1.09, about 1.0001 to about 1.08, about 1.0001 to about 1.07, about 1.0001 to about 1.06, about 1.0001 to about 1 .05, from about 1.0001 to about 1.04, from about 1.0001 to about 1.03, about 1.0001 to about 1.02, about 1.0001 to about 1.01, about 1.0001 to about 1.005, about 1.0001 to about 1.004, about 1.0001 to about 1.003, about 1.0001 to about 1.002, or about 1.0001 to about 1.005).

In contrast, the term "polydisperse" means that a sample of the HA polymer or HA conjugate has a PDI greater than 1.1, e.g., about 1.3, about 1.4, about 1.5, about 1.6, about 1, 7, about 1.8 or higher. For example, in some embodiments, a polydisperse sample of the HA polymer or HA conjugate has a PDI in the range of about 1.3 to about 2, about 1.4 to about 2, about 1.5 to about 2, about 1.6 to about 2, from about 1.7 to about 2, from about 1.8 to about 2, or from about 1.9 to about 2.

The terms "hyaluronic acid", "hyaluronan" and "HA", which are used interchangeably herein, refer to a polymeric glycosaminoglycan (GAG) that contains repeating disaccharide units of N-acetylglucosamine and glucuronic acid. HA is an anionic, non-sulfated GAG that can be found, for example, in the extracellular matrix (eg, in the vitreous humor of the eye), connective tissue, epithelial and nervous tissue.

The term "polyethylene glycol" or "PEG" as used herein refers to a polyester compound, which is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular weight. The PEG may have the structure H-(O-CH ₂ -CH ₂ ) _n -OH, where n is any suitable integer. The PEG may be a branched PEG, a star PEG, or a comb PEG. The PEG may be, for example, a PEG tetramer, a PEG hexamer, or a PEG octamer.

The term "clearance" as used herein refers to the volume of a substance (e.g., anti-VEGF antibody, antibody conjugate, fusion protein (e.g., Fab fusion protein), or polymeric drug) that is cleared from a compartment (e.g., eye ( for example, the vitreous body)) per unit of time.

The term "half-life" refers to the time required to halve the concentration of a substance (e.g., anti-VEGF antibody, antibody conjugate, fusion protein (e.g., Fab fusion protein), or polymeric drug), in vivo (e.g., in the eye ( e.g. vitreous body)) or in vitro.

II. COMPOSITIONS AND METHODS

The invention provides antibody conjugates that include polymers (e.g., monodisperse HA polymers) covalently linked to antibodies (e.g., anti-VEGF antibodies, including the anti-VEGF antibody described herein), cysteine-engineered antibodies that can be used , for example, in the preparation of antibody conjugates, compositions that contain antibody conjugates (eg, pharmaceutical compositions), as well as methods for their preparation and use, for example, for therapeutic use (eg, for the treatment of eye diseases).

A. Exemplary Antibodies for Use in Conjugates of the Invention

The invention provides antibody conjugates that include antibodies (eg, anti-VEGF antibodies) covalently linked to polymers (eg, monodisperse polymers). Any suitable antibody may be used (eg, anti-VEGF antibodies). For example, an antibody can specifically bind to an antigen selected from the group consisting of VEGF; interleukin-1 beta (IL-1β); interleukin-6 (IL-6); interleukin-6 receptor (IL-6R); interleukin-13 (IL-13); IL-13 receptor (IL-13R); TsFR (for example, PDGF-BB); angiopoietin; angiopoietin-2 (Ang2); Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin; apelina/APJ; erythropoietin; complement factor D; TNF-α; HtrA1; a VEGF receptor (for example, VEGF1, VEGF2, VEGF3, membrane-bound VEGF receptor (mbVESR), or soluble VEGF receptor (eVEGF)); ST-2 receptor; and a protein genetically associated with the risk of age-related macular degeneration (AMD) (eg, components of the C2 complement pathway, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; interleukin-8 ( IL-8); CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; and TNFRSF10A). Such antibodies may be useful, for example, to reduce angiogenesis and/or to treat or slow the progression of a disorder associated with abnormal angiogenesis (eg, eye disease). Illustrative, non-limiting anti-VEGF antibodies that can be used in antibody conjugates of the invention are described below.

In some instances, the anti-VEGF antibody may comprise at least one, two, three, four, five, or six HVRs selected from: (a) an HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GX ₁ TPX ₂ GGX ₃ X ₄ X ₅ YX ₆ DSVX ₇ X ₈ (SEQ ID NO: 2), where X ₁ is IIe or His, X ₂ is Ala or Arg , X ₃ is Tyr or Lys, X ₄ is Thr or Glu, X ₅ is Arg, Tyr, GIn, or Glu, X ₆ is Ala or Glu, X ₇ is Lys or Glu, and X ₈ is is Gly or Glu; (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQX- ₁ VSTAVA (SEQ ID NO: 4), where X ₁ represents Asp or Arg; (e) HVR-L2 containing the amino acid sequence X ₁ ASFLYS (SEQ ID NO: 5), where X ₁ represents Ser or Met; and (f) an HVR-L3 containing the amino acid sequence X ₁ QGYGX ₂ PFT (SEQ ID NO: 6), where X ₁ is GIn, Asn, or Thr and X ₂ is Ala, Asn, Gin, or Arg, or a combination of one or more of the above HVRs and one or more variants thereof having at least about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) with any of SEQ ID NOs: 1-6.

For example, an anti-VEGF antibody may comprise at least one, two, three, four, five, or six HVRs selected from: (a) an HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYTRYADSVKG (SEQ ID NO: 7), GITPAGGYEYYADSVKG (SEQ ID NO: 21), or GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) an HVR-L3 comprising the amino acid sequence QQGYGAPFT (SEQ ID NO: 10) or QQGYGNPFT (SEQ ID NO: 23), or a combination of one or more of the above HVRs and one or more variants thereof, having at least about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98% or 99% identity) with any of SEQ ID NOs: 1, 3, 7-10, or 21-23.

For example, in some instances, the anti-VEGF antibody may comprise at least one, two, three, four, five, or six HVRs selected from: (a) an HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYTRYADSVKG (SEQ ID NO: 7); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) an HVR-L3 comprising the amino acid sequence QQGYGAPFT (SEQ ID NO: 10), or a combination of one or more of the above HVRs and one or more variants thereof, having at least about 80% sequence identity (e.g., 81% , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% identity) with any of SEQ ID NOs: 1, 3, or 7-10. In a specific example, in some cases, the anti-VEGF antibody contains the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYTRYADSVKG (SEQ ID NO: 7); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGAPFT (SEQ ID NO: 10).

In some cases, any of the prior anti-VEGF antibodies may contain one, two, three, or four of the following heavy chain variable domain (FR) framework regions: (a) FR-H1 containing the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTIS (SEQ ID NO: 13) ; (b) FR-H2 containing the amino acid sequence WVRQAPGKGLEWVA (SEQ ID NO: 14); (c) FR-H3 containing the amino acid sequence RFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO: 15); and (d) FR-H4 containing the amino acid sequence WGQGTLVTVSS (SEQ ID NO: 16).

In some instances, any of the prior anti-VEGF antibodies may contain one, two, three, or four of the following light chain variable domain (FR) framework regions: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17) ; (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20).

For example, in some cases, the anti-VEGF antibody contains the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYTRYADSVKG (SEQ ID NO: 7); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGAPFT (SEQ ID NO: 10). In some cases, the anti-VEGF antibody contains the following heavy chain variable domain FRs: (a) FR-H1 containing the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTIS (SEQ ID NO: 13); (b) FR-H2 containing the amino acid sequence WVRQAPGKGLEWVA (SEQ ID NO: 14); (c) FR-H3 containing the amino acid sequence RFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO: 15); and (d) FR-H4 containing the amino acid sequence WGQGTLVTVSS (SEQ ID NO: 16). In additional instances, the anti-VEGF antibody contains the following light chain variable domain FRs: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20). In some cases, the anti-VEGF antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 11, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 12.

For example, in some instances, the anti-VEGF antibody may comprise at least one, two, three, four, five, or six HVRs selected from: (a) an HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) an HVR-L3 comprising the amino acid sequence QQGYGNPFT (SEQ ID NO: 23), or a combination of one or more of the above HVRs and one or more variants thereof, having at least about 80% sequence identity (e.g., 81% , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% identity) with any of SEQ ID NOs: 1, 3, 8, 9, 22, or 23. In a specific example, in some cases, the anti-VEGF antibody contains the following six HVRs: (a) HVR-H1, containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGNPFT (SEQ ID NO: 23).

In some instances, any of the prior anti-VEGF antibodies may contain one, two, three, or four of the following heavy chain variable domain (FR) framework regions: (a) FR-H1 containing the amino acid sequence EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29) or EEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 52); (b) FR-H2 containing the amino acid sequence WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) FR-H3 containing the amino acid sequence RFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) FR-H4 containing the amino acid sequence WGQGELVTVSS (SEQ ID NO: 32).

In some instances, any of the prior anti-VEGF antibodies may contain one, two, three, or four of the following light chain variable domain (FR) framework regions: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17) ; (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 24); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20).

For example, in some cases, the anti-VEGF antibody contains the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGNPFT (SEQ ID NO: 23). In some cases, the anti-VEGF antibody contains the following heavy chain variable domain FRs: (a) FR-H1 containing the amino acid sequence EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29); (b) FR-H2 containing the amino acid sequence WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) FR-H3 containing the amino acid sequence RFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) FR-H4 containing the amino acid sequence WGQGELVTVSS (SEQ ID NO: 32). In additional instances, the anti-VEGF antibody contains the following light chain variable domain FRs: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 24); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20). In some cases, the anti-VEGF antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 33, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 38.

In some cases, the anti-VEGF antibody contains the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGNPFT (SEQ ID NO: 23). In some cases, the anti-VEGF antibody contains the following heavy chain variable domain FRs: (a) FR-H1 containing the amino acid sequence EEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 52); (b) FR-H2 containing the amino acid sequence WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) FR-H3 containing the amino acid sequence RFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) FR-H4 containing the amino acid sequence WGQGELVTVSS (SEQ ID NO: 32). In additional instances, the anti-VEGF antibody contains the following light chain variable domain FRs: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 24); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20). In some cases, the anti-VEGF antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 51, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 38.

For example, in some instances, the anti-VEGF antibody may comprise at least one, two, three, four, five, or six HVRs selected from: (a) an HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) an HVR-L3 comprising the amino acid sequence QQGYGAPFT (SEQ ID NO: 10), or a combination of one or more of the above HVRs and one or more variants thereof, having at least about 80% sequence identity (e.g., 81% , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% identity) with any of SEQ ID NOs: 1, 3, 8-10, or 22. In a specific example, in some cases, the anti-VEGF antibody contains the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGAPFT (SEQ ID NO: 10).

In some instances, any of the prior anti-VEGF antibodies may contain one, two, three, or four of the following heavy chain variable domain (FR) framework regions: (a) FR-H1 containing the amino acid sequence EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29) or EEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 52); (b) FR-H2 containing the amino acid sequence WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) FR-H3 containing the amino acid sequence RFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) FR-H4 containing the amino acid sequence WGQGELVTVSS (SEQ ID NO: 32).

In some instances, any of the prior anti-VEGF antibodies may contain one, two, three, or four of the following light chain variable domain (FR) framework regions: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17) , DIQMTQSPESLSASVGDEVTITC (SEQ ID NO: 25), or DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18) or WYQQKPGEAPKLLIY (SEQ ID NO: 27); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19) or GVPSRFSGSGSGTDFTLTIESLQPEDAATYYC (SEQ ID NO: 28); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20).

For example, in some cases, the anti-VEGF antibody contains the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGAPFT (SEQ ID NO: 10). In some cases, the anti-VEGF antibody contains the following heavy chain variable domain FRs: (a) FR-H1 containing the amino acid sequence EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29); (b) FR-H2 containing the amino acid sequence WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) FR-H3 containing the amino acid sequence RFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) FR-H4 containing the amino acid sequence WGQGELVTVSS (SEQ ID NO: 32). In additional cases, the anti-VEGF antibody contains the following light chain variable domain FRs: (a) FR-L1 containing the amino acid sequence DIQMTQSPESLSASVGDEVTITC (SEQ ID NO: 25); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20). In some cases, the anti-VEGF antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 33, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 34.

For example, in other cases, the anti-VEGF antibody contains the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGAPFT (SEQ ID NO: 10). In some cases, the anti-VEGF antibody contains the following heavy chain variable domain FRs: (a) FR-H1 containing the amino acid sequence EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29); (b) FR-H2 containing the amino acid sequence WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) FR-H3 containing the amino acid sequence RFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) FR-H4 containing the amino acid sequence WGQGELVTVSS (SEQ ID NO: 32). In additional instances, the anti-VEGF antibody contains the following light chain variable domain FRs: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) FR-L2 containing the amino acid sequence WYQQKPGEAPKLLIY (SEQ ID NO: 27); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTIESLQPEDAATYYC (SEQ ID NO: 28); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20).

In some cases, the anti-VEGF antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 33, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 35.

For example, in other cases, the anti-VEGF antibody contains the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGAPFT (SEQ ID NO: 10). In some cases, the anti-VEGF antibody contains the following heavy chain variable domain FRs: (a) FR-H1 containing the amino acid sequence EEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 52); (b) FR-H2 containing the amino acid sequence WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) FR-H3 containing the amino acid sequence RFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) FR-H4 containing the amino acid sequence WGQGELVTVSS (SEQ ID NO: 32). In additional instances, the anti-VEGF antibody contains the following light chain variable domain FRs: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) FR-L2 containing the amino acid sequence WYQQKPGEAPKLLIY (SEQ ID NO: 27); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTIESLQPEDAATYYC (SEQ ID NO: 28); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20). In some cases, the anti-VEGF antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 51, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 35.

For example, in still other cases, the anti-VEGF antibody contains the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGAPFT (SEQ ID NO: 10). In some cases, the anti-VEGF antibody contains the following heavy chain variable domain FRs: (a) FR-H1 containing the amino acid sequence EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29); (b) FR-H2 containing the amino acid sequence WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) FR-H3 containing the amino acid sequence RFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) FR-H4 containing the amino acid sequence WGQGELVTVSS (SEQ ID NO: 32). In additional cases, the anti-VEGF antibody contains the following light chain variable domain FRs: (a) FR-L1 containing the amino acid sequence DIQMTQSPESLSASVGDEVTITC (SEQ ID NO: 25); (b) FR-L2 containing the amino acid sequence WYQQKPGEAPKLLIY (SEQ ID NO: 27); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20). In some cases, the anti-VEGF antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 33, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 36.

For example, in yet additional cases, the anti-VEGF antibody contains the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGAPFT (SEQ ID NO: 10). In some cases, the anti-VEGF antibody contains the following heavy chain variable domain FRs: (a) FR-H1 containing the amino acid sequence EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29); (b) FR-H2 containing the amino acid sequence WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) FR-H3 containing the amino acid sequence RFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) FR-H4 containing the amino acid sequence WGQGELVTVSS (SEQ ID NO: 32). In additional cases, the anti-VEGF antibody contains the following light chain variable domain FRs: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20). In some cases, the anti-VEGF antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 33, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 37.

In some cases, the anti-VEGF antibody contains the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGAPFT (SEQ ID NO: 10). In some cases, the anti-VEGF antibody contains the following heavy chain variable domain FRs: (a) FR-H1 containing the amino acid sequence EEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 52); (b) FR-H2 containing the amino acid sequence WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) FR-H3 containing the amino acid sequence RFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) FR-H4 containing the amino acid sequence WGQGELVTVSS (SEQ ID NO: 32). In additional cases, the anti-VEGF antibody contains the following light chain variable domain FRs: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20). In some cases, the anti-VEGF antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 51, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 37.

For example, in other cases, the anti-VEGF antibody contains the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGAPFT (SEQ ID NO: 10). In some cases, the anti-VEGF antibody contains the following heavy chain variable domain FRs: (a) FR-H1 containing the amino acid sequence EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29); (b) FR-H2 containing the amino acid sequence WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) FR-H3 containing the amino acid sequence RFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) FR-H4 containing the amino acid sequence WGQGELVTVSS (SEQ ID NO: 32). In additional instances, the anti-VEGF antibody contains the following light chain variable domain FRs: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20). In some cases, the anti-VEGF antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 33, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 12.

In some cases, the anti-VEGF antibody contains the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGAPFT (SEQ ID NO: 10). In some cases, the anti-VEGF antibody contains the following heavy chain variable domain FRs: (a) FR-H1 containing the amino acid sequence EEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 52); (b) FR-H2 containing the amino acid sequence WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) FR-H3 containing the amino acid sequence RFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) FR-H4 containing the amino acid sequence WGQGELVTVSS (SEQ ID NO: 32). In additional instances, the anti-VEGF antibody contains the following light chain variable domain FRs: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20). In some cases, the anti-VEGF antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 51, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 12.

In some cases, the anti-VEGF antibody contains (a) a heavy chain variable domain (VH) containing an amino acid sequence having at least 90% sequence identity with (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity), or the sequence of any of SEQ ID NO: 11, 40 or 42; (b) a light chain variable domain (VL) comprising an amino acid sequence having at least 90% sequence identity with (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, or 99% sequence identity), or the sequence of any of SEQ ID NO: 12, 41, or 46; or (c) a VH domain as in (a) and a VL domain as in (b). For example, in some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 11 and a VL domain containing the amino acid sequence of SEQ ID NO: 12. In some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 40, and a VL domain containing the amino acid sequence of SEQ ID NO: 12. In some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 42, and a VL domain containing the amino acid sequence of SEQ ID NO: 12. In some cases, the antibody contains a VH domain, containing the amino acid sequence of SEQ ID NO: 42, and a VL domain containing the amino acid sequence of SEQ ID NO: 41. In some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 11, and a VL domain containing the amino acid sequence of SEQ ID NO: 46.

In some instances, any of the prior anti-VEGF antibodies may contain one, two, three, or four of the following heavy chain variable domain (FR) framework regions: (a) FR-H1 containing the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTIS (SEQ ID NO: 13) ; (b) FR-H2 containing the amino acid sequence WVRQAPGKGLEWVA (SEQ ID NO: 14) or WVRQEPGKGLEWVA (SEQ ID NO: 39); (c) FR-H3 containing the amino acid sequence RFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO: 15); and (d) FR-H4 containing the amino acid sequence WGQGTLVTVSS (SEQ ID NO: 16).

In some instances, any of the prior anti-VEGF antibodies may contain one, two, three, or four of the following light chain variable domain (FR) framework regions: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17) or DIQMTQSPSSLSASVGDRVTIDC (SEQ ID NO: 45); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19), GVPSRFSGSGSGTDFTLTISSLQPEDSATYYC (SEQ ID NO: 44), or GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO: 54); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20) or FGQGTKVEVK (SEQ ID NO: 55).

In some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 11, and a VL domain containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGNPFTFGQGTKVEIK (SEQ ID NO: 59).

In some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 33, and a VL domain containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGNPFTFGQGTKVEIK (SEQ ID NO: 59).

In some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 40 and a VL domain containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGNPFTFGQGTKVEIK (SEQ ID NO: 59).

In some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 42, and a VL domain containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGNPFTFGQGTKVEIK (SEQ ID NO: 59).

In some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 51 and a VL domain containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGNPFTFGQGTKVEIK (SEQ ID NO: 59).

In some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 11 and a VL domain containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGAPFTFGQGTKVEIK (SEQ ID NO: 60).

In some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 33, and a VL domain containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGTDFTLTISSLQPEDVATYYCQQGYGAPFTFGQGTKVEIK (SEQ ID NO: 60).

In some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 40 and a VL domain containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGAPFTFGQGTKVEIK (SEQ ID NO: 60).

In some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 42, and a VL domain containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGAPFTFGQGTKVEIK (SEQ ID NO: 60).

In some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 51, and a VL domain containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGAPFTFGQGTKVEIK (SEQ ID NO: 60). For example, in some instances, an anti-VEGF antibody may comprise (a) a VH domain containing an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) with, or sequence, SEQ ID NO: 11; (b) a VL domain containing an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 % sequence identity) c, or sequence, SEQ ID NO: 11; or (c) a VH domain as in (a) and a VL domain as in (b). In some cases, the anti-VEGF antibody may comprise (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYTRYADSVKG (SEQ ID NO: 7); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGAPFT (SEQ ID NO: 10). In some cases, the anti-VEGF antibody contains the following heavy chain framework regions: (a) FR-H1 containing the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTIS (SEQ ID NO: 13); (b) FR-H2 containing the amino acid sequence WVRQAPGKGLEWVA (SEQ ID NO: 14); (c) FR-H3 containing the amino acid sequence RFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO: 15); and (d) FR-H4 containing the amino acid sequence WGQGTLVTVSS (SEQ ID NO: 16). In some cases, the anti-VEGF antibody contains the following light chain framework regions: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20). In some cases, the anti-VEGF antibody contains a binding domain containing (a) a VH domain containing the amino acid sequence of SEQ ID NO: 11, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 12. In some cases, an exemplary anti-VEGF is N94A.F83A.N82aR.Y58R (also referred to as G6.31AARR or G6.31.AARR).

In some instances, the anti-VEGF antibody may comprise (a) a VH domain containing an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, or 99% sequence identity) with, or sequence, SEQ ID NO: 33 or 51; (b) a VL domain containing an amino acid sequence having at least 90% sequence identity with (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity), or the sequence of any of SEQ ID NO: 12, 34, 35, 36, 37, or 38; or (c) a VH domain as in (a) and a VL domain as in (b). For example, in some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 33 and a VL domain containing the amino acid sequence of SEQ ID NO: 12. In some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 33, and a VL domain containing the amino acid sequence of SEQ ID NO: 34. In some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 33, and a VL domain containing the amino acid sequence of SEQ ID NO: 35. In some cases, the antibody contains a VH domain, containing the amino acid sequence of SEQ ID NO: 33, and a VL domain containing the amino acid sequence of SEQ ID NO: 36. In some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 33, and a VL domain containing the amino acid sequence of SEQ ID NO: 37. In some cases, the antibody contains a VH domain containing the amino acid sequence be SEQ ID NO: 33, and a VL domain containing the amino acid sequence of SEQ ID NO: 38. In some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 51 and a VL domain containing the amino acid sequence of SEQ ID NO: 38. In some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 51 and a VL domain containing the amino acid sequence of SEQ ID NO: 35. In some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 51 and a VL domain , containing the amino acid sequence of SEQ ID NO: 37. In some cases, the antibody contains a VH domain containing the amino acid sequence of SEQ ID NO: 51 and a VL domain containing the amino acid sequence of SEQ ID NO: 12.

In some instances, any of the prior anti-VEGF antibodies may contain one, two, three, or four of the following heavy chain variable domain (FR) framework regions: FR-H1 containing the amino acid sequence EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29) or EEQLVEEGGGLVQPGESLRLSCAASGFEIS ( SEQ ID NO: 52); (b) FR-H2 containing the amino acid sequence WVRQEPGEGLEWVA (SEQ ID NO: 30) or WVRQEPGKGLEWVA (SEQ ID NO: 39); (c) FR-H3 containing the amino acid sequence RFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) FR-H4 containing the amino acid sequence WGQGELVTVSS (SEQ ID NO: 32).

In some cases, any of the prior anti-VEGF antibodies may contain one, two, three, or four of the following light chain variable domain (FR) framework regions: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17) , DIQMTQSPESLSASVGDEVTITC (SEQ ID NO: 25), or DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18) or WYQQKPGEAPKLLIY (SEQ ID NO: 27); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19), GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 24), or GVPSRFSGSGSGTDFTLTIESLQPEDAATYYC (SEQ ID NO: 28); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20).

In some instances, the invention provides an antibody comprising (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 48 and/or (b) a light chain comprising the amino acid sequence of SEQ ID NO: 50. In some embodiments, the antibody is G6.31 AARR expressed in Fab format.

In some instances, the invention provides an antibody comprising (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49 and/or (b) a light chain comprising the amino acid sequence of SEQ ID NO: 50. In some embodiments, the antibody is a variant version of G6 .31 AARR that lacks reactivity to anti-human IgG.

In a further aspect, an antibody (e.g., an anti-VEGF antibody) according to any of the above embodiments may include any of the features, alone or in combination, as described in sections 1-8 below:

1. Antibody affinity

In some embodiments, an antibody provided herein has a dissociation constant (Kd) ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM ( eg 10 -8 M or less, eg 10 ^-8 M to 10 ^-13 M, eg 10 ^-9 M to 10 ^-13 M). For example, in some instances, an antibody provided herein binds an antigen (eg, human VEGF (hVESF)) with a Kd of about 10 nM or less. In some instances, an antibody provided herein binds an antigen (eg, hVEGF) with a Kd of about 5 nM or less. In some instances, an antibody provided herein binds an antigen (eg, human VEGF (hVESF)) with a Kd of about 2 nM or less. For example, in some cases, an antibody binds an antigen (e.g., hVEGF) with a Kd of about 25 pM to about 2 nM (e.g., about 25 pM, about 50 pM, about 75 pM, about 100 pM, about 125 pM, about 150 pM, about 175 pM, about 200 pM, about 225 pM, about 250 pM, about 275 pM, about 300 pM, about 325 pM, about 350 pM, about 375 pM, about 400 pM, about 425 pM, about 450 pM, about 475 pM, about 500 pM, about 525 pM, about 550 pM, about 575 pM, about 600 pM, about 625 pM, about 650 pM, about 675 pM, about 700 pM, about 725 pM, about 750 pM, about 775 pM, about 800 pM, about 825 pM, about 850 pM, about 875 pM, about 900 pM, about 925 pM, about 950 pM, about 975 pM, about 1 nM, about 1.1 nM, about 1.2 nM, about 1 .3 nM, about 1.4 nM, about 1.5 nM, about 1.6 nM, about 1.7 nM, about 1.8 nM, about 1.9 nM, or about 2 nM). In some cases, an antibody binds an antigen (eg, hVEGF) with a Kd of about 75 pM to about 600 pM (eg, about 75 pM, about 100 pM, about 125 pM, about 150 pM, about 175 pM, about 200 pM, about 225 pM, about 250 pM, about 275 pM, about 300 pM, about 325 pM, about 350 pM, about 375 pM, about 400 pM, about 425 pM, about 450 pM, about 475 pM, about 500 pM, about 525 pM, about 550 pM, about 575 pM, about 600 pM). In some instances, an antibody binds an antigen (eg, hVEGF) with a Kd of about 75 pM to about 500 pM. In some instances, an antibody binds an antigen (eg, hVEGF) with a Kd of about 75 pM to about 400 pM. In some instances, an antibody binds an antigen (eg, hVEGF) with a Kd of about 75 pM to about 300 pM. In some instances, an antibody binds an antigen (eg, hVEGF) with a Kd of about 75 pM to about 200 pM. In some instances, an antibody binds an antigen (eg, hVEGF) with a Kd of about 75 pM to about 150 pM. In some cases, an antibody binds an antigen (eg, hVEGF) with a Kd of about 75 pM to about 125 pM. In some instances, an antibody binds an antigen (eg, hVEGF) with a Kd of about 75 pM to about 100 pM. In some cases, an antibody binds an antigen (eg, hVEGF) with a Kd of about 80 pM. In some cases, an antibody binds an antigen (eg, hVEGF) with a Kd of about 60 pM. In some cases, an antibody binds an antigen (eg, hVEGF) with a Kd of about 40 pM.

In one embodiment, Kd is measured using a radiolabelled antigen (RIA) binding assay. In one embodiment, RIA was performed using the Fab version of the antibody of interest and its antigen. For example, the binding affinity of a Fab in solution for an antigen is measured by equilibrating the Fab with a minimum concentration of ( ¹²⁵ I)-labeled antigen in the presence of a series of titers of unlabeled antigen, then immobilizing the bound antigen on an anti-Fab coated plate (see, e.g., Chen et al ., J. Moi, B/o/, 293:865-881 (1999)). To determine assay conditions, MICROTITER® multiwell plates (Thermo Scientific) were coated overnight with anti-Fab capture antibody (Cappel Labs) at 5 µg/mL in 50 µM sodium carbonate solution (pH 9.6) and then blocked with 2% ( w/v) with a solution of bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for two to five hours at room temperature (about 23°C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [ ¹²⁵ I] antigen was mixed with serial dilutions of the target Fab (e.g., as assessed by the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest was then incubated overnight; however, incubation may be continued for a longer period (eg, about 65 hours) to ensure equilibrium is reached. The mixture was then transferred to a capture plate and incubated at room temperature (eg, one hour). The solution is then removed and the plate washed eight times with a 0.1% solution of polysorbate 20 (TWEEN-20®) in PBS. After the plates were dry, 150 μl/well of scintillator (MICROSCINT-20™; Packard) was added and the plates were read on a TOPCOUNT™ gamma counter (Packard) for ten minutes. Concentrations of each Fab that provided less than or equal to 20% of maximum binding were selected for use in a competitive binding assay.

According to another embodiment, Kd is measured by the BIACORE® Surface Plasma Resonance method. For example, assay using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) was performed at 25° C. with immobilized antigen CM5 chips at 10 response units (RU). In one embodiment, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions. The antigen is diluted with 10 μM sodium acetate, pH 4.8, to 5 μg/ml (~0.2 μM) prior to injection at a flow rate of 5 μl/minute to achieve approximately 10 response units (RU) of bound protein. After the introduction of the antigen, a 1 M solution of ethanolamine was introduced to block unreacted groups. For kinetic measurements, two-fold dilution series of Fab (from 0.78 nM to 500 nM) were injected into PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately 25 μl/min. Association (k _on ) and dissociation (k _off ) rates were calculated as simple unambiguous Langmuir binding models (BIACORE® Evaluation Software version 3.2) by simultaneously fitting association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) was calculated as the ratio k _off /k _on . See, for example, Chen et al., J. Mol. In/. 293:865-881 (1999). If the rate exceeds 10 ⁶ M ^-1 s ^-1 from the above surface plasmon resonance method, then the forward reaction rates can be determined using the fluorescence decay method, which measures the increase or decrease in the intensity of fluorescent emission (excitation = 295 nm; emission = 340 nm, 16 nm bandwidth) at 25° C. 20 nM anti-antigen (Fab form) in PBS, at pH 7.2, in the presence of increasing concentrations of antigen, as measured by a spectrometer such as a spectrophotometer with flow stop device (Aviv Instruments) or SLM-AMINCO™ 8000 series spectrometer (ThermoSpectronic) with stirring cell.

2. Antibody stability

In some instances, the antibody used in the antibody conjugates of the invention or compositions thereof has improved stability, for example, compared to an anti-VEGF antibody, for example, G6.31 (see, for example, US Pat. No. 7,758,859 and International Publication No. WO 2005/012359, which are incorporated herein by reference in their entirety). Antibody stability can be determined using any method known in the art, e.g. differential scanning fluorimetry (DSF), circular dichroism (CD), protein intrinsic fluorescence, differential scanning calorimetry, spectroscopy, light scattering (e.g. dynamic light scattering (DLS) and Static Light Scattering (SLS), Interacting Molecule Chromatography (SIC) An anti-VEGF antibody may have, for example, an improved melting point (T _m ), aggregation temperature (T _agg ), or other stability characteristics compared to an anti-VEGF antibody, for example, G6.31.

In some embodiments, an antibody provided herein has a T _m that is greater than or equal to about 80°C (e.g., about 81°C, about 82°C, about 83°C, about 84°C, about 85°C , about 86°C, about 87°C, about 88°C, about 89°C, about 90°C, about 91°C, about 92°C or about 93°C). For example, in some instances, the anti-VEGF antibody has a T _m that is greater than or equal to about 83.5°C (e.g., about 83.5°C, about 84°C, about 85°C, about 86°C, about 87 °C, about 88°C, about 89°C, about 90°C, about 91°C, about 92°C or about 93°C). In some cases, the anti-VEGF antibody has a T _m of about 82°C to about 92°C (e.g., about 82°C, about 83°C, about 84°C, about 85°C, about 86°C, about 87 °C, about 88°C, about 89°C, about 90°C, about 91°C, or from about 92°C). In some cases, the anti-VEGF antibody has a T _m of about 82°C. In some cases, any of the previous T _m values of the anti-VEGF antibody is determined using DSF. In some embodiments, the T _m value of an anti-VEGF antibody is determined as described, for example, in Example 1 of International Patent Application PCT/US2016/053454, which is incorporated herein by reference in its entirety.

3. Antibody fragments

, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); см. также WO 93/16185; патенты США №№5571894 и 5587458. Обсуждение фрагментов Fab и F(ab')₂, содержащих остатки эпитопа связывания рецептора реутилизации и имеющих увеличенное in vivo время полужизни, смотрите в патенте США №5869046.In certain embodiments, an antibody provided herein is an antibody fragment. Antibody fragments include, without limitation, Fab, Fab', Fab-C, Fab'-SH, F(ab') ₂ , Fv, and scFv fragments and other fragments described below. For a review of some antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For an overview of scFv fragments see, for example,

, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; US _Pat .

Diabodies are antibody fragments with two antigen-binding sites, which can be divalent or bispecific. See, for example, EP 404097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. sci. USA 90: 6444-6448 (1993). Triatebodies and tetrabodies are also described in Hudson et al. Nat. Med. 9:129-134 (2003).

Single domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain, or all or part of the light chain variable domain of an antibody. In some embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Massachusetts; see, for example, US Pat. No. 6,248,516 B1).

Antibody fragments can be obtained by various methods, including, without limitation, proteolytic cleavage of intact antibodies, as well as production using recombinant host cells (eg, E. coli or phage), as described in this document.

4. Chimeric and humanized antibodies

In certain embodiments, an antibody provided herein is a chimeric antibody. Some chimeric antibodies are described, for example, in US patent No. 4816567 and Morrison et al., Proc. Natl. Acad. sci. USA 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (eg, a variable domain derived from an antibody from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant domain. In a further example, the chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from the parent antibody. The term "chimeric antibodies" includes antigen-binding fragments of such antibodies.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce human immunogenicity while retaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody contains one or more variable domains in which HVRs, for example, CDRs (or fragments thereof) are derived from a non-human antibody, and FRs (or fragments thereof) are derived from human antibody sequences. In some instances, the humanized antibody also contains at least a human constant region fragment. In some embodiments, certain FR residues of a humanized antibody are replaced with corresponding residues from a non-human antibody (eg, an antibody from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their preparation are discussed, for example, in Almagro and Fransson, Front. biosci. 13:1619-1633 (2008), and are further described in, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. sci. USA 86:10029-10033 (1989); US patents No. 5821337, 7527791, 6982321 and 7087409; Kashmiri et al., Methods 36:25-34 (2005) (which describes grafting a specificity-determining region (SDR)); Padlan, Mol. Immunol. 28:489-498 (1991) (which describes "surface change"); Dall'Acqua et al., Methods 36:43-60 (2005) (which describes "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (which describes a "directed selection" approach to FR shuffling).

Human frameworks that can be used for humanization include, but are not limited to, frameworks selected using a "best fit" method (see, for example, Sims et al., J. Immunol. 151:2296 (1993) ); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. , J. Immunol., 151:2623 (1993)); mature (containing somatic mutations) human frameworks or human embryonic frameworks (see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions resulting from screening of FR libraries (see, for example, Baca et al. J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611- 22618 (1996)).

5. Human antibodies

In certain embodiments, the antibody provided herein is a human antibody. Human antibodies can be obtained using various methods known in the art. Human antibodies are outlined in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

Human antibodies can be generated by administering an immunogen to a transgenic animal modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci that replace endogenous immunoglobulin loci or that are extrachromosomal or randomly integrated into the animal's chromosomes. As a rule, endogenous immunoglobulin loci are inactivated in such transgenic mice. For a review of methods for generating human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, US Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; US patent No. 5770429 describing HUMAB® technology; US Pat. No. 7,041,870 describing K-M MOUSE® technology and US Patent Application Publication No. US 2007/0061900 describing VELOCIMOUSE® technology). Human variable regions from intact antibodies generated by such animals can be further modified, for example by combining with different human conserved regions.

Human antibodies can also be generated using hybridoma-based methods. Cell lines of human myeloma and murine-human heteromyeloma have been described for the production of human monoclonal antibodies. (See, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeuret al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies prepared using human B-cell hybridoma technology are described in Li et al., Proc. Natl. Acad. sci. USA 103:3557-3562 (2006). Additional methods include those described in, for example, US Pat. No. 7,189,826 (describes the production of human IgM monoclonal antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describes human-type hybridomas -human). Technology using human hybridomas (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005).

Human antibodies can also be generated by isolating the Fv clone variable domain sequences from phage display libraries of human origin. Such variable domain sequences can then be combined with the desired human constant domain. Methods for selecting human antibodies from antibody libraries are described below.

6. Antibodies obtained from libraries

Antibodies of the invention can be isolated by screening combinatorial libraries of antibodies with the desired activity or properties. For example, many methods are known in the art for obtaining phage display libraries and screening such libraries for antibodies having the desired binding characteristics. Such methods are discussed, for example, in Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and are further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597, (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mot. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In some phage display methods, VH and VL gene repertoires are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al. Ann. Rev. Immunol., 12: 433-455 (1994). The phage typically presents antibody fragments, both single chain Fv (scFv) fragments and Fab fragments. Libraries from immunized sources allow the production of antibodies with high affinity for the immunogen without the need for hybridoma construction. Alternatively, a naive repertoire (eg, from a human) can be cloned to obtain a single source of antibodies to a wide range of foreign antigens and self-antigens without immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be obtained by synthesis by cloning unrearranged segments of stem cell V genes and using PCR primers containing random sequences to encode the CDR3 hypervariable region and perform rearrangement in vitro, as described in Hoogenboom and Winter. , J. Mol. Biol., 227: 381-388 (1992). Patent publications that describe human antibody phage libraries include, for example: US Patent No. 5,750,373, and US Patent Publications Nos. 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments can be obtained from phage libraries as described in International Patent Application PCT/US2016/053454.

Antibodies or antibody fragments isolated from human antibody libraries are referred to herein as human antibodies or human antibody fragments.

7. Multispecific antibodies

In some embodiments, an antibody provided herein is a multispecific antibody, such as a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In some embodiments, one of the binding specificities for VEGF and others for any other antigen (e.g., a second biological molecule, e.g., interleukin-1 beta (IL-1β); interleukin-6 (IL-6); interleukin-6 receptor (IL -GR); interleukin-13 (IL-13); IL-13 receptor (IL-13R); TcFR (e.g., PDGF-BB); angiopoietin; angiopoietin-2 (Ang2); Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin; apelin/APJ; erythropoietin; complement factor D; TNF-α; HtrA1; VEGF receptor (eg, VEGF1, VEGF2, VEGF3, membrane-bound VEGF receptor (mbVEGF), or soluble VEGF receptor (sVEGF)); ST- 2 receptor, and proteins genetically associated with the risk of age-related macular degeneration (AMD), such as components of the C2 complement pathway, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; interleukin-8 (IL-8); CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; and TNFRSF10A). Accordingly, bispecific antibodies may have binding specificity for VEGF and IL-1β; FRES and IL-6; FRES and IL-6R; FRES and IL-13; FRES and IL-13R; VEGF and TsFR (for example, PDGF-BB); VEGF and angiopoietin; VEGF and Ang2; VEGF and Tie2; VEDF and S1P; VEGF and integrin αvβ3; VEGF and integrin αvβ5; VEGF and integrin α5β1; VEGF and betacellulin; VEGF and apelina/APJ; VEGF and erythropoietin; VEGF and complement factor D; VEGF and TNF-α; VEGF and HtrA1; VEGF and VEGF receptor (eg, VEGF1, VEGF2, VEGF3, mbVEGF, or sVEGF); VEGF and ST-2 receptor; FRES and C2; VEGF and factor B; VEGF and factor H; VEGF and CFHR3; VEGF and C3b; FRES and C5; VEGF and C5a; VEGF and C3a; FRES and ARMS2; VEGF and TIMP3; VEGF and HLA; FRES and IL-8; VEGF and CX3CR1; VEGF and TLR3; VEGF and TLR4; FRES and CETR; FRES and LIPC; VEGF and COL10A1; or VEGF and TNFRSF10A. In some embodiments, the bispecific antibodies can bind to two different VEGF epitopes. Bispecific antibodies can also be used to move cytotoxic agents into cells that express VEGF. Bispecific antibodies can be prepared as full length antibodies or as antibody fragments (eg, Fab, Fab', or Fab-C fragments).

In some instances, the bispecific antibody is the anti-VEGF/anti-angiopoietin 2 (Ang2) bispecific antibody disclosed in US Patent Application No. US 2014/0017244, which is incorporated herein by reference in its entirety. For example, an anti-VEGF/anti-Ang2 bispecific antibody may comprise a first binding domain that binds to VEGF (e.g., any of the anti-VEGF antibodies described herein) and a second binding domain that binds to ANG2, which contains (a) HVR-H1 containing the amino acid sequence GYYMH (SEQ ID NO: 62); (b) HVR-H2 containing the amino acid sequence WINPNSGGTNYAQKFQG (SEQ ID NO: 63); (c) HVR-H3 containing the amino acid sequence SPNPYYYDSSGYYYPGAFDI (SEQ ID NO: 64); (d) HVR-L1 containing the amino acid sequence GGNNIGSKSVH (SEQ ID NO: 65); (e) HVR-L2 containing the amino acid sequence DDSDRPS (SEQ ID: 66); and (f) an HVR-L3 containing the amino acid sequence QVWDSSSSDHWV (SEQ ID NO: 67), or a combination of one or more of the above HVRs and one or more variants thereof, having at least about 80% sequence identity (e.g., 81% , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% identity) with any of SEQ ID NOs: 62-67.

In some cases, an anti-VEGF/anti-Ang2 bispecific antibody may contain a first binding domain that binds VEGF (e.g., any of the anti-VEGF antibodies described herein) and a second binding domain that binds Ang2 and contains ( a) a VH domain containing an amino acid sequence having at least 80% sequence identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) c, or sequence, SEQ ID NO: 68, (b) VL domain, containing an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity) with, or sequence, SEQ ID NO: 69; or (c) a VH domain as in (a) and a VL domain as in (b). In some instances, the anti-VEGF/anti-Ang2 bispecific antibody may contain a first binding domain that binds to VEGF (e.g., any of the anti-VEGF antibodies described herein) and a second binding domain that specifically binds to Ang2, wherein the second binding domain is any antibody-binding domain described in International Patent Application Publication No. WO 2010/069532, which is incorporated herein by reference in its entirety, or a variant thereof.

In other cases, the anti-VEGF/anti-Ang2 bispecific antibody is any anti-VEGF/anti-Ang2 bispecific antibody described in International Patent Application Publication No. WO 2016/073157.

In some instances, the bispecific antibody is an anti-VEGF/anti-IL-6 bispecific antibody. In some cases, an anti-VEGF/anti-IL-6 antibody may contain a first binding domain that binds VEGF (eg, any of the anti-VEGF antibodies described herein) and a second binding domain that binds IL-6. The second binding domain may be the binding domain of any anti-IL-6 antibody known in the art, such as EBI-031 (Eleven Biotherapeutics; see, for example, WO 2016/073890, which is incorporated herein by reference in its entirety). volume), siltuximab (SYLVANT®), olokizumab, clasakizumab, sirukumab, elsilimomab, gerilimzumab, OPR-003, MEDI-5117, PF-04236921, or a variant thereof.

In some instances, the bispecific antibody is an anti-VEGF/anti-IL-6R bispecific antibody. In some instances, an anti-VEGF/anti-IL-6R antibody may contain a first binding domain that binds to VEGF (eg, any of the anti-VEGF antibodies described herein) and a second binding domain that binds to IL-6R. The second binding domain can be any anti-IL-6R antibody binding domain known in the art, such as tocilizumab (ACTEMRA®) (see, for example, WO 1992/019579, which is incorporated herein by reference in its entirety. ), sarilumab, vobarilizumab (ALX-0061), SA-237, or a variant thereof.

Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO 93/08829, and Traunecker et al ., EMBO J. 10: 3655 (1991)), and a ridge-to-bottom design method (see, for example, US Pat. No. 5,731,168). Multispecific antibodies can also be obtained by design using the effect of electrostatic interaction to obtain molecules of Fc-heterodimeric antibodies (WO 2009/089004 A1); by cross-linking two or more antibodies or fragments (see, for example, US patent No. 4676980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to create bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using diabody technology to generate bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single chain Fv (sFv) dimers (see, for example, Gruber et al., J. Immunol., 152:5368 (1994)); and obtaining trispecific antibodies, as described, for example, in Tutt et al., J. immunoi. 147:60 (1991).

Engineered antibodies with three or more functional antigen-binding sites, including "octopus antibodies", are also included in this document (see, for example, US 2006/0025576A1).

As used herein, an antibody or fragment also includes a "dual acting FAb" or "DAF" which contains an antigen binding site that binds to VEGF as well as another different antigen (see, for example, US 2008/0069820).

8. Antibody variants

In some embodiments, amino acid sequence variants (eg, antibody variants comprising one or more amino acid residue changes) of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Variants of the amino acid sequences of an antibody can be obtained by making appropriate modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequences of the antibody. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, as long as the final construct has the desired characteristics, such as antigen binding.

a) Options for substitutions, insertions and deletions

In some embodiments, the antibody variants have one or more amino acid substitutions. Sites of interest for substitution mutagenesis include HVR and FR. Conservative substitutions are shown in Table 1 under the heading "preferred substitutions". More significant changes are presented in Table 1 under the heading "illustrative substitutions", and as further described below with reference to classes of amino acid side chains. Amino acid substitutions can be made to the antibody of interest and the resulting products screened for desired activity, eg, maintaining/improving antigen binding, reducing immunogenicity, or improving ADCC or CDC.

Amino acids can be divided into groups according to the general properties of the side chains:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) main: His, Lys, Arg;

(5) residues that affect chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions result in replacing a member of one of these classes with a member of another class.

One type of substitution variant involves substitution of one or more hypervariable region residues and/or FR residues of the parent antibody (eg, humanized or human antibody). Typically, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, increased stability, increased expression, altered pI, and/or reduced immunogenicity) compared to the original antibody, and/or will have substantially retained certain biological properties of the parent antibody. An exemplary replacement variant is an affinity matured antibody, which can traditionally be generated, for example, using phage display maturation techniques such as those described herein. Briefly, one or more HVR amino acid residues are mutated and the variant antibodies are displayed on the surface of the phage and screened for a particular biological activity (eg, binding affinity).

Changes (eg substitutions) can be made to the HVR, eg to improve the affinity of the antibody. Such changes can be made at HVR hotspots, i.e. residues encoded by codons that undergo mutation at a high rate during the somatic maturation process (see, for example, Chowdhury, Methods Mol. Biol, 207:179-196 (2008)), and/or residues that come into contact with an antigen, with testing the resulting VH or VL variant for binding affinity. Affinity maturation by construction and reselection from secondary libraries has been described, for example, in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, the diversity is introduced into variable genes selected for maturation by any of a variety of methods (eg, reduced precision PCR, strand shuffling, or oligonucleotide directed mutagenesis). Then a secondary library is created. The library is then screened to identify any antibody variants with the desired affinity. Another way to introduce diversity includes approaches directed to HVR, in which several amino acid residues of HVR (4-6 amino acid residues in a row) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine-scanning mutagenesis or modeling. Targets in particular are CDR-H3 and CDR-L3.

In some embodiments, substitutions, insertions, or deletions may be made within one or more HVRs, provided that such changes do not significantly reduce the ability of the antibody to bind antigen. For example, conservative substitutions (eg, conservative substitutions provided herein) can be made in HVR that do not significantly reduce binding affinity. Such changes may be, for example, beyond antigen-contacting residues in the HVR. In some embodiments, in the VH and VL sequence variants above, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

In some embodiments, substitutions, insertions, or deletions may be made within one or more FRs, provided that such changes do not significantly reduce the ability of the antibody to bind an antigen. Such changes may, for example, improve the affinity and/or stability of the antibody (eg, as assessed by elevated melting point).

A convenient method for identifying antibody residues or regions that can be targets for mutagenesis is called "alanine scanning mutagenesis", described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, an amino acid residue or group of target amino acid residues (e.g., charged amino acid residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine the effect on antibody interaction. with antigen. Additional substitutions may be made at those amino acid arrangements that were functionally sensitive to the original substitutions. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify points of contact between the antibody and antigen. Such contact residues and adjacent residues can be used as targets or removed from candidates for replacement. Variants can be screened to determine whether they have the desired properties.

Amino acid inserts include amino- and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as insertions of one or more amino acid residues within a sequence. Examples of end inserts include an antibody with an N-terminal methionyl residue. Other insertion variants of an antibody molecule include fusion at the N- or C-terminus of the antibody with an enzyme (eg, for ADEPT therapy) or a polypeptide that increases serum half-life.

b) Alicosylation variants

In some embodiments, an antibody provided herein is modified to increase or decrease the degree of glycosylation of the antibody. The addition or removal of glycosylation sites in an antibody is easily accomplished by altering the amino acid sequence resulting in the creation or removal of one or more glycosylation sites.

If the antibody contains an Fc region, the carbohydrate attached to it can be changed. Native antibodies produced by mammalian cells typically contain a branched, biantennary oligosaccharide that is generally N-linked to the Asn297 CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide may contain various carbohydrates, such as mannose, N-acetyl glucose min (GlcNAc), galactose and sialic acid, as well as fucose attached to GlcNAc in the stem of the biantennary oligosaccharide structure. In some embodiments, modifications can be made to the oligosaccharide in an antibody of the invention in order to create antibody variants with certain improved properties.

In one embodiment, antibody variants are provided in which the carbohydrate structure attached (either directly or indirectly) to the Fc region has a reduced fucose content. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the Asn297 sugar chain relative to the total number of glycostructures attached to Asn297 (e.g., complex, hybrid, or mannose-rich structures) according to MALDI-TOF mass spectrometry, as described, for example, in WO 2008/077546. Asn297 refers to an asparagine residue located at position 297 in the Fc region (following the EU numbering of Fc region residues); while Asn297 can also be located about ±3 amino acids upstream or downstream of position 297, i.e. between positions 294 and 300 due to minor sequence variations in the antibodies. Such fucosylated variants may have improved ADCC function. See, for example, US Patent Nos. US 2003/0157108; US 2004/0093621. Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO2003/085119; WO2003/084570; WO2005/035586; W02005/035778; W02005/053742; W02002/031140; Okazaki et al, J. Mol. In/. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include protein fucosylation deficient CHO Lec13 cells (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US 2003/0157108 A1, Presta, L, and WO 2004/056312 A1, Adams et al., especially in Example 11), and knockout cell lines such as CHO knockout cells for the alpha-1,6-fucosyltransferase gene, FUT8, (see, for example, Yamane- Ohnuki et al Biotech Bioeng 87:614 (2004), Kanda et al Biotechnol Bioeng 94(4):680-688 (2006) and WO 2003/085107).

In addition, variants of antibodies with oligosaccharides bisected are provided, for example, in which the biantenna oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced levels of fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878; in US patent No. 6602684; and US 2005/0123546 (Umana et al.). Additionally, antibody variants are provided that contain at least one galactose residue in an oligosaccharide affixed to an Fc region. Such antibody variants may have improved CSC function. Such antibody variants are described, for example, in WO 1997/30087; WO 1998/58964; and WO 1999/22764 (Raju, S.).

c) Fc region options

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby providing a variant Fc region. A variant Fc region may comprise a sequence of a human Fc region (eg, human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid change (eg, substitution) at one or more amino acid positions. In certain embodiments, the invention provides an antibody variant that has some, but not all, effector functions, making it a suitable candidate for applications where the in vivo half-life of the antibody is important, although certain effector functions (such as complement and ADCC ) are not necessary or harmful. Cytotoxic assays can be performed in vitro and/or in vivo to confirm recovery/attenuation of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that an antibody does not bind to FcγR (and therefore does not have ADCC activity) while retaining FcRn binding ability. The primary cells to mediate ADCC, NK cells, only express FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).

Non-limiting examples of in vitro assays for evaluating ADCC activity of a molecule of interest are described in US Pat. I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985), U.S. Patent No. 5,821,337, and Bruggemann et al., J. Exp. Med. ). Alternatively, non-radioactive assay methods may be used (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wisconsin). Effector cells suitable for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model, as disclosed in Clynes et al. ., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998) A C1q binding assay can also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CSC activity. , C1q and C3c binding assays by ELISA in WO 2006/029879 and WO 2005/100402. l., J. Immunol. Methods 202:163 (1996); Cragg et al., Blood 101:1045-1052 (2003); and Cragg et al., Blood 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance/half-life can also be performed using methods known in the art (see, for example, Petkova et al., Int'l. Immunol. 18(12): 1759-1769 ( 2006)).

Antibodies with reduced effector function include those containing substitutions for one or more of residues 238, 265, 269, 270, 297, 327, and 329 of the Fc region (US Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutants with substitutions of residues 265 and 297 for alanine (US Pat. No. 7,332,581).

Certain modifications of the antibody with improved or reduced FcR binding have been described. (See, for example, US patent No. 6737056;

International patent application WO 2004/056312 and the publication of Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

In some embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and/or 334 of the Fc region (EU residue numbering).

In some embodiments, changes are made in the Fc region that result in a change (i.e., either improvement or deterioration) in C1q binding and/or complement dependent cytotoxicity (CDC), e.g., as described in US Pat. No. 6,194,551, WO 99/51642 , and Idusogie et al., J. Immunol. 164:4178-4184(2000).

Antibodies with extended half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kirn et al., J. Immunol. 24:249 (1994)), described in US 2005/0014934 A1 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include those with substitutions of one or more Fc region residues: 382, 413, 424 or 434, for example, substitution of amino acid residue 434 in the Fc region (US patent No. 7371826). See also Duncan & Winter, Nature 322:738-40 (1988); US patent No. 5648260; US patent No. 5624821; and international patent application WO 94/29351 for other examples of Fc region variants.

d) Antibody variants engineered by injection into the cyst and onto

The invention provides cysteine-engineered antibodies in which one or more amino acids of the wild-type or parent antibody (e.g., anti-VEGF antibodies, including any anti-VEGF antibodies described herein) are replaced (i.e., "replace" or "mutated") with the amino acid cysteine (i.e., "engineered cysteine"). Any form of antibody can be constructed in this way, i.e. mutated. Thus, for example, a parent monoclonal antibody can be engineered to form a "THIOMAB™ antibody". One example of a THIOMAB™ antibody is an antibody fragment (ie, Fab) that has an engineered cysteine. This Fab THIOMAB™ antibody may be referred to as "thio-Fab". It should be noted that a single site mutation produces a single engineered cysteine residue in the thio-Fab, while a single site mutation produces two engineered cysteine residues in the THIOMAB™ antibody, due to the dimeric nature of the IgG antibody.

Mutants with engineered cysteine residues (Cys) can be evaluated for the reactivity of newly introduced engineered cysteine thiol groups. The thiol reactivity value is a relative, numerical value ranging from 0 to 1.0 and can be measured for any antibody engineered by introducing cysteine. The thiol reactivity values of the cysteine-engineered antibodies according to the invention range from 0.0 to 1.0. In general, the thiol reactivity values of the cysteine-engineered antibodies according to the invention range from 0.1 to 1.0. In certain embodiments, thiol reactivity values according to the invention range from 0.0 to 0.1, 0.1 to 0.5, 0.1 to 0.6, 0.1 to 0.7, 0.1 to 0.8, 0.1 to 0.9, or 0.1 to 1.0. In certain embodiments, thiol reactivity values according to the invention range from 0.2 to 1.0, 0.3 to 1.0, 0.4 to 1.0, 0.5 to 1.0, from 0.6 to 1.0, 0.7 to 1.0, or 0.8 to 1.0. In certain embodiments, the thiol reactivity values of cysteine-engineered antibodies of the invention range from 0.6 to 1.0. In certain embodiments, the thiol reactivity values of cysteine-engineered antibodies of the invention range from 0.7 to 1.0. In certain embodiments, the thiol reactivity values of the cysteine-engineered antibodies of the invention are in the range of 0.8 to 10. In certain embodiments, the thiol reactivity values of the cysteine-engineered antibodies of the invention are in the range of 0.5 to 0.8. In certain embodiments, the thiol reactivity values of cysteine-engineered antibodies of the invention range from 0.5 to 0.9. In some embodiments, the thiol reactivity values of cysteine-engineered antibodies of the invention range from 0.5 to 0.7. In certain embodiments, thiol reactivity values of cysteine-engineered antibodies of the invention range from 0.5 to 1.0.

The methods of design, selection, and production of the invention enable the production of cysteine-engineered antibodies that are reactive with electrophilic functionality. In addition, these methods make it possible to prepare antibody conjugate compounds, such as, for example, antibody-polymer conjugates with polymer fragments conjugated at predetermined, designed, selective sites. Reactive cysteine residues on the surface of the antibody allow specific conjugation of the polymer with a thiol reactive group such as maleimide or haloacetyl. The nucleophilic reactivity of the thiol functional group of a Cys residue to the maleimide group is about 1000 times greater than any other amino acid functional group in the protein, such as the amino group of lysine residues or the M-terminal amino group. The thiol-specific functional group in the iodoacetyl and maleimide reagents can react with amino groups, but higher pH (>9.0) and longer reaction times are required (Garman, 1997, Non-Radioactive Labeling: A Practical Approach, Academic Press, London) .

The cysteine-engineered antibody of the invention preferably retains the antigen-binding capacity of their parental wild-type antibody counterparts. Thus, cysteine-engineered antibodies are able to preferentially bind specifically to antigens. Illustrative, non-limiting antigens include VEGF; IL-1β; IL-6; IL-6R; IL-13; IL-13R; TsFR (for example, PDGF-BB); angiopoietin; angiopoietin 2 (Ang2); Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin; apelin/APJ; erythropoietin; complement factor D; TNF-α; HtrA1; VEGF receptor (eg, VEGF1, VEGF2, VEGF3, mbVEGF, or sVECP); ST-2 receptor; and a protein genetically associated with the risk of AMD (eg, complement pathway components C2, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4 ; CETP; LIPC; COL10A1; and TNFRSF10A).

Any of the antibodies described herein (eg, any of the anti-VEGF antibodies described above) can be the parent antibody used to create the antibody, engineered by introducing cysteine. The exemplary methods described herein can generally be used to identify and generate antibodies by applying the design and screening steps described herein.

Cysteine-engineered antibodies of the invention can be site-specifically and efficiently coupled to a thiol-reactive reagent. The thiol-reactive reagent can be, for example, a clearance-altering agent such as a polymer (e.g., a polymer of HA or various polyethylene glycol isomers), a peptide that binds to a third component, or another carbohydrate or lipophilic agent, a multifunctional linker reagent, a labeled capture reagent. , i.e. affinity (eg, biotin linker reagent), detection label (eg, fluorophore reagent), solid phase immobilization reagent (eg, SEPHAROSE™, polystyrene, or glass), or drug-linker intermediate. One example of a thiol-reactive reagent is N-ethyl maleimide (NEM). In an exemplary embodiment, reaction of the THIOMAB™ antibody with a biotin linker reagent provides a biotilinated THIOMAB™ antibody, whereby the presence and reactivity of the engineered cysteine residue can be detected and measured. Reaction of a THIOMAB™ antibody with a multifunctional linker reagent provides a THIOMAB™ antibody with a functionalized linker that can then be reacted with a polymer, drug moiety reagent, or other label. The reaction of the THIOMAB™ antibody with the drug-linker intermediate provides a drug conjugate with the THIOMAB™ antibody. In some embodiments, the THIOMAB™ antibody is a thio-Fab.

An antibody constructed by introducing cysteine can be conjugated with thiol-reactive agents in which the reactive group is, for example, maleimide, iodoacetamide, pyridyl disulfide, or other thiol-reactive conjugation partner (see, for example, Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1 :2; Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671). The partner may be a cytotoxic agent (eg, a toxin such as doxorubicin or pertussis toxin), fluorophores such as a fluorescent dye like fluorescein or rhodamine, a chelating agent for metals used in diagnostic imaging or radiotherapy, a peptidyl or non-peptidyl tag or a detection tag , or a clearance-modifying agent such as a polymer (eg, a HA polymer or various polyethylene glycol isomers), a peptide that binds to the third component, or another carbohydrate or lipophilic agent.

The PHESELECTOR assay (phage ELISA for the selection of reactive thiols) makes it possible to determine groups of reactive cysteine groups in antibodies in phage ELISA format. See US Patent No. 7,521,541 and US Patent Publication No. 20110301334, which are incorporated herein by reference in their entirety. In particular, the PHESESLECTOR assay involves the process of coating a protein of interest (eg, an antibody) on a surface, followed by incubation with phage particles and then with a secondary antibody labeled with horseradish peroxidase (HRP), with optical density determination. It is possible to screen for phage-exposed mutant proteins with a fast, reliable, high throughput method. Libraries of cysteine-engineered antibodies can be generated and selected for binding activity using the same approach as for identifying the appropriate incorporation of free Cys reactive sites from random phage protein libraries of antibodies or other proteins. This technique involves reacting the cysteine of phage-exposed mutant proteins with an affinity reagent or reporter group that is also a reactive cthiol group.

In certain embodiments, the PHESELECTOR assay comprises the following steps: (1) bovine serum albumin (BSA), some or all of the target protein (e.g., VEGF), and streptavidin (100 μl 2 μg/ml) were individually applied to 96-well MAXISORB plates ®; (2) after blocking with 0.5% Tween-20 (in PBS), biotinylated and non-biotinylated THIOMAB™-phage antibody (2×10 ¹⁰ phage particles) were incubated for 1 hour at room temperature; (3) incubation with phage followed by incubation with an HRP-labeled secondary antibody (anti-M13 phage coat protein, pVIII protein); (4) run standard HRP reactions and measure absorbance at 450 nm; (5) thiol reactivity is measured by calculating the ratio between streptavidin OD450/target protein OD450 (eg, VEGF), such that the thiol reactivity value of 1 indicates complete biotinylation of the cysteine thiol.

DNA encoding a cysteine-engineered antibody is readily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes that are capable of specifically binding to genes encoding heavy and light chains of mouse antibodies). Hybridoma cells serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors which are then transfected into host cells such as E. coli cells, monkey COS cells, Chinese Hamster Ovary (CHO) cells, HEK293T cells, or other mammalian host cells, such as myelomas (US patent No. 5807715; US 2005/0048572; US 2004/0229310), which do not otherwise produce antibody protein, to achieve the synthesis of monoclonal antibodies in recombinant host cells. In most cases, the resulting cysteine-constructed antibodies are similar to wild-type antibodies.

Once constructed and selected, cysteine-engineered antibodies, e.g., the THIOMAB™ antibody, with high reactivity of unpaired Cys residues, can be generated by: (i) expression in a bacterial, e.g., E. coli, or mammalian cell culture system (WO 01 /00245), for example, in Chinese Hamster Ovary (CHO) cells or HEK293 cells (eg, HEK293T cells); and (ii) purification using general protein purification technologies (eg, Lowman et al (1991) J. Biol. Chem. 266(17):10982-10988). In specific embodiments, THIOMAB™ antibodies are expressed in a mammalian cell expression system. In specific embodiments, the mammalian cell expression system is HEK293T cells.

Structural positions of the engineered heavy and light chain Cys residues are numbered according to the sequential numbering system. This sequential numbering system is consistent with the Kabat numbering system (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.) for the 4D5 antibody in FIG. 13A and 13B. When using the Kabat numbering system, the actual linear amino acid sequence may contain fewer or more amino acids due to shortening or insertions in the FR or CDR of the variable domain. Light chain variant sites and heavy chain variant sites engineered by cysteine insertion are identified by the sequential numbering and Kabat numbering schemes in FIG. 13A and 13B.

Thiol reactivity can also be generalized to specific antibody domains such as the light chain constant domain (CL) and the heavy chain constant domains, CH1, CH2 and CH3. Cysteine substitutions resulting in thiol reactivity values of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 0, 95 and above can be made in the α, δ, ε, γ, and μ heavy chain constant domains of intact antibodies: IgA, IgD, IgE, IgG, and IgM, respectively, including IgG subclasses: IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

Constructed antibodies can be prepared as described, for example, in US Pat. No. 7,521,541 or International Patent Publication No. WO 2006/034488, which are incorporated herein by reference in their entirety. In some embodiments, the cysteine engineered antibody variant is the cysteine engineered antibody variant described in US Pat. No. 7,521,541 or International Patent Publication No. WO 2006/034488. In some instances, the cysteine engineered antibody variant is the cysteine engineered antibody variant described in International Patent Application Publication No. WO 2011/156328 or US Pat. No. 9,000,130, which are incorporated herein by reference in their entirety. In some embodiments, the cysteine engineered antibody variant is the cysteine engineered antibody variant described in International Patent Application Publication No. WO 2016/040856, which is incorporated herein by reference in its entirety, e.g., in Tables 1-4 in WO 2016/040856.

For example, in certain embodiments, the cysteine mutation is selected from the group consisting of HC-1195C, HC-S420C, HC-Y432C, and LC-G64C (according to Kabat numbering). In certain embodiments, the cysteine mutation is selected from the group consisting of HC-Y432C and LC-G64C (according to Kabat numbering). In certain embodiments, the cysteine mutation is a heavy chain mutation and is selected from the group consisting of Y33C, G162C, V184C, I195C, S420C, Y432C, and Q434C (according to Kabat numbering). In certain embodiments, the cysteine mutation is a heavy chain mutation and is selected from the group consisting of R19C, E46C, T57C, Y59C, A60C, M100cC, W103C, G162C, I195C, V258C, S420C, H425C, and N430C (numbered by Kabat). In certain embodiments, the cysteine mutation is a heavy chain mutation and is selected from the group consisting of Y33C, G162C, V184C, and I195C (according to Kabat numbering). In certain embodiments, the cysteine mutation is a heavy chain mutation and is selected from the group consisting of R19C, E46C, Y59C, A60C, M100cC, W103C, V258C, H425C, and N430C (according to Kabat numbering).

In certain embodiments, the cysteine mutation is a light chain mutation and is selected from the group consisting of Y55C, G64C, T85C, T180C, and N430C (according to Kabat numbering). In certain embodiments, the cysteine mutation is a light chain mutation and is selected from the group consisting of T31C, S52C, G64C, R66C, A193C, and N430C (according to Kabat numbering). In certain embodiments, the cysteine mutation is a light chain mutation and is selected from the group consisting of G64C, T85C, T180C, and N430C (according to Kabat numbering). In certain embodiments, the cysteine mutation is a light chain mutation and is selected from the group consisting of S52C, G64C, R66C, A193C, and N430C (according to Kabat numbering). In specific embodiments, the cysteine mutation in the light chain is selected from the group of cysteine mutations consisting of LC-I106C, LC-R108C, LC-R142C, and LC-K149C (according to Kabat numbering) (see Fig. 13A; Table 2 ). In a specific embodiment, the cysteine mutation in the light chain is LC-K149C (according to Kabat numbering) (see Fig. 13A). In a specific embodiment, the cysteine mutation in the light chain is LC-V205C (according to Kabat numbering).

In specific embodiments, the heavy chain cysteine mutation is selected from the group of cysteine mutations consisting of HC-T114C, HC-A140C, HC-L174C, HC-L179C, HC-T187C, HC-T209C, HC-V262C, HC-G371C, HC -Y373C, HC-E382C, HC-S424C, HC-N434C, and HC-Q438C (according to EU numbering) (see Fig. 13B; Table 3). In a specific embodiment, the heavy chain cysteine mutation is HC-A143C according to Kabat numbering (ie, HC-A140C according to EU numbering) (see Fig. 13B; Table 3). In a specific embodiment, the heavy chain cysteine mutation is HC-A174C according to EU numbering (see FIG. 13B; Table 3). In a specific embodiment, the heavy chain cysteine mutation is HC-A118C according to EU numbering (ie, HC-A114C according to Kabat numbering).

In a specific embodiment, any antibody engineered by introducing cysteine as described herein has one of the following cysteine mutations: LC-K149C according to Kabat numbering and HC-A140C according to EU numbering (see Tables 2 and 3 and Fig. 13A and 13B).

In some embodiments, one or more of the following residues can be replaced with cysteine: V205 (Kabat numbering) in the light chain, AH 8 (EU numbering) in the heavy chain, and S400 (EU numbering) in the Fc region of the heavy chain.

In some embodiments, a cysteine-engineered antibody may contain one or more cysteine heavy chain mutations selected from the group consisting of V2C, L4C, V5C, L11C, R19C, F27C, I29C, T32C, Y33C, Q39C, A40C, K43C, L45C, E46C, T53C, G55C, T57C, R58C, Y59C, A60C, T68C, N76C, Y79C, Q81 C, W95C, G96C, D101 C, W103C, T116C, K117C, T135C, N155C, A158C, G162C, G174C, L174C, L174C, Т183С, V184C, 1195С, N199C, S203C, F239C, М248С, Е254С, V258C, N272C, V278C, L305C, Т331С, S333C, R340C, Q343C, K356С, Е384С, S399C, K410С, Q414C, G416C, N417С, Y432C, Т433С, K435C, S438C, L439C, M100cC, and N82aC (according to Kabat numbering). See also Table 3 of international patent application WO 2016/040856.

In some embodiments, a cysteine-engineered antibody may contain one or more cysteine light chain mutations selected from the group consisting of S12C, S14C, G16C, R18C, T22C, R24C, Q27C, T31C, A32C, Q38C, K39C, G41C, K42C, P44C, Y49C, S50C, S52C, F53C, L54C, Y55C, S63C, G64C, R66C, D70C, T72C, T74C, S76C, Q79C, T85C, H91C, Y92C, P95C, T97C, F98C, K103C, E105C, K107C, P119C, K126C, T129C, S131C, Q147C, W148C, A153C, Q155C, S156C, S159C, Q160C, S162C, Q166C, T172C, T180C, V191C, A193C, E195C, V205C, T206C, and N210C in accordance with the numbering) . See also Table 4 of international patent application WO 2016/040856.

In some embodiments, a cysteine-engineered antibody may contain one or more cysteine mutations selected from the group consisting of HC-I195C, HC-S420C, HC-Y432C, and LC-G64C (according to Kabat numbering). See also Table 5 of international patent application WO 2016/040856.

In some embodiments, the cysteine-engineered antibody contains cysteine light chain mutations selected from the group of sites consisting of LC-T22C, LC-K39C, LC-Y49C, LC-Y55C, LC-T85C, LC-T97C, LC-I106C , LC-R108C, LC-R142C, LC-K149C, and LC-V205C (according to Kabat numbering).

A cysteine engineered antibody may contain any suitable number of engineered cysteine residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more engineered residues cysteine. In some embodiments, a cysteine engineered antibody may contain 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, or 1-10 engineered cysteine residues. In some embodiments, a cysteine engineered antibody may contain 1 engineered cysteine residue. In some embodiments, a cysteine engineered antibody may contain 2 engineered cysteine residues. In some embodiments, a cysteine engineered antibody may contain 3 engineered cysteine residues.

In any of the previous embodiments, a cysteine engineered antibody may contain the engineered cysteine at an equivalent position as any of the cysteine mutations described above. For example, if the antibody contains a native serine at position 118 (EU numbering), the serine can be changed to a cysteine to form the S118C mutation.

For example, the invention provides a cysteine engineered anti-VEGF antibody comprising a heavy chain cysteine mutation selected from the group consisting of HC-A118C, HC-A140C and HC-L174C (EU numbering) or a light chain cysteine mutation selected from the group consisting of LC-V205C and LC-K149C (Kabat numbering), wherein the anti-VEGF antibody is any anti-VEGF antibody described herein, e.g., any anti-VEGF antibody described in Tables 8-10 . In some embodiments, the anti-VEGF antibody is N94A.F83A.N82aR.Y58R (G6.31 AARR). In some embodiments, the anti-VEGF antibody is G6.31 DT. In some embodiments, the anti-VEGF antibody is LC-N94A. In some embodiments, the anti-VEGF antibody is LC-N94A.LC-F83A. In some embodiments, the anti-VEGF antibody is LC-N94A.LC-F83A. In some embodiments, the anti-VEGF antibody is HC-A40E.HC-T57E (G6.31 AAEE). In some embodiments, the anti-VEGF antibody is HCcombo. In some embodiments, the anti-VEGF antibody is HCLC2. In some embodiments, the anti-VEGF antibody is HCLC4. In some embodiments, the anti-VEGF antibody is HCLC5. In some embodiments, the anti-VEGF antibody is HCLC3. In some embodiments, the anti-VEGF antibody is HCLC1. In some embodiments, the anti-VEGF antibody is R19HC combo. In some embodiments, the anti-VEGF antibody is R19HCLC2. In some embodiments, the anti-VEGF antibody is R19HCLC4. In some embodiments, the anti-VEGF antibody is R19HCLC5.

In a specific example, the invention provides a cysteine engineered anti-VEGF antibody comprising a heavy chain cysteine mutation selected from the group consisting of HC-A118C, HC-A140C and HC-L174C (EU numbering) or a light chain cysteine mutation, selected from the group consisting of LC-V205C and LC-K149C (Kabat numbering), said antibody containing the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYTRYADSVKG (SEQ ID NO: 7); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGAPFT (SEQ ID NO: 10). In some cases, the cysteine-engineered anti-VEGF antibody contains the following heavy chain variable domain FRs: (a) FR-H1 containing the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTIS (SEQ ID NO: 13); (b) FR-H2 containing the amino acid sequence WVRQAPGKGLEWVA (SEQ ID NO: 14); (c) FR-H3 containing the amino acid sequence RFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO: 15); and (d) FR-H4 containing the amino acid sequence WGQGTLVTVSS (SEQ ID NO: 16). In additional instances, the cysteine-engineered anti-VEGF antibody contains the following light chain variable domain FRs: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20). In some instances, the cysteine-engineered anti-VEGF antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 11, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 12. In some cases, the parent antibody is G6.31 AARR. In some embodiments, the cysteine mutation is HC-A118C. In other embodiments, the cysteine mutation is HC-A140C. In other embodiments, the cysteine mutation is HC-L174C (EU numbering). In other embodiments, the cysteine mutation is LC-V205C (Kabat numbering). In other embodiments, the cysteine mutation is LC-K149C (Kabat numbering).

In some instances, the invention provides an antibody comprising (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 90 and/or (b) a light chain comprising the amino acid sequence of SEQ ID NO: 89.

In some instances, the invention provides an antibody comprising (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and/or (b) a light chain comprising the amino acid sequence of SEQ ID NO: 91.

In some instances, the invention provides an antibody comprising (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 94 and/or (b) a light chain comprising the amino acid sequence of SEQ ID NO: 93.

f) Antibody derivatives

In certain embodiments, an antibody provided herein may be further modified to include additional non-protein components that are known in the art and are commonly available. Fragments suitable for derivatization of antibodies include, without limitation, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic copolymer anhydride, polyamino acids (either homopolymers or random copolymers), dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, oxide/ethylene oxide copolymers, polyoxyethylene polyols (eg, glycerol), polyvinyl alcohol, and mixtures thereof. Propionaldehyde polyethylene glycol may be advantageous in production due to its stability in water. The specified polymer may have any molecular weight and be branched or unbranched. The number of polymers attached to an antibody may vary and, if more than one polymer is attached, they may be the same or different molecules. In general, the amount and/or type of polymers used for derivatization can be determined based on factors including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative is to be used in therapy under certain conditions, and the like. . Additional antibody conjugates are described herein, for example, in section G below and in Examples 1 and 2.

In another embodiment, the invention provides conjugates of an antibody and a non-protein component that can be selectively heated by irradiation. In one embodiment, the non-protein component is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength and includes, without limitation, wavelengths that do not harm normal cells but that heat the non-protein component to a temperature at which cells close to the antibody-non-protein component die.

f) Isoelectric point options

The invention relates to variants of antibodies with altered isoelectric points. For example, the invention relates to antibody variants with a reduced isoelectric point (pI), eg compared to an anti-VEGF antibody, eg G6.31. In some cases, the surface charge decreases at physiological pH. In some instances, the anti-VEGF antibody has a pI equal to or less than about 8 (eg, about 8, about 7, about 6, about 5, or about 4). In some instances, the antibody has a pI of about 4 to about 8 (eg, about 4, about 5, about 6, about 7, or about 8). In some instances, the anti-VEGF antibody has a pI of about 5 to about 7 (eg, about 5, about 6, or about 7). In some instances, the anti-VEGF antibody has a pI of about 5 to about 6 (e.g., about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, or about 6).

Antibodies of the invention can be engineered to have a reduced pI, for example by replacing wild-type amino acid residues at a given position with an amino acid having a lower pI. The pI of an amino acid can be determined based on the pKa values of the amine (-NH ₂ ), carboxylic acid (-COOH), and amino acid side chain, which are known in the art. In some embodiments, surface-exposed amino acid residues may be substituted to reduce the pI of the antibody. In one embodiment, the surface-exposed amino acid residues may be substituted with glutamate (E). In one embodiment, the surface-exposed amino acid residues may be replaced with aspartate (D).

B. Recombinant Methods and Compositions

Any of the antibodies (e.g., anti-VEGF antibodies, including cysteine-engineered anti-VEGF antibodies) described herein can be made using recombinant methods and compositions, for example, as described in US Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-VEGF antibody described herein is provided. Such a nucleic acid may encode an amino acid sequence containing the VL and/or an amino acid sequence containing the VH of an antibody (eg, an antibody light and/or heavy chain). In a further embodiment, one or more vectors (eg, expression vectors) containing such nucleic acids are provided. In a further embodiment, a host cell containing such a nucleic acid is provided. In one such embodiment, the host cell contains (eg, transformed): (1) a vector containing a nucleic acid that encodes an amino acid sequence containing the VL of the antibody, and an amino acid sequence containing the VH of the antibody; or (2) a first vector containing a nucleic acid that encodes an amino acid sequence containing the VL of an antibody and a second vector containing a nucleic acid that encodes an amino acid sequence containing the VH of an antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (eg, a Y0, NS0, Sp20 cell). In one embodiment, a method is provided for producing an anti-VEGF antibody, the method comprising culturing a host cell containing a nucleic acid encoding an antibody as defined above under conditions suitable for expression of the antibody, and, in some cases, isolating the antibody from the cell. host (or from host cell culture medium).

For recombinant production of an antibody (e.g., an anti-VEGF antibody, including an anti-VEGF antibody engineered by introducing cysteine), the nucleic acid encoding the antibody, for example, as described previously, is isolated and inserted into one or more vectors for further cloning and /or expression in the host cell. Such nucleic acid can be readily isolated and sequenced using conventional techniques (eg, using oligonucleotide probes that are capable of specifically binding to genes that encode for heavy and light chains of an antibody).

Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, especially if glycosylation and Fc effector function are not needed. For the expression of antibody fragments and polypeptides in bacteria, see, for example, US Pat. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli. After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes suitable for cloning or expression of vectors encoding antibodies, hosts are eukaryotic microorganisms such as filamentous fungi or yeasts, including strains of fungi and yeasts with "humanized" glycosylation pathways that result in antibodies with partial or fully human glycosylation profile. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for expression of the glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used in combination with insect cells, especially for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. See, for example, US Pat. Nos. 5,959,177; 6,040,498; 6,420,548;

Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be suitable. Other examples of suitable mammalian cell lines are monkey kidney line CV1 transformed with SV40 (COS-7); human kidney embryonic cell line (293 or 293 cells as described, for example, in Graham et al., J. Gen Virol. 36:59 (1977)); newborn hamster kidney cells (BHK); mouse sertoli cells (TM4 cells described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma (HELA) cells; dog kidney cells (MDCK, Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells as described, for example, in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982) MRC 5 cells and FS4 cells Other suitable mammalian cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR ^- CHO cells (Urlaub et al ., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2/0. For a review of some mammalian host cell lines suitable for antibody production, see , for example, Yazaki and Wu, Methods in Molecular Biology, Vol.248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

C. Analyzes

Antibodies (for example, anti-VEGF antibodies described herein, including anti-VEGF antibodies engineered by introducing cysteine), as well as antibody conjugates (for example, antibody conjugates that contain anti-VEGF antibodies (for example, any anti-VEGF antibody provided herein)) can be identified, screened, or characterized for their physicochemical properties and/or biological activity using various assays known in the art.

1. Binding assays and other assays

In one aspect, an antibody (e.g., an anti-VEGF antibody, including an anti-VEGF antibody engineered by introducing cysteine), or a conjugate of such an antibody, is tested for antigen-binding activity, e.g., by known methods such as ELISA, Western blotting. , etc.

In another aspect, competition assays can be used to identify an antibody that competes with an antibody as described herein, or a conjugate of such an antibody, for binding to an antigen (eg, VEGF). In some embodiments, such a competing antibody binds to the same epitope (eg, a linear or conformational epitope) that binds to the antibody, as described herein. A detailed description of exemplary methods for mapping an epitope to which an antibody binds is provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

In an exemplary competition assay, immobilized VEGF is incubated in a solution containing a first labeled antibody that binds to VEGF and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to VEGF. The second antibody may be present in the hybridoma supernatant. As a control, immobilized VEGF is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that ensure the binding of the first antibody to VEGF, the excess of unbound antibody is removed and the amount of label associated with the immobilized VEGF is measured. If the amount of label bound to the immobilized VEGF is significantly reduced in the test sample relative to the control sample, then this indicates that the second antibody is competing with the first antibody for binding to VEGF. Similar analyzes can be performed for other antigens. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

2. Activity assays

In one aspect, assays are provided to identify antibodies (eg, anti-VEGF antibodies, including cysteine-engineered anti-VEGF antibodies) or conjugates of such antibodies having biological activity. Biological activity may include, for example, binding to an antigen (eg, VEGF (eg, VEGF in the bloodstream)), or a peptide fragment thereof, or in vivo, in vitro, or ex vivo. In some embodiments, the biological activity may include blocking or neutralizing an antigen. For example, in some embodiments, the biological activity may include blocking or neutralizing VEGF or preventing the binding of VEGF to a ligand, for example, a receptor such as KDR or Flt-1. Also provided are antibodies or conjugates of such antibodies having such biological activity in vivo and/or in vitro. In some embodiments, an antibody of the invention, or conjugates of such antibodies, is tested for such biological activity.

3. Stability analyzes

In one aspect, assays are provided to determine the stability (eg, thermal stability) of an antibody (eg, an anti-VEGF, including an anti-VEGF antibody, engineered antibody) or a conjugate of such an antibody. For example, the stability of an antibody or such antibody conjugate can be determined using any method known in the art, e.g., differential scanning fluorimetry (DSF), circular dichroism (CD), protein intrinsic fluorescence, differential scanning calorimetry, spectroscopy, light scattering (e.g. , Dynamic Light Scattering (DLS) and Static Light Scattering (SLS), Interacting Molecule Chromatography (SIC) Stability of an antibody or such antibody conjugate can be determined as described herein, e.g. and 2 International Patent Application No. PCT/US 2016/053454 In some cases, the stability of an antibody conjugate can be determined using size exclusion chromatography with built-in refractive index and multi-angle laser light scattering (SEC-RI-MALS) detector, for example, as described in Example 1.

D. Pharmaceutical formulations

Pharmaceutical formulations of an antibody (e.g., an anti-VEGF antibody, including an anti-VEGF antibody engineered by introducing cysteine) or a conjugate of such an antibody provided herein are prepared by mixing such an antibody or antibody conjugate having the desired purity with one or more optional pharmaceutically acceptable carriers Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffering agents such as phosphate, citrate, and other organic salts; antioxidants including ascorbic acid and methionine; preservatives (eg octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkyl parabens, eg methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (containing less than 10 residues) polypeptides; proteins, eg serum albumin, gelatin or immunoglobulins; hydrophilic polymers, for example polyvinylpyrrolidone; amino acids, for example glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars, for example sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal-containing complexes (eg Zn-protein complexes) and/or non-ionic surfactants such as polyethylene glycol (PEG). Examples of pharmaceutically acceptable carriers herein further include interstitial distribution agents such as soluble neutrally active hyaluronidase-containing glycoproteins (sHASEGP), e.g., hyaluronidase-containing soluble human PH-20 glycoproteins such as Baxter International Inc.). Some examples of sHASEGP and uses, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

Exemplary lyophilized antibody formulations are described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO 2006/044908, the latter containing a histidine acetate buffer.

The dosage form described herein may also contain more than one active compound required for the particular indication being treated, preferably with complementary activities that do not adversely affect each other. For example, there may be a need to provide an immunosuppressive agent. Such molecules are present in proper combination in amounts that are effective for the intended use.

The active ingredients can be placed in microcapsules obtained, for example, by coacervation or interfacial polymerization, for example, microcapsules of hydroxymethylcellulose or gelatin and microcapsules of poly(methyl methacrylate), respectively; in colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such methods are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Formulations intended for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through sterile filter membranes.

In certain embodiments, the implementation of the pharmaceutical composition contains one or more additional compounds. In certain embodiments, the additional compound binds to a second biological molecule selected from the group consisting of: IL-1β; IL-6; IL-6R; IL-13; IL-13R; TCFR; angiopoietin; angiopoietin 2; Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin; apelina/APJ; erythropoietin; complement factor D; TNF-α; HtrA1; VEGF receptor; ST-2 receptor; and proteins genetically associated with the risk of age-related macular degeneration (AMD), such as components of the C2 complement pathway, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; SETP; LIPC; COL10A1; and TNFRSF10A. In some embodiments, the additional compound is an antibody or an antigen-binding fragment thereof.

For example, in some instances, the additional compound is a bispecific antibody (e.g., an anti-VEGF/anti-Ang2 bispecific antibody such as RG-7716 or any anti-VEGF/anti-Ang2 bispecific antibody disclosed in WO 2010/069532 or WO 2016 /073157.

In another example, in some instances, the additional compound is an anti-IL-6 antibody, e.g. EBI-031 (Eleven Biotereutics; See e.g. WO 2016/073890), siltuximab (SYLVANT®), olokizumab, clasakizumab, sirukumab, elsilimomab, gerilimzumab, OPR-003, MEDI-5117, PF-04236921, or a variant thereof.

In yet another example, in some cases, the additional compound is an anti-IL-6R antibody, e.g. his option.

E. Therapeutic Methods and Compositions

Any of the antibodies (eg, anti-VEGF, including cysteine-engineered anti-VEGF antibodies) or conjugates thereof (eg, monodisperse HA conjugates) provided herein can be used in therapeutic methods.

In one aspect, an anti-VEGF antibody (eg, an anti-VEGF antibody engineered by introducing cysteine) is provided for use as a drug. In another aspect, an antibody conjugate (eg, a monodisperse HA conjugate) is provided for use as a drug. In other aspects, the invention provides an anti-VEGF antibody (eg, an anti-VEGF antibody engineered by introducing cysteine) for use in the treatment of a disorder associated with abnormal angiogenesis. In other aspects, the invention provides an anti-VEGF antibody (eg, an anti-VEGF antibody engineered by introducing cysteine) for use in the treatment of a disorder associated with abnormal angiogenesis. In some embodiments, the disorder associated with abnormal angiogenesis is an eye disease. In some cases, the eye disease is AMD (eg, wet AMD, dry AMD, intermediate AMD, advanced AMD, or geographic retinal atrophy (GA)), macular degeneration, macular edema, DME (eg, focal DME). extracentral or diffuse DME involving the central retina), retinopathy, diabetic retinopathy (DR) (eg, proliferative DR (PDR), non-proliferative DR (NPDR), or high altitude DR), other ischemia-related retinopathies, ROP, occlusion retinal vein occlusion (RVO) (e.g., forms of central retinal vein occlusion (CRVO) and branches of the central retinal vein (RVO))), CNV (e.g., myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases, associated with retinal neovascularization/choroidal neovascularization, pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, EVRP, Coats disease, Norrie disease, OPPG, subconjugation nctival hemorrhage, rubeosis, neovascular eye disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, macular telangiectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystic macular edema (CM), vasculitis, papilledema, retinitis, conjunctivitis (eg, infectious and noninfectious (eg, allergic) conjunctivitis), Leber's congenital amaurosis, uveitis (including infectious and noninfectious uveitis), choroiditis (eg, multifocal choroiditis), ocular histoplasmosis, blepharitis, dry eye syndrome, traumatic eye damage, Sjögren's syndrome.

In another aspect, an anti-VEGF antibody (eg, an anti-VEGF antibody engineered by introducing cysteine) is provided for use in a method of treatment. In another aspect, an antibody conjugate (eg, a monodisperse HA conjugate) is provided for use in a method of treatment. In some instances, the invention provides an anti-VEGF antibody (e.g., an anti-VEGF antibody engineered by introducing cysteine) for use in a method of treating a subject having a disorder associated with abnormal angiogenesis, comprising administering to the subject an effective amount of the anti-VEGF antibody. The invention also relates to an antibody conjugate (eg, a monodisperse HA conjugate) for use in a method of treating a subject having a disorder associated with pathological angiogenesis, comprising administering to the individual an effective amount of the antibody conjugate. In some cases, the disorder associated with pathological angiogenesis is an eye disease. In some cases, the eye disease is AMD (eg, wet AMD, dry AMD, intermediate AMD, advanced AMD, or geographic retinal atrophy (GA)), macular degeneration, macular edema, DME (eg, focal DME). extracentral or diffuse DME involving the central retina), retinopathy, diabetic retinopathy (DR) (eg, proliferative DR (PDR), non-proliferative DR (NPDR), or high altitude DR), other ischemia-related retinopathies, ROP, occlusion retinal vein occlusion (RVO) (e.g., forms of central retinal vein occlusion (CRVO) and branches of the central retinal vein (RVO))), CNV (e.g., myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases, associated with retinal neovascularization/choroidal neovascularization, pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, EVRP, Coats disease, Norrie disease, OPPG, subconjugation nctival hemorrhage, rubeosis, neovascular eye disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, macular telangiectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystic macular edema (CM), vasculitis, papilledema, retinitis, conjunctivitis (eg, infectious and noninfectious (eg, allergic) conjunctivitis), Leber's congenital amaurosis, uveitis (including infectious and noninfectious uveitis), choroiditis (eg, multifocal choroiditis), ocular histoplasmosis, blepharitis, dry eye syndrome, traumatic eye damage, Sjögren's syndrome.

In some instances, the invention provides an anti-VEGF antibody (eg, an anti-VEGF antibody engineered by introducing cysteine) for use in reducing or inhibiting angiogenesis in a subject. In another aspect, an antibody conjugate (eg, a monodisperse HA conjugate) is provided for use in reducing or inhibiting angiogenesis in a subject. In some embodiments, the invention provides an anti-VEGF antibody (eg, an anti-VEGF antibody) for use in a method for reducing or inhibiting angiogenesis in a subject, comprising administering to the subject an effective anti-VEGF antibody to reduce or inhibit angiogenesis. The invention also provides an antibody conjugate (eg, a monodisperse HA conjugate) for use in a method for reducing or inhibiting angiogenesis in a subject, comprising administering to the subject an effective amount of the antibody conjugate. A "subject" in any of the above uses may be a human.

The invention relates to the use of an anti-VEGF antibody (eg, an anti-VEGF antibody engineered by introducing cysteine) in the manufacture or preparation of a medicament. The invention also relates to the use of an antibody conjugate (eg, a monodisperse HA conjugate) in the manufacture or preparation of a drug. For example, in one example, the drug is for the treatment of a disorder associated with abnormal angiogenesis. In an additional example, the drug is for use in a method of treating a disorder associated with pathological angiogenesis, comprising administering to a subject having a disorder associated with pathological angiogenesis, an effective amount of the drug. In some cases, the disorder associated with pathological angiogenesis is an eye disease. In some cases, the eye disease is AMD (eg, wet AMD, dry AMD, intermediate AMD, advanced AMD, or geographic retinal atrophy (GA)), macular degeneration, macular edema, DME (eg, focal DME). extracentral or diffuse DME involving the central retina), retinopathy, diabetic retinopathy (DR) (eg, proliferative DR (PDR), non-proliferative DR (NPDR), or high altitude DR), other ischemia-related retinopathies, ROP, occlusion retinal vein occlusion (RVO) (e.g., forms of central retinal vein occlusion (CRVO) and branches of the central retinal vein (RVO))), CNV (e.g., myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases, associated with retinal neovascularization/choroidal neovascularization, pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, EVRP, Coats disease, Norrie disease, OPPG, subconjugation nctival hemorrhage, rubeosis, neovascular eye disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, macular telangiectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystic macular edema (CM), vasculitis, papilledema, retinitis, conjunctivitis (eg, infectious and noninfectious (eg, allergic) conjunctivitis), Leber's congenital amaurosis, uveitis (including infectious and noninfectious uveitis), choroiditis (eg, multifocal choroiditis), ocular histoplasmosis, blepharitis, dry eye syndrome, traumatic eye damage, Sjögren's syndrome. In another example, the drug is for reducing or inhibiting angiogenesis in a subject. In a further example, the drug is for use in a method for reducing or inhibiting angiogenesis in a subject, comprising administering to the subject an amount effective to reduce or inhibit angiogenesis. In any of the prior drug uses, the method may include administering to the subject an effective amount of at least one additional therapeutic agent, for example, as described below.

The invention provides a method for treating a disorder associated with pathological angiogenesis. In one embodiment, the method comprises administering to an individual having a disorder associated with abnormal angiogenesis an effective amount of an anti-VEGF antibody (eg, an anti-VEGF antibody engineered by the administration of cysteine). In another example, the method includes administering to an individual having a disorder associated with pathological angiogenesis an effective amount of an antibody conjugate (eg, a monodisperse HA conjugate). In some cases, the disorder associated with pathological angiogenesis is an eye disease. In some cases, the eye disease is AMD (eg, wet AMD, dry AMD, intermediate AMD, advanced AMD, or geographic retinal atrophy (GA)), macular degeneration, macular edema, DME (eg, focal DME). extracentral or diffuse DME involving the central retina), retinopathy, diabetic retinopathy (DR) (eg, proliferative DR (PDR), non-proliferative DR (NPDR), or high altitude DR), other ischemia-related retinopathies, ROP, occlusion retinal vein occlusion (RVO) (e.g., forms of central retinal vein occlusion (CRVO) and branches of the central retinal vein (RVO))), CNV (e.g., myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases, associated with retinal neovascularization/choroidal neovascularization, pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, EVRP, Coats disease, Norrie disease, OPPG, subconjugation nctival hemorrhage, rubeosis, neovascular eye disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, macular telangiectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystic macular edema (CM), vasculitis, papilledema, retinitis, conjunctivitis (eg, infectious and noninfectious (eg, allergic) conjunctivitis), Leber's congenital amaurosis, uveitis (including infectious and noninfectious uveitis), choroiditis (eg, multifocal choroiditis), ocular histoplasmosis, blepharitis, dry eye syndrome, traumatic eye damage, Sjögren's syndrome. In additional cases, the method further includes administering to the individual an effective amount of at least one additional therapeutic agent, for example, as described below. The "subject" according to any of the above methods may be a human.

It is contemplated that an antibody (eg, an anti-VEGF antibody engineered by introducing cysteine) or an antibody conjugate (eg, a monodisperse HA conjugate) of the present invention can be used to treat a mammal. In one embodiment, an antibody (eg, an anti-VEGF antibody engineered by introducing cysteine) or an antibody conjugate (eg, a monodisperse HA conjugate) is administered to a non-human mammal, for example, in order to obtain, for example, preclinical data. Exemplary non-human mammals to be treated include non-human primates, dogs, cats, rodents (eg, mice and rats), and other mammals subject to preclinical studies. Such mammals may be standard animal models for the disease being treated with the antibody, or may be used to study the toxicity or pharmacokinetics of the antibody of interest. In each of these embodiments, dose escalation studies may be conducted in a mammal. An antibody (eg, an anti-VEGF antibody engineered by introducing cysteine) or an antibody conjugate (eg, a monodisperse HA conjugate) can be administered to a rodent host, eg, in a solid tumor model. The antibody or antibody conjugate can be administered to a host (eg, a rodent, eg, a rabbit) for ocular pharmacokinetic studies, eg, by intravitreal administration (eg, intravitreal injection) or via an implantable delivery device.

In a further aspect, the invention provides pharmaceutical formulations comprising any of the antibodies (e.g., cysteine engineered anti-VEGF antibodies) or antibody conjugates (e.g., monodisperse HA conjugates) provided herein, for example, for use in any of the above therapeutic methods. . In one embodiment, the pharmaceutical formulation comprises any of the antibodies (eg, cysteine engineered anti-VEGF antibodies) or antibody conjugates (eg, monodisperse HA conjugates) provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the antibodies (e.g., cysteine engineered anti-VEGF antibodies) or antibody conjugates (e.g., monodisperse HA conjugates) provided herein and at least one additional therapeutic agent, such as described below. In certain embodiments, the implementation of the pharmaceutical composition contains one or more additional compounds. In certain embodiments, the additional compound binds to a second biological molecule selected from the group consisting of: IL-1β; IL-6; IL-6R; IL-13; IL-13R; TCFR; angiopoietin; ang2; Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin; apelina/APJ; erythropoietin; complement factor D; TNF-α; HtrA1; VEGF receptor; ST-2 receptor; and proteins genetically associated with the risk of age-related macular degeneration (AMD), such as components of the C2 complement pathway, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; interleukin-8 (IL-8); CX3CR1; TLR3; TLR4; SETP; LIPC, COL10A1; and TNFRSF10A. In some embodiments, the additional compound is an antibody or an antigen-binding fragment thereof. For example, in some instances, the additional compound is a bispecific antibody (e.g., an anti-VEGF/anti-Ang2 bispecific antibody such as RG-7716 or any anti-VEGF/anti-Ang2 bispecific antibody disclosed in WO 2010/069532 or WO 2016 /073157, or a variant thereof In another example, in some cases, the additional compound is an anti-IL-6 antibody, e.g. EBI-031 (Eleven Biotereutics; See e.g. , clazakizumab, sirukumab, elsilimomab, gerilimzumab, OPR-003, MEDI-5117, PF-04236921, or a variant thereof In yet another example, in some instances, the additional compound is an anti-IL-6R antibody, such as tocilizumab (ACTEMRA®) ( see, for example, WO 1992/019579), sarilumab, vobarilizumab (ALX-0061), SA-237, or a variant thereof.

Antibodies (eg, cysteine-engineered anti-VEGF antibodies) or antibody conjugates (eg, monodisperse HA conjugates) may be used either alone or in combination with other agents in therapy. For example, an antibody (eg, an anti-VEGF antibody engineered by introducing cysteine) or an antibody conjugate (eg, a monodisperse HA conjugate) can be co-administered with at least one additional therapeutic agent. In some embodiments, the additional therapeutic agent is another antibody, an antiangiogenic agent, an immunosuppressive agent, a cytokine, a cytokine antagonist, a corticosteroid, an antiemetic, a cancer vaccine, an analgesic, or combinations thereof.

For example, in some embodiments, any of the preceding methods further comprises administering one or more additional compounds. In some embodiments, the antibody (eg, anti-VEGF antibody) or antibody conjugate (eg, monodisperse HA conjugate) is administered simultaneously with the additional compound(s). In some embodiments, the antibody or antibody conjugate is administered before or after the additional compound(s). In certain embodiments, the additional compound binds to a second biological molecule selected from the group consisting of: IL-1β; IL-6; IL-6R; IL-13; IL-13R; TCFR; angiopoietin; ang2; Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin; apelina/APJ; erythropoietin; complement factor D; TNF-α; HtrA1; VEGF receptor; ST-2 receptor; and proteins genetically associated with the risk of AMD, such as components of the C2 complement pathway, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; interleukin-8 (IL-8); CX3CR1; TLR3; TLR4; SETP; LIPC; COL10A1; and TNFRSF10A. In some embodiments, the additional compound is an antibody or an antigen-binding fragment thereof.

In some embodiments, in accordance with (in relation to) any of the embodiments described above, the eye disease is an intraocular neovascular disease selected from the group consisting of proliferative retinopathy, choroidal neovascularization (CNV), age-related macular degeneration (AMD), diabetic and other ischemia-related retinopathy, diabetic macular edema, pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, retinal vein occlusion (RVO), including RCVO and RVO, corneal neovascularization, retinal neovascularization, and retinopathy of prematurity (RP). For example, in some instances, the additional compound is a bispecific antibody (e.g., an anti-VEGF/anti-Ang2 bispecific antibody such as RG-7716 or any anti-VEGF/anti-Ang2 bispecific antibody disclosed in WO 2010/069532 or WO 2016 /073157, or a variant thereof In another example, in some cases, the additional compound is an anti-IL-6 antibody, e.g. EBI-031 (Eleven Biotereutics; See e.g. , clazakizumab, sirukumab, elsilimomab, gerilimzumab, OPR-003, MEDI-5117, PF-04236921, or a variant thereof In yet another example, in some instances, the additional compound is an anti-IL-6R antibody, such as tocilizumab (ACTEMRA®) ( see, for example, WO 1992/019579), sarilumab, vobarilizumab (ALX-0061), SA-237, or a variant thereof.

In some instances, an antibody (e.g., an anti-VEGF antibody engineered by the introduction of cysteine) or an antibody conjugate (e.g., a monodisperse HA conjugate) of the invention may be administered in combination with at least one additional therapeutic agent for the treatment of an ocular disease, e.g., eye disease, described in this document (for example, AMD (eg, wet AMD), DMO, DR, or OBC). Illustrative additional therapeutic agents for combination therapy for the treatment of eye diseases include, without limitation, anti-angiogenic agents such as VEGF antagonists, including, for example, anti-VEGF antibodies (eg, anti-VEGF Fab LUCENTIS® (ranibizumab)), soluble receptor fusion proteins (e.g., recombinant EYLEA® soluble receptor fusion protein (aflibercept, also known as VEGF Trap Eye; Regeneron/Aventis)), aptamers (e.g., anti-VEGF pegylated MACUGEN® aptamer (pegaptanib sodium; NeXstar Pharmaceuticals/OSI Pharmaceuticals)), and VEGF tyrosine kinase inhibitors (e.g., 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline (ZD6474), 4-(4-fluoro-2-methylindole-5 -yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline (AZD2171), vatalanib (PTK787), semaxaminib (SU5416; SUGEN), and SUTENT® (sunitinib)); tryptophanyl-tRNA synthetase (TrpRS); squalamine; RETAANE® (anecortave acetate for depot suspension; Alcon, Inc.); prodrug combretastatin A4, (CA4P); MIFEPREX® (mifepristone-ru486); subtenon triamcinolone acetonide; intravitreal crystalline triamcinolone acetonide; matrix metalloproteinase inhibitors (eg, primostat (AG3340; Pfizer)); fluocinolone acetonide (including intraocular fluocinolone implant; Bausch & Lomb/Control Delivery Systems); linomide; β3 integrin function inhibitors; angiostatin, as well as their combinations. These and other therapeutic agents that can be administered in combination with an antibody conjugate of the invention are described, for example, in US Patent Application No. US 2014/0017244, which is incorporated herein by reference in its entirety.

Other examples of additional therapeutic agents that can be used in combination with an antibody (e.g., an anti-VEGF antibody engineered with cysteine) or an antibody conjugate (e.g., a monodisperse HA conjugate) of the invention for the treatment of eye disease (e.g., AMD, DME, DR) or OBC) include, but are not limited to, VISUDYNE® (verteporfin; a light-activated drug that is typically used in combination with non-thermal laser photodynamic therapy), PKC412, Endovion (NS 3728; NeuroSearch A/S), neurotrophic factors (eg, glial neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF)), diltiazem, dorzolamide, PHOTOTROP®, 9-cis-retinal, ophthalmic drugs (eg, phospholine iodide, echothiophate, or carbonic anhydrase inhibitors), neovastat (AE- 941; AEterna Laboratories, Inc.), Sirna-027 (AGF-745; Sima Therapeutics, Inc.), neurotrophins (including, by way of example only, NT-4/5, Genentech) , Cand5 (Acuity Pharmaceuticals), INS-37217 (Inspire Pharmaceuticals), integrin antagonists (including integrin antagonists from Jerini AG and Abbott Laboratories), EG-3306 (Ark Therapeutics Ltd.), BDM-E (BioDiem Ltd.), thalidomide ( used e.g. EntreMed, Inc.), cardiotrophin-1 (Genentech), 2-methoxyestradiol (Allergan/Oculex), DL-8234 (Toray Industries), NTC-200 (Neurotech), tetrathiomolybdate (University of Michigan), LYN-002 (Lynkeus Biotech), microalgae compound (Aquasearch/Albany, Mera Pharmaceuticals), D-9120 (Celltech Group plc), ATX-S10 (Hamamatsu Photonics), TGF-beta 2 (Genzyme/Celtrix), tyrosine kinase inhibitors (e.g., from Allergan, SUGEN, or Pfizer), NX-278-L (NeXstar Pharmaceuticals/Gilead Sciences), Opt-24 (OPTIS France SA), retinal ganglion cell neuroprotectors (Cogent Neurosciences), N-nitropyrazole derivatives (Texas A&M University System), KP-102 (Krenitsky Pharmaceuticals), cyclosporine A, therapeutic agents used in photodynamic therapy ii (e.g. VISUDYNE®; receptor-targeted PDT, Bristol-Myers Squibb, Co.; sodium porfimer for injection with PDT; verteporfin, QLT Inc.; growthporphin with PDT, Miravent Medical Technologies; thalaporfin sodium with PDT, Nippon Petroleum; and lutetium motexafine, Pharmacyclics, Inc.), antisense oligonucleotides (including, by way of example only, the products tested by Novagali Pharma SA and ISIS-13650, Isis Pharmaceuticals), and combinations thereof.

An antibody (e.g., an anti-VEGF antibody engineered with cysteine) or an antibody conjugate (e.g., a monodisperse HA conjugate) of the invention can be used in combination with a therapy or surgical procedure for the treatment of an eye disease (e.g., AMD, DME, DR, and RVO) including, for example, laser photocoagulation (eg, panretinal photocoagulation (PRP)), laser treatment of drusen, macular hole surgery, macular translocation surgery, implantable miniature telescopes, Phi motion angiography (also known as microlaser therapy and or feeder vessel angiography) , proton beam bombardment, microstimulation therapy, retinal detachment surgery and vitreous surgery, sphere indentation surgery, submacular surgery, transpupillary thermotherapy, photosystem I therapy, RNA interference (RNAi), extracorporeal reopheresis (also known as membrane differential filtration and reotherapy ii), microchip implantation, stem cell therapy, gene replacement therapy, ribozyme-based gene therapy (including gene therapy using the hypoxia response element, Oxford Biomedica; Lentipak, Genetix; and PDEF gene therapy, GenVec), photoreceptor/retinal cell transplantation (including transplantable retinal epithelial cells, Diacrin, Inc.; retinal cell transplant, Cell Genesys, Inc.), acupuncture, and combinations thereof.

In some instances, an antibody (e.g., an anti-VEGF antibody engineered with cysteine) or an antibody conjugate (e.g., a monodisperse HA conjugate) of the invention may be administered in combination with an anti-angiogenic agent to treat an ocular disease (e.g., AMD, DME, DR, or OBC) . Any suitable anti-angiogenic agent may be used in combination with an antibody (eg, an anti-VEGF antibody engineered with cysteine) or an antibody conjugate of the invention, including, but not limited to, those listed in Carmeliet et al. Nature 407:249-257, 2000. In some embodiments, the anti-angiogenic agent is a VEGF antagonist, including but not limited to an anti-VEGF antibody (e.g., anti-VEGF Fab LUCENTIS® (ranibizumab), RTH-258 (formerly ESBA -1008, anti-VEGF single chain antibody fragment; Novartis), or a bispecific anti-VEGF antibody (e.g., a bispecific anti-VEGF/anti-angiopoietin 2 antibody such as RG-7716; Roche)), a recombinant soluble receptor fusion protein (e.g. , EYLEA® (aflibercept)), VEGF variant, soluble VEGF fragment, VEGF-blocking aptamer (eg, pageptanib) or VEGF, anti-VEGF neutralizing antibody, small molecule tyrosine kinase inhibitor VEGF, anti-VEGF DARPin® (eg, abitsipar pegol) , small interfering RNAs that inhibit VEGF or VEGF expression, VEGF tyrosine kinase inhibitor (e.g. 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline (ZD6474), 4-(4-fluoro-2-methylindol-5-yloxy )-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline (AZD2171), vatalanib (PTK787), semaxaminib (SU5416; SUGEN), and SUTENT® (sunitinib)), and combinations thereof. In some cases, the bispecific anti-VEGF antibody binds to a second biological molecule, including, but not limited to, IL-1β; IL-6; IL-6R; TsFR (for example, PDGF-BB); angiopoietin; angiopoietin 2; Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin; apelin/APJ; erythropoietin; complement factor D; TNF-α; HtrA1; VEGF receptor (eg, VEGF1, VEGF2, VEGF3, mbVEGF, or sVEGF); ST-2 receptor; and proteins genetically associated with the risk of age-related macular degeneration (AMD), such as complement pathway components C2, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; SETP; LIPC; COL10A1; and TNFRSF10A. For example, in some instances, the additional compound is a bispecific antibody (e.g., an anti-VEGF/anti-Ang2 bispecific antibody such as RG-7716 or any anti-VEGF/anti-Ang2 bispecific antibody disclosed in WO 2010/069532 or WO 2016 /073157, or its variant.

Other suitable anti-angiogenic agents that can be administered in combination with an antibody (eg, an anti-VEGF antibody engineered by cysteine injection) or an antibody conjugate (eg, a monodisperse HA conjugate) of the invention for the treatment of an ocular disease (eg, AMD, DME, DR, or OBC) include corticosteroids, angiostatic steroids, anecortava acetate, angiostatin, endostatin, tyrosine kinase inhibitors, matrix metalloproteinase (MMP) inhibitors, insulin-like growth factor-binding protein-3 (IGFBP3), stromal cell factor (SDF-1) antagonists (e.g. , anti-SDF-1 antibodies), pigment epithelial factor (PEDF), gamma-secretase, delta-like ligand-4, integrin antagonists, hypoxia-inducible factor (HIF)-1α antagonists, CK2 protein kinase antagonists, agents that inhibit stem cells (eg, endothelial progenitor cells) at the site of neovascularization (eg, antibody against vascular endothelial cadherin (CD-144) and/or anti-SDF-1 antibody), and combinations thereof.

In another example, in some cases, an antibody (e.g., an anti-VEGF antibody engineered by introducing cysteine) or an antibody conjugate (e.g., a monodisperse HA conjugate) of this invention may be administered in combination with an agent that has anti-neovascularization activity to treat a disease. eye (eg, AMD, DMO, DR, or OBC) such as an anti-inflammatory drug, mammalian target of rapamycin (mTOR) inhibitor (eg, rapamycin, AFINITOR® (everolimus), and TORISEL® (temsirolimus)), cyclosporine, antagonist tumor necrosis factor (TNF) (eg, anti-TNF-α antibody or antigen-binding fragment thereof (eg, infliximab, adalimumab, certolizumab pegol, and golimumab) or soluble receptor fusion protein (eg, etanercept)), anti-complement agent, non-steroidal anti-inflammatory drug ( NSAIDs), or combinations thereof.

As another example, in some instances, an antibody (eg, an anti-VEGF antibody engineered by introducing cysteine) or an antibody conjugate (eg, a monodisperse HA conjugate) of the invention may be administered in combination with an agent that is neuroprotective and can potentially reduce progression of dry AMD to wet AMD, such as a class of drugs called "neurosteroids" which include drugs such as dehydroepiandrosterone (DEA) (trade names: PRASTERA™ and FIDELIN®), dehydroepiandrosterone sulfate, and pregnenolone sulfate.

Any suitable AMD therapeutic agent may be administered as an additional therapeutic agent in combination with an antibody (eg, cysteine engineered anti-VEGF antibody) or antibody conjugate (eg, monodisperse HA conjugate) of the invention for the treatment of an ocular disease (eg, AMD). , DMO, DR, or OBC), including, but not limited to, a VEGF antagonist, for example, an anti-VEGF antibody (eg, LUCENTIS® (ranibizumab), RTH-258 (formerly ESBA-1008, an anti-VEGF single chain antibody fragment; Novartis), or an anti-VEGF bispecific antibody (eg, anti-VEGF/anti-angiopoietin 2 bispecific antibody such as RG-7716; Roche)), soluble VEGF receptor fusion protein (eg, EYLEA® (aflibercept)), anti- VEGF DARPin® (eg, abicipar pegol; Molecular Partners AG/Allergan), or anti-VEGF aptamer (eg, MACUGEN® (pegaptanib sodium)); platelet-derived growth factor (TcFR) antagonist, eg, anti-TsFR antibody, anti-PDGFR antibody (eg, REGN2176-3), anti-PDGF-BB pegylated aptamer (eg, FOVISTA®; Ophthotech/Novartis), soluble PDGFR receptor fusion protein , or a dual TcFR/VEGF antagonist (eg, a small molecule inhibitor (eg, DE-120 (Santen) or X-82 (TyrogeneX)) or a bispecific anti-TcFR/anti-VEGF antibody)); VISUDYNE® (verteporfin) in combination with photodynamic therapy; antioxidant; complement system antagonist, eg, complement factor C5 antagonist (eg, small molecule inhibitor (eg, ARC-1905; Opthotech) or anti-C5 antibody (eg, LFG-316; Novartis), properdin antagonist (eg, anti-properdin antibody, eg, CLG-561; Alcon), or a complement factor D antagonist (eg, anti-complement factor D antibody, eg lampalizumab; Roche)); rhodopsin cycle modifier (eg, emixustat hydrochloride); squalamine (eg, OHR-102; Ohr Pharmaceutical); vitamin and mineral supplements (eg, those described in Study 1 (AREDS1; zinc and/or antioxidants) and Age-Related Eye Disease Study 2 (AREDS2; zinc, antioxidants, lutein, zeaxanthin, and/or omega-3 fatty acids)) ; cell therapy, for example, NT-501 (Renexus); PH-05206388 (Pfizer), huCNS-SC cell transplant (StemCells), CNTO-2476 (Janssen), OpRegen (Cell Cure Neurosciences), or MA09-hRPE cell transplant (Ocata Therapeutics); tissue factor antagonist (eg, hl-con1; Iconic Therapeutics); an alpha-adrenergic receptor agonist (eg brimonidine tartrate); a peptide vaccine (eg S-646240; Shionogi); an amyloid-beta antagonist (eg, an anti-amyloid-beta monoclonal antibody, eg, GSK-933776); an S1P antagonist (eg, an anti-S1P antibody, eg iSONEP™; Lpath Inc); ROBO4 antagonist (eg, anti-ROBO4 antibody, eg DS-7080a; Daiichi Sankyo); a lentiviral vector expressing endostatin and angiostatin (eg, RetinoStat); and any combination of them. In some cases, AMD therapeutic agents (including any of the previous AMD therapeutic agents) may be co-formulated. For example, the anti-PDGFR antibody REGN2176-3 can be co-formulated with aflibercept (EYLEA®). In some instances, such a co-formulation may be administered in combination with an antibody of the invention. In some cases, the eye disease is AMD (eg, wet AMD).

An antibody (eg, an anti-VEGF antibody engineered with cysteine) or an antibody conjugate (eg, a monodisperse HA conjugate) of the invention may be administered in combination with LUCENTIS® (ranibizumab) to treat an ocular disorder (eg, AMD, DME, DR, or RVO). ). LUCENTIS® (ranibizumab) can be administered, for example, at a dose of 0.3 mg/eye or 0.5 mg/eye by intravitreal injection, for example, every month. In some cases, the eye disease is AMD (eg, wet AMD).

An antibody (for example, an anti-VEGF antibody engineered with cysteine) or an antibody conjugate (for example, a monodisperse HA conjugate) of the invention can be administered in combination with EYLEA® (aflibercept) to treat an eye disease (for example, AMD, DME, DR, or RVO). ). EYLEA® (aflibercept) can be administered, for example, at a dose of 2 mg/eye by intravitreal injection, for example, every four weeks (Q4W), or Q4W for the first three months, followed by bimonthly injections as a maintenance dose . In some cases, the eye disease is AMD (eg, wet AMD).

An antibody (eg, an anti-VEGF antibody engineered with cysteine) or an antibody conjugate (eg, a monodisperse HA conjugate) of the invention can be administered in combination with MACUGEN® (pegaptanib sodium) to treat an ocular disorder (eg, AMD, DME, DR, or OVS). MACUGEN® (pegaptanib sodium) can be administered, for example, at a dose of 0.3 mg/eye by intravitreal injection every six weeks. In some cases, the eye disease is AMD (eg, wet AMD).

An antibody (cysteine-engineered anti-VEGF antibody) or antibody conjugate (e.g. monodisperse HA conjugate) of the invention can be administered in combination with VISUDYNE® (verteporfin) in combination with photodynamic therapy for the treatment of eye disease (e.g. AMD, DME, DR or OBC). VISUDYNE® may be administered, for example, by intravenous infusion at any suitable dose (eg, 6 mg/m ² body surface area) and may be administered once every three months (eg, over a 10 minute infusion). In some cases, the eye disease is AMD (eg, wet AMD).

An antibody (eg, an anti-VEGF antibody engineered with cysteine) or an antibody conjugate (eg, a monodisperse HA conjugate) of the invention may be administered in combination with a TcFR antagonist to treat an ocular disease (eg, AMD, DME, DR, or RVO). Exemplary TsFR antagonists that can be used in combination with an antibody of the invention include an anti-TsFR antibody, an anti-PDGFR antibody, a small molecule inhibitor (e.g. squalamine), an anti-PDGF-B pegylated aptamer such as FOVISTA® (E10030; Ophthotech/ Novartis), or a dual TcFR/VEGF antagonist (eg, a small molecule inhibitor (eg, DE-120 (Santen) or X-82 (TyrogeneX)) or a bispecific anti-TcFR/anti-VEGF antibody). For example, FOVISTA® can be administered as adjuvant therapy with an antibody of the invention. FOVISTA® can be administered at any suitable dose, for example, from 0.1 mg/eye to 2.5 mg/eye, for example, 0.3 mg/eye or 1.5 mg/eye, for example, by intravitreal injection, for example, every four weeks (Q4W). OHR-102 (squalamine lactate ophthalmic solution, 0.2%) can be administered via eye drops, for example twice a day. OHR-102 may be administered in combination with VEGF antagonists such as LUCENTIS® or EYLEA®. In some embodiments, an antibody conjugate of the invention may be administered in combination with OHR-102, LUCENTIS®, and/or EYLEA®. In some cases, the eye disease is AMD (eg, wet AMD).

An antibody (eg, an anti-VEGF antibody engineered with cysteine) or an antibody conjugate (eg, a monodisperse HA conjugate) of the invention can be administered in combination with RTH-258 to treat an ocular disorder (eg, AMD, DME, DR, or RVO). PTH-258 can be administered, for example, by intravitreal injection or ophthalmic infusion. For intravitreal injection, RTH-258 may be administered at any suitable dose (eg, 3 mg/eye or 6 mg/eye), eg, once every four weeks (Q4W) for the first three months, as a loading dose, with subsequent injection every 12 weeks (Q12W) or every eight weeks (Q8W) as a maintenance dose. In some cases, the eye disease is AMD (eg, wet AMD).

An antibody (eg, an anti-VEGF antibody engineered with cysteine) or an antibody conjugate (eg, a monodisperse HA conjugate) of the invention may be administered in combination with pegol abicipar to treat an eye disease (eg, AMD, DME, DR, or OBC). Abicipar pegol can be administered, for example, by intraviteral injection. Abicipar pegol can be administered at any suitable dose (eg, 1 mg/eye, 2 mg/eye, 3 mg/eye, 4 mg/eye, or 4.2 mg/eye), eg, once every four weeks (Q4W) during the first three months, as a loading dose, followed by an injection every 12 weeks (Q12W) or every eight weeks (Q8W) as a maintenance dose. In some cases, the eye disease is AMD (eg, wet AMD).

Any suitable DME and/or DR therapeutic agent may be administered in combination with an antibody (e.g., cysteine engineered anti-VEGF antibody), antibody conjugate (e.g., monodisperse HA conjugate) of the invention for the treatment of an ocular disease (e.g., AMD, DME). , DR, or OBC), including but not limited to a VEGF antagonist (eg, LUCENTIS® or EYLEA®), a corticosteroid (eg, a corticosteroid implant (eg, OZURDEX® (intravitreal dexamethasone implant) or ILUVIEN® (intravitreal fluocinolone acetonide implant) ) or a corticosteroid formulated for administration by intravitreal injection (eg, triamcinolone acetonide)), or combinations thereof. In some cases, the eye disease is DMO and/or DR.

An antibody (eg, an anti-VEGF antibody engineered with cysteine) or an antibody conjugate (eg, a monodisperse HA conjugate) of the invention can be administered in combination with LUCENTIS® (ranibizumab) to treat DME and/or DR (eg, NPDR or PDR). ). LUCENTIS® (ranibizumab) can be administered, for example, at a dose of 0.3 mg/eye or 0.5 mg/eye by intravitreal injection, for example, every four weeks (Q4W).

An antibody (e.g. an anti-VEGF antibody engineered with cysteine) or an antibody conjugate (e.g. a monodisperse HA conjugate) of the invention can be administered in combination with EYLEA® (aflibercept) to treat DME and/or DR (e.g. NPDR or PDD) . EYLEA® (aflibercept) can be administered, for example, at a dose of 2 mg/eye by intravitreal injection, for example, every four weeks (Q4W), or Q4W for the first five months, followed by injections once every eight weeks (Q8W) in maintenance dose.

An antibody (eg, an anti-VEGF antibody engineered with cysteine) or an antibody conjugate (eg, a monodisperse HA conjugate) of the invention may be administered in combination with an OZURDEX® (dexamethasone intravitreal implant) for the treatment of DME and/or DR. OZURDEX® can be administered as an intravitreal implant with 0.7 mg dexamethasone, which can be administered every six months.

An antibody (eg, cysteine engineered anti-VEGF antibody) or antibody conjugate (eg, monodisperse HA conjugate) of the invention can be administered in combination with ILUVIEN® (dexamethasone intravitreal implant) to treat DME and/or DR. OZURDEX® can be administered as an intravitreal implant with 0.19 mg of fluorocinolone acetonide, which can be released at a rate of 0.25 µg/day for up to about 36 months.

In some cases, a TAO/PRN (treat and observe/as needed) regimen or a TAE (dose interval treatment) regimen may be used to administer an anti-AMD therapeutic agent (eg, ranibizumab or aflibercept) into combination with an antibody (eg, an anti-VEGF antibody engineered with cysteine) or an antibody conjugate (eg, a monodisperse HA conjugate) of the invention. For the TAO/PRN regimen, after initial intravitreal injections every four weeks (Q4W) (typically for about 3 months), the subject was observed monthly or once a month (or even at longer intervals) with injections administered when signs appeared. disease activity (eg, decreased visual acuity or fluid as assessed by optical coherence tomography (OCT)). For the TAE regimen, the subject may receive treatment every four weeks (Q4W), followed by an extension of the treatment interval at a fixed number of weeks (e.g., 2 weeks) for each subsequent visit up to a maximum interval (e.g., every 6 weeks, every 8 weeks, every 10 weeks, or every 12 weeks). The eye(s) can be observed and treated at each visit, even if there is no evidence of disease activity. If the macula becomes moist (eg by OCT), the injection interval may be shortened (eg -2 weeks) until the macula is dry again. In some cases, the eye disease is AMD (eg, wet AMD).

Such combination therapies as previously mentioned encompass combined administration (in which two or more therapeutic agents are included in the same or separate compositions) as well as separate administration, in which case administration of an antibody or antibody conjugate of the invention may occur before, concomitantly and/or after administration of an additional therapeutic agent or agents. In one embodiment, administration of the antibody or antibody conjugate and administration of an additional therapeutic agent occurs every about one, two, three, four, or five months, or every about one, two, or three weeks, or every about one, two, three, four, five or six days.

An antibody (e.g., an anti-VEGF antibody engineered with cysteine) or an antibody conjugate (e.g., a monodisperse HA conjugate) of the invention (and any additional therapeutic agent) for the prevention or treatment of an eye disease or condition may be administered by any suitable route, including , but not limited to, for example, ocular, intraocular, and / or intravitreal injection, and / or injection into the periscleral region, and / or injection into the sub-Tenon space, and / or injection into the suprachoroidal space, and / or topical application in the form of eye drops and/or ointments. Such antibodies or antibody conjugates can be delivered by various methods, for example, intravitreally in the form of a device, and/or depot, which provides a slow release of the compound into the vitreous body, including those methods described in the references, such as Intraocular Drug Delivery, Jaffe, Jaffe, Ashton, and Pearson, editors, Taylor & Francis (March 2006). In one example, the device may be in the form of a mini-pump and/or matrix and/or passive diffusion system and/or encapsulated cells that release compounds over an extended period of time (Intraocular Drug Delivery, Jaffe, Jaffe, Ashton, and Pearson, editors, Taylor & Francis (March 2006) Additional approaches that may be used are described in section G below.

Compositions for ocular, intraocular or intravitreal administration can be obtained using methods and using excipients known in the art. An important feature for effective treatment is proper penetration through the eye. Unlike diseases of the anterior eye, where drugs can be delivered locally, retinal disease generally has the advantage of a more site-specific approach. Eye drops and ointment rarely penetrate the back of the eye, and the blood-ophthalmic barrier prevents the penetration of systemically administered drugs into the ocular tissue. Accordingly, the preferred method for delivering drugs for the treatment of retinal diseases such as AMD and CNV is generally direct intravitreal injection. Intravitreal injections are usually repeated at intervals that depend on the condition of the patient and the properties and half-life of the drug delivered. Additional approaches that may be used are described in section G below.

The amount of antibody or antibody conjugate that will be effective in treating a particular disease or condition of the eye will depend on the nature of the disease or condition, and can be determined by standard clinical methods. Where possible, it is desirable to determine the dose-response curve of the pharmaceutical compositions of the invention first in vitro and then in suitable animal model systems prior to testing in humans.

Additional suitable routes of administration include parenteral, intrapulmonary and intranasal and, if necessary for topical therapy, intralesional administration. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal and subcutaneous administration. Dosing may be by any convenient means, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is transient or continuous. This document discusses various dosing regimens, including, without limitation, single or multiple administrations at time intervals, in the form of a single dose of administration and pulsed infusion. In some cases, the antibody conjugate of the invention may be administered intravenously, intramuscularly, intradermally, transdermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, into the prostate, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, rectally, topically, intratumorally, peritoneally, intraventricularly , subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraorbitally, orally, topically, transdermally, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by localized perfusion directly through target cells, using a catheter, by lavage, in creams or in lipid formulations.

For the prevention or treatment of a disease, the appropriate dose of an antibody (eg, an anti-VEGF antibody engineered by introducing cysteine) or an antibody conjugate (eg, a monodisperse HA conjugate) of the invention (whether used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered prophylactically or therapeutically, previous therapy, the patient's medical history, sensitivity to the antibody, and the prescription of the attending physician. An antibody (eg, an anti-VEGF antibody engineered by introducing cysteine) or an antibody conjugate (eg, a monodisperse HA conjugate) is suitably administered to a patient at one time or during a course of treatment. Depending on the type and severity of the disease, from about 1 mcg/kg to 15 mg/kg (eg, 0.1 mg/kg, 0.2 mg/kg, 0.4 mg/kg, 0.6 mg/kg , 0.8 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg /kg or 10 mg/kg) of the antibody or antibody conjugate may be the initial intended dosage for administration to the patient, for example, by one or more separate injections, or by continuous infusion. In some embodiments, the amount of antibody or antibody conjugate used is from about 0.01 mg/kg to about 45 mg/kg, from about 0.01 mg/kg to about 40 mg/kg, from about 0.01 mg/kg to about 35 mg/kg, about 0.01 mg/kg to about 30 mg/kg, about 0.01 mg/kg to about 25 mg/kg, about 0.01 mg/kg to about 20 mg/kg , about 0.01 mg/kg to about 15 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.01 mg/kg to about 5 mg/kg, or about 0 .01 mg/kg to about 1 mg/kg. To prepare antibody conjugates, dose selection may be based on the weight of the antibody component in the conjugate. One typical daily dose may range from about 1 μg/kg to 100 mg/kg or more, depending on the above factors. In the case of repeated administrations over several days or more, depending on the condition, the treatment is usually carried out until the desired degree of suppression of the symptoms of the disease is achieved.

In some embodiments, the implementation of the methods may further include additional therapy. Complementary therapy may be radiation therapy, surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the above. Additional therapy may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, the implementation of additional therapy is the introduction of a small molecular weight enzymatic inhibitor or anti-metastatic agent. In some embodiments, the adjunctive therapy is the administration of agents that limit side effects (eg, agents designed to reduce the occurrence and/or severity of side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the implementation of additional therapy is a surgical operation. In some embodiments, the complementary therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy may be a separate administration of one or more of the therapeutic agents described above.

It should be understood that any of the above compositions or any of the therapeutic methods can be carried out using the immunoconjugate of the invention instead of or in addition to the anti-VEGF antibody.

It should be understood that any of the above compositions or any of the therapeutic methods can be carried out using an antibody conjugate of the invention (eg, any described herein, for example, in section G below).

F. Industrial Products

In another aspect of the invention, there is provided an article of manufacture containing materials useful in the treatment and/or prevention of the disorders described above. The article of manufacture comprises a container and a label or package insert applied to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, and the like. Containers can be made from various materials such as glass or plastic. The container contains a composition, alone or in combination with another composition, effective in the treatment, prevention and/or diagnosis of a pathological condition, and may have a sterile inlet (for example, the container may be a bag for solutions for intravenous administration or a vial with a stopper pierceable with using a hypodermic needle). At least one active agent in the composition is an antibody composition (eg, an antibody (eg, an anti-VEGF antibody engineered by introducing cysteine) or a conjugate of such an antibody) according to the invention. The label or package insert indicates that the composition is used for the selected pathological condition. In addition, the industrial product may contain (a) the first container containing the composition, while the composition contains an antibody or a composition of such antibodies according to the invention; and (b) a second container containing the composition, wherein the composition contains an additional cytotoxic or other therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert stating that the composition(s) may be used to treat a particular pathological condition. Alternatively or additionally, the product may further comprise a second (or third) container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BVDI), phosphate buffered saline, Ringer's solution, and dextrose solution. Additionally, other substances desirable from a commercial and consumer point of view may be included, including other buffers, diluents, filters, needles and syringes.

It is understood that any of the above industrial products may contain any of the antibodies or conjugates of such antibodies described in this document, and/or any additional therapeutic agents.

G. Delivery approaches for extended-release ophthalmic drugs

The invention provides ophthalmic compositions that can be used to deliver sustained release antibodies (eg, anti-VEGF antibodies (including any anti-VEGF antibody described herein, such as G6.31 AARR)) to the eye. For example, the invention provides antibody conjugates (e.g., monodisperse HA conjugates) that contain an anti-VEGF antibody as described herein (e.g., Fab, Fab-C, or cysteine engineered antibody conjugates (e.g., thio-Fab) ). The invention also relates to devices that can be used for ocular administration of the antibody or antibody conjugate described herein. In addition, the invention provides pharmaceutical compositions that contain the antibodies or antibody conjugates described herein. These compositions can be used in any of the therapeutic methods described herein, for example, methods for treating eye disease (e.g., AMD (e.g., wet AMD), DME, DR (e.g., NPDR or PDR), or RVO (e.g., RCVO or OVVS)).

1. Antibody conjugates

The invention provides antibody conjugates that contain an antibody (eg, an anti-VEGF antibody) and a monodisperse polymer covalently attached to the antibody. An antibody (eg, an anti-VEGF antibody) may be covalently attached to the monodisperse polymer in an irreversible or reversible manner. Any suitable monodisperse polymer may be used, including those described herein or others known in the art.

The invention provides an antibody conjugate that contains an antibody and a monodisperse polymer (eg, monodisperse HA polymer) covalently attached to the antibody. The polymer may have a polydispersity index (PDI) of about 1.1 or less. It should be understood that the PDI value may refer to the PDI value of the polymer used to prepare the antibody conjugate. For example, in some embodiments, the polymer has a PDI of 1.0 to about 1.1 (e.g., 1 to about 1.1, 1 to about 1.09, 1 to about 1 to about 1.07, from 1 to about 1.06, 1 to about 1.05, 1 to about 1.04, 1 to about 1.03, 1 to about 1.02, 1 to about 1.01, 1 to about 1.005, about 1.001 to about 1.1, about 1.001 to about 1.1, about 1.001 to about about 1.001 to about 1.08, about 1.001 to about 1.07, about 1.001 to about 1 .06, about 1.001 to about 1.05, about 1.001 to about 1.04, about 1.001 to about 1.03, about 1.001 to about 1.02, about 1.001 to about 1.01, from about 1.001 to about 1.005, about 1.001 to about 1.004, about 1.001 to about 1.003, about 1.001 to about 1.002, about 1.0001 to about 1.1, about 1.0001 to about 1.09, from about 1.0001 to about 1.08, about 1.0001 to about 1.07, about 1.0001 to about 1.06, about 1.0001 to about 1.05, about 1.0001 to about 1 .04, from about 1.0001 to about 1.03 , from about 1.0001 to about 1.02, from about 1.0001 to about 1.01, from about 1.0001 to about 1.005, from about 1.0001 to about 1.004, from about 1.0001 to about 1.003, about 1.0001 to about 1.002, or about 1.0001 to about 1.005).

For example, in some embodiments, the monodisperse polymer (eg, monodisperse HA polymer) has a PDI of 1.001, about 1.0001, about 1.00001, about 1.000001, about 1.0000001, or less. In some embodiments, the monodisperse polymer (e.g., monodisperse HA polymer) has a PDI of 1.0, about 1.001, about 1.002, about 1.003, about 1.004, about 1.005, about 1.006, about 1.007, about 1.008, about 1.009, about 1 .01, about 1.011, about 1.012, about 1.013, about 1.014, about 1.015, about 1.016, about 1.017, about 1.018, about 1.019, about 1.02, about 1.021, about 1.022, about 1.023, about 1.024, about 1.025, about 1.026, about 1.027, about 1.028, about 1.029, about 1.03, about 1.031, about 1.032, about 1.033, about 1.034, about 1.035, about 1.036, about 1.037, about 1.038, about 1.039, about 1.04, about 1.041, about 1.042, about 1.043, about 1.044, about 1.045, about 1.046, about 1.047, about 1.048, about 1.049, about 1.05, about 1.051, about 1.052, about 1.053, about 1.054, about 1.055, about 1.056, about 1.057, about 1.058, about 1.059, about 1.06, about 1.061, about 1.062, about 1.063, about 1.064, about 1.065, about 1.066, about 1.067, about 1.068, ok about 1.069, about 1.07, about 1.071, about 1.072, about 1.073, about 1.074, about 1.075, about 1.076, about 1.077, about 1.078, about 1.079, about 1.08, about 1.081, about 1.082, about 1.083, about or about 1.1. In some embodiments, the polymer (eg, HA polymer) has a PDI of about 1.001.

The monodisperse polymer may be a hydrophilic polymer or a hydrophobic polymer. It should be understood that the hydrophilic polymer may be a water-soluble polymer. Any suitable hydrophilic polymer can be used, for example, the hydrophilic polymer is described in International Patent Application Publication No. WO 2011/066417. and/or Pelegri-O'Day et al. J. Am. Chem. soc. 136:14323-14332, 2014, which are incorporated herein by reference in their entirety. Illustrative non-limiting hydrophilic polymers that can be used include hyaluronic acid, (HA), polyethylene glycol (PEG; also known as poly(ethylene glycol)) (e.g., linear PEG, branched PEG, comb PEG, and dendritic PEG), poly[ethylene oxide]. )-co-(methylene-ethylene oxide)], poly(poly(ethylene glycol) methyl ether methacrylate) (pPEGMA), agarose, alginate, carrageenans, carboxymethylcellulose, cellulose, cellulose derivatives, chitosan, chondroitin sulfate, collagen, dermatan sulfate, dextran, dextran sulfate , fibrin, fibrinogen, fibronectin, fucoidan, gelatin, glycosaminoglycans (GAGs), glycopolymer, heparin, heparin sulfate, highly branched polysaccharide (e.g. galactose dendrimer), keratan sulfate, methylcellulose, hydroxypropyl methylcellulose (HPMC), poly(N-(2-hydroxypropyl )methacrylamide) (pHPMA), pectins, pectin derivatives, pentosan polysulfate, starch, hydroxyethyl starches (HES), styrene, vitronectin, poly(acrylic acid), poly(meth acrylic acid), poly(acrylamide), poly(acrylic acid), poly(amines), poly(amino acids), poly(carboxybetaine) (PCB), polyelectrolytes, poly(glutamic acid) (PGA), poly(glycerol) (PG ) (for example, linear, medium functional, hyperbranched, or linear hyperbranched PG), poly(maleic acid), poly(2-oxazoline) (POZ), poly(2-ethyl-2-oxazoline, polysialic acid (PSA), polystyrene, polystyrene derivatives (eg charged polystyrene derivatives), poly(styrenesulfonate-co-RESMA), polyvinylpyrrolidone (PVP), poly(N-acryloylmorpholine) (pNAcM), and their copolymers. In some cases, the polymer is a hydrophobic polymer, such as polylactide co-glycolide (PLGA), polylactide (PLA), and polyglycolide (PGA). The polymer may be biodegradable and/or biocompatible. In particular embodiments, the polymer is HA.

By way of example, a monodisperse polymer (e.g., HA polymer) may contain any suitable number of monomers, e.g., 2 to about 1×10 ⁴ monomers (e.g., about 10, about 50, about 100, about 200, about 300, about 400 , about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000 or about 1×10 ⁴ monomers) , or more. For example, a polymer (e.g., a HA polymer) may contain from about 50 to about 250 monomers, from about 50 to about 500 monomers, from about 50 to about 1000 monomers, from about 50 to about 2000 monomers, from about 50 to about 3000 monomers. , from about 50 to about 4000 monomers, from about 50 to about 5000 monomers, from about 50 to about 6000 monomers, from about 50 to about 7000 monomers, from about 50 to about 8000 monomers, from about 50 to about 9000 monomers, from about 50 to about 10,000 monomers, about 100 to about 250 monomers, about 100 to about 500 monomers, about 100 to about 1000 monomers, about 100 to about 2000 monomers, about 100 to about 3000 monomers, about 100 up to about 4000 monomers, from about 100 to about 5000 monomers, from about 100 to about 6000 monomers, from about 100 to about 7000 monomers, from about 100 to about 8000 monomers, from about 100 to about 9000 monomers, from about 100 to about 10,000 monomers, about 250 to about 500 monomers, from about 250 to about 1000 monomers, about 250 to about 2000 monomers, about 250 to about 3000 monomers, about 250 to about 4000 monomers, about 250 to about 5000 monomers, about 250 to about 6000 monomers, about 250 to about 7000 monomers about 250 to about 8000 monomers about 250 to about 9000 monomers about 250 to about 10000 monomers about 500 to about 1000 monomers about 500 to about 2000 monomers about 500 to about 3000 monomers, from about 500 to about 4000 monomers, from about 500 to about 5000 monomers, from about 500 to about 6000 monomers, from about 500 to about 7000 monomers, from about 500 to about 8000 monomers, from about 500 to about 9000 monomers, or from about 500 to about 10,000 monomers. In some cases, the polymer (eg, HA polymer) may contain about 500 monomers.

The invention provides an antibody conjugate that contains an antibody (eg, an anti-VEGF antibody such as G6.31 AARR) covalently attached to a monodisperse HA polymer. Such antibody conjugates are sometimes referred to herein as "monodisperse HA conjugates". The monodisperse HA polymer may have a polydispersity index (PDI) of about 1.1 or less. For example, in some embodiments, the monodisperse HA polymer has a PDI of 1.0 to about 1.1 (e.g., 1 to about 1.1, 1 to about 1.09, 1 to about 1.08, 1 to about 1.07, 1 to about 1.06, 1 to about 1.05, 1 to about 1.04, 1 to about 1.03, 1 to about 1.02, 1 to about 1 .01, 1 to about 1.005, about 1.001 to about 1.1, about 1.001 to about 1.1, about 1.001 to about 1.09, about 1.001 to about 1.08, about 1.001 to about 1.07, from about 1.001 to about 1.06, from about 1.001 to about 1.05, from about 1.001 to about 1.04, from about 1.001 to about 1.03, from about 1.001 to about 1.02, from about 1.001 to about 1.01, about 1.001 to about 1.005, about 1.001 to about 1.004, about 1.001 to about 1.003, about 1.001 to about 1.002, about 1.0001 to about 1.1, from about 1 .0001 to about 1.09, about 1.0001 to about 1.08, about 1.0001 to about 1.07, about 1.0001 to about 1.06, about 1.0001 to about 1.05, from about 1.0001 to about 1.04, about 1.0001 to about 1.03, about 1.0001 to about 1.02, about 1.0001 to about 1.01, about 1.0001 to about 1.005, about 1.0001 to about 1.004 , about 1.0001 to about 1.003, about 1.0001 to about 1.002, or about 1.0001 to about 1.005).

For example, in some embodiments, the monodisperse HA polymer has a PDI of 1.001, about 1.0001, about 1.00001, about 1.000001, about 1.0000001, or less. In some embodiments, the monodisperse HA polymer has a PDI of 1.0, about 1.001, about 1.002, about 1.003, about 1.004, about 1.005, about 1.006, about 1.007, about 1.008, about 1.009, about 1.01, about 1.011, About 1.012 About 1.013 About 1.014 About 1.015 About 1.016 About 1.017 About 1.018 About 1.019 About 1.02 About 1.021 About 1.022 About 1.023 About 1.024 About 1.025 About 1.026 About 1.027 about 1.028, about 1.029, about 1.03, about 1.031, about 1.032, about 1.033, about 1.034, about 1.035, about 1.036, about 1.037, about 1.038, about 1.039, about 1.04, about 1.041, about 1.042, about 1.043, approx. 1.044, approx. 1.045, approx. 1.046, approx. 1.047, approx. 1.048, approx. 1.049, approx. 1.05, approx. 1.051, approx. 1.059, about 1.06, about 1.061, about 1.062, about 1.063, about 1.064, about 1.065, about 1.066, about 1.067, about 1.068, about 1.069, about 1.07, about 1.071, About 1.072 About 1.073 About 1.074 About 1.075 About 1.076 About 1.077 About 1.078 About 1.079 About 1.08 About 1.081 About 1.082 About 1.083 About 1.084 About 1.085 about 1088, about 1089, about 109, about 1091, about 1092, about 1093, about 1094, about 1095, about 1096, about 1097, about 1098, about 1099, or about 1.1. In some embodiments, the monodisperse HA polymer has a PDI of about 1.001.

In some instances, the monodisperse HA polymer has a molecular weight of about 2.5 megadaltons (MDa) or less (e.g., about 2.5 MDa or less, about 2.4 MDa or less, about 2.3 MDa or less, about 2.2 MDa or below, about 2.1 MDa or below, about 2.0 MDa or below, about 1.9 MDa or below, about 1.8 MDa or below, about 1.7 MDa or below, about 1.6 MDa or less, about 1.5 MDa or less, about 1.4 MDa or less, about 1.3 MDa or less, about 1.2 MDa or less, about 1.1 MDa or less, about 1.0 MDa or less , about 900 kDa or less, about 800 kDa or less, about 700 kDa or less, about 600 kDa or less, about 500 kDa or less, about 400 kDa or less, about 300 kDa or less, about 200 kDa or less, or about 100 kDa or less). In some instances, the HA polymer has a molecular weight of about 1 MDa or less (e.g., about 1.0 MDa or less, about 900 kDa or less, about 800 kDa or less, about 700 kDa or less, about 600 kDa or less, about 500 kDa or less, about 400 kDa or less, about 300 kDa or less, about 200 kDa or less, about 100 kDa or less). In some instances, the HA polymer has a molecular weight of about 25 kDa to about 2.5 MDa (e.g., about 25 kDa to about 2.5 MDa, about 25 kDa to about 2 MDa, about 25 kDa to about 1.5 MDa, from about 25 kDa to about 1 MDa, from about 25 kDa to about 900 kDa, from about 25 kDa to about 800 kDa, from about 25 kDa to about 700 kDa, from about 25 kDa to about 600 kDa, from about 25 kDa to about 500 kDa, from about 100 kDa to about 2.5 MDa, from about 100 kDa to about 2 MDa, from about 100 kDa to about 1.5 MDa, from about 100 kDa to about 1 MDa, from about 100 kDa up to about 900 kDa, from about 100 kDa to about 800 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 500 kDa, from about 250 kDa to about 2.5 MDa, about 250 kDa to about 2 MDa, about 250 kDa to about 1.5 MDa, about 250 kDa to about 1 MDa, about 250 kDa to about 900 kDa, from about 250 kDa to about 800 kDa, from about 250 kDa to about 700 kDa, from about 250 kDa to about 600 kDa Yes, about 250 kDa to about 500 kDa, about 500 kDa to about 2.5 MDa, about 500 kDa to about 2 MDa, about 500 kDa to about 1.5 MDa, about 500 kDa to about 1 MDa , from about 500 kDa to about 900 kDa, from about 500 kDa to about 800 kDa, from about 500 kDa to about 700 kDa, from about 500 kDa to about 600 kDa, from about 1 MDa to about 2.5 MDa, from about 1 MDa to about 2 MDa, from about 1 MDa to about 1.5 MDa, from about 1 MDa to about 1.25 MDa, from about 1.25 MDa to about 2.5 MDa, from about 1.25 MDa to about 2 MDa, from about 1.25 MDa to about 1.5 MDa, from about 1.5 MDa to about 2.5 MDa, from about 1.5 MDa to about 2 MDa, from about 1.5 MDa to about 1, 75 MDa, from about 1.75 MDa to about 2.5 MDa).

In some instances, the HA monodisperse polymer has a molecular weight of about 25 kD to about 500 kD (e.g., about 25 kD to about 500 kD, about 25 kD to about 450 kD, about 25 kD to about 400 kD, about 25 kD). kDa to about 350 kDa, about 25 kDa to about 300 kDa, about 25 kDa to about 300 kDa, about 25 kDa to about 250 kDa, about 25 kDa to about 200 kDa, about 25 kDa to about 150 kDa , about 25 kDa to about 100 kDa, about 25 kDa to about 50 kDa, about 40 kDa to about 500 kDa, about 40 kDa to about 450 kDa, about 40 kDa to about 400 kDa, from about 40 kDa up to about 350 kDa, from about 40 kDa to about 300 kDa, from about 40 kDa to about 300 kDa, from about 40 kDa to about 250 kDa, from about 40 kDa to about 200 kDa, from about 40 kDa to about 150 kDa, about 40 kDa to about 100 kDa, about 40 kDa to about 50 kDa, about 50 kDa to about 500 kDa, about 50 kDa to about 450 kDa, about 50 kDa to about 400 kDa, from about 50 kDa to about 350 kDa, from about 50 kDa to about 300 kDa, about 50 kDa to about 300 kDa, about 50 kDa to about 250 kDa, about 50 kDa to about 200 kDa, about 50 kDa to about 150 kDa, about 50 kDa to about 100 kDa, about 50 kDa to about 75 kDa, about 100 kDa to about 500 kDa, about 100 kDa to about 450 kDa, about 100 kDa to about 400 kDa, about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, about 100 kDa to about 300 kDa, about 100 kDa to about 250 kDa, about 100 kDa to about 200 kDa, about 100 kDa to about 150 kDa, about 150 kDa to about 500 kDa, about 150 kDa to about 450 kDa, about 150 kDa to about 400 kDa, about 150 kDa to about 350 kDa, about 150 kDa to about 300 kDa, about 150 kDa to about 300 kDa, from about 150 kD to about 250 kD, about 150 kD to about 200 kD, about 175 kD to about 500 kD, about 175 kD to about 450 kD, about 175 kD to about 400 kD, about 175 kD to about 350 kDa, from about 175 kDa to about 300 kDa, from about 175 kDa to about 300 kDa, from about 175-200 to about 250 kDa, from about 175 kDa to about 225 kDa, from about 200 kDa to about 500 kDa, from about 200 kDa to about 450 kDa, from about 200 kDa to about 400 kDa, about 200 kDa to about 350 kDa, about 200 kDa to about 300 kDa, about 200 kDa to about 300 kDa, about 200 kDa to about 250 kDa, or about 200 kDa to about 225 kDa).

In some instances, the monodisperse HA polymer has a molecular weight of about 100 kDa to about 250 kDa (e.g., about 100 kDa, about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa, about 150 kDa, about 160 kDa, about 170 kDa, about 180 kDa, about 190 kDa, about 200 kDa, about 210 kDa, about 220 kDa, about 230 kDa, about 240 kDa, or about 250 kDa). In specific cases, the HA polymer has a molecular weight of about 200 kDa.

Any of the preceding molecular weights may be the weight average molecular weight (also known as the weight average molecular weight).

In some instances, any of the prior monodisperse HA polymers is linear, ie, not cross-linked.

In other instances, the invention provides an antibody conjugate that contains an antibody (eg, an anti-VEGF antibody such as G6.31 AARR) covalently attached to a monodisperse HA polymer. Such antibody conjugates are sometimes referred to herein as "PEG conjugates". Any suitable PEG monodisperse polymer may be used. It should be understood that monodisperse PEG polymers may have different PDI values compared to monodisperse HA polymers. For example, commercially available PEG polymers may have a PDI below 1.1; thus, a PEG monodisperse polymer will have a different range of PDI values compared to a HA monodisperse polymer. For example, a PEG monodisperse polymer may have a PDI of about 1 to about 1.02 (e.g., a PDI of 1, about 1.001, about 1.002, about 1.003, about 1.004, about 1.005, about 1.006, about 1.007, about 1.008, about 1.009 , about 1.01, about 1.011, about 1.012, about 1.013, about 1.014, about 1.015, about 1.016, about 1.017, about 1.018, about 1.019, or about 1.02). The PEG may be a branched PEG, a star PEG, or a comb PEG. The PEG polymer may be, for example, a PEG tetramer, a PEG hexamer, or a PEG octamer. In some cases, the antibody conjugate contains an anti-VEGF antibody (eg, an anti-VEGF antibody described herein, such as G6.31 AARR) covalently attached to a PEG dendrimer. PEG polymers are commercially available from, for example, JenKem Technology, Quanta BioDesign, NOF America Corporation and others.

In some instances, the PEG monodisperse polymer has a molecular weight of about 1 kDa to about 500 kDa (e.g., about 1 kDa to about 500 kDa, about 1 kDa to about 450 kDa, about 1 kDa to about 400 kDa, about 1 kDa up to about 350 kDa, from about 1 kDa to about 300 kDa, from about 1 kDa to about 300 kDa, from about 1 kDa to about 250 kDa, from about 1 kDa to about 200 kDa, from about 1 kDa to about 150 kDa, about 1 kDa to about 100 kDa, about 1 kDa to about 50 kDa, about 10 kDa to about 500 kDa, about 10 kDa to about 450 kDa, about 10 kDa to about 400 kDa, from about 10 kDa to about 350 kDa, from about 10 kDa to about 300 kDa, from about 10 kDa to about 300 kDa, from about 10 kDa to about 250 kDa, from about 10 kDa to about 200 kDa, from about 10 kDa to about 150 kDa, from about 10 kDa to about 100 kDa, about 10 kDa to about 50 kDa, about 20 kDa to about 500 kDa, about 20 kDa to about 450 kDa, about 20 kDa to about 400 kDa, about 20 kDa to about 350 kDa, from about 20 kDa up to about 300 kDa, from about 20 kDa to about 300 kDa, from about 20 kDa to about 250 kDa, from about 20 kDa to about 200 kDa, from about 20 kDa to about 150 kDa, from about 20 kDa to about 100 kDa, about 20 kDa to about 75 kDa, about 30 kDa to about 500 kDa, about 30 kDa to about 450 kDa, about 30 kDa to about 400 kDa, about 30 kDa to about 350 kDa, from about 30 kDa to about 300 kDa, from about 30 kDa to about 300 kDa, from about 30 kDa to about 250 kDa, from about 30 kDa to about 200 kDa, from about 30 kDa to about 150 kDa, from about 40 kDa to about 500 kDa, from about 40 kDa to about 450 kDa, about 40 kDa to about 400 kDa, about 40 kDa to about 350 kDa, about 40 kDa to about 300 kDa, about 40 kDa to about 300 kDa, about 40 kDa to about 250 kDa, about 40 kDa to about 200 kDa, about 50 kDa to about 500 kDa, about 50 kDa to about 450 kDa, about 50 kDa to about 400 kDa, about 50 kDa to about 350 kDa, from about 50 kDa to about 300 kDa, from about 50 kDa to about 300 kDa Yes, from 50-200 kDa to about 250 kDa, or from about 50 kDa to about 225 kDa).

In some instances, the PEG monodisperse polymer has a molecular weight of about 5 kDa to about 250 kDa (e.g., about 1 kDa, about 5 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, 100 kDa, about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa, about 160 kDa, about 170 kDa, about 180 kDa, about 190 kDa, about 200 kDa, about 210 kDa, about 220 kDa, about 230 kDa, about 240 kDa, or about 250 kDa). In specific cases, the PEG polymer has a molecular weight of about 20 kDa. In other cases, the HA polymer has a molecular weight of about 40 kDa.

Any of the preceding molecular weights may be the weight average molecular weight (also known as the weight average molecular weight).

In some cases, the PEG monodisperse polymer is a PEG tetramer. PEG tetramers are commercially available from, for example, OF America SUNBRIGHT® PTE-400MA, PTE-200MA, PTE-100MA, and JenKem Technology USA 4-arm PEG maleimide (Cat. No. 4ARM-MAL). In some cases, the PEG tetramer has a pentaerythritol core. For example, in some cases, the PEG tetramer includes a structure of formula (I), where n is independently any suitable integer:

Formula I:

In another example, in some instances, the PEG monodisperse polymer is a PEG hexamer. PEG hexamers are commercially available, for example from JenKem Technology USA 6-arm PEG-amine (Cat. No. 6ARM(DP)-NH2HCl), or PEG hexamers from Quanta BioDesign. In some cases, PEG hexamers contain a dipentaerythritol core.

In some cases, the PEG monodisperse polymer is a PEG octamer. PEG octamers are commercially available, for example from the NOF America SUNBRIGHT® HGEO series or from JenKem Technology USA 8-arm PEG maleimide (Cat. No. 8ARM(TP)-MAL). In some cases, the PEG octamer may contain a core of three pentaerythritol. For example, in some cases, the PEG octamer includes the structure of formula (I), where n is independently any suitable integer:

Formula II:

In yet another example, in some cases, the PEG octamer contains a tripentaerythritol core.

It should be understood that any suitable conjugation approach, including those described herein and others known in the art, can be used to conjugate an anti-VEGF antibody of the invention to a monodisperse polymer. For example, the monodisperse polymer may be conjugated to any suitable protein functional group, including a primary amino group, a carboxyl group, a sulfhydryl group, or a carbonyl group. Any suitable reactive group can be used to target a functional group on a protein, e.g., carbodiimide (e.g., EDC), NHS ester, imidoester, pentafluorophenyl ether, hydroxymethyl phosphine, maleimide, haloacetyl (e.g., bromoacetyl or iodoacetyl), pyridyl disulfide, thiosulfonate, vinyl sulfone, hydrazine, alkoxyamine, diazirine, aryl azide, isocyanate, or others known in the art. See, e.g., Hermanson, ^Bioconjugate Techniques, 3rd Edition, 2013. In specific embodiments, HA (e.g., monodisperse HA) is modified using maleimide groups (HA-maleimide), and second, an antibody that contains a free thiol on cysteine ( e.g., Fab-C or a cysteine variant (e.g., THIOMAB™ or thio-Fab)) is contacted with HA-maleimide to form covalent HA-Fab conjugates, for example, as described in Example 1.

Any of the preceding antibody conjugates may have a hydrodynamic radius of about 5 nm to about 200 nm (e.g., about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 approx. 80 nm, approx. 90 nm, approx. 100 nm, approx. 110 nm, approx. 120 nm, approx. 130 nm, approx. 140 nm, approx. about 200 nm). In some cases, the antibody conjugate has a hydrodynamic radius of about 5 nm to about 150 nm (e.g., about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm , about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm or 150 nm). In some cases, the antibody conjugate has a hydrodynamic radius of about 5 nm to about 100 nm (e.g., about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm or about 100 nm). In some instances, the antibody conjugate has a hydrodynamic radius of about 5 nm to about 60 nm (eg, about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, or about 60 nm). In some cases, the antibody conjugate has a hydrodynamic radius of about 25 nm to about 35 nm (e.g., about 25 nm, about 26 nm, about 27 nm, about 28 nm, about 29 nm, about 30 nm, about 31 nm, about 32 nm, about 33 nm, about 34 nm or about 35 nm). In some cases, the hydrodynamic radius is about 28 nm.

In some instances, the antibody conjugate has a hydrodynamic radius of about 10 nm to about 200 nm, about 10 nm to about 180 nm, about 10 nm to about 160 nm, about 10 nm to about 140 nm, about 10 nm to about 120 nm, about 10 nm to about 100 nm, about 10 nm to about 80 nm, about 10 nm to about 60 nm, about 10 nm to about 50 nm, about 10 nm to about 40 nm, from about 10 nm to about 30 nm, about 20 nm to about 200 nm, about 20 nm to about 180 nm, about 20 nm to about 160 nm, about 20 nm to about 140 nm, about 20 nm to about 120 nm, from about 20 nm to about 100 nm, from about 20 nm to about 80 nm, from about 20 nm to about 60 nm, from about 20 nm to about 50 nm, from about 20 nm to about 40 nm, from about 20 about 30 nm to about 30 nm, about 30 nm to about 200 nm, about 30 nm to about 180 nm, about 30 nm to about 160 nm, about 30 nm to about 140 nm, about 30 nm to about 120 nm , from about 30 nm to about 100 nm, from about 30 nm to about 80 nm, from about 30 nm up to about 60 nm, from about 30 nm to about 50 nm, from about 30 nm to about 40 nm, from about 40 nm to about 200 nm, from about 40 nm to about 180 nm, from about 40 nm to about 160 nm, about 40 nm to about 140 nm, about 40 nm to about 120 nm, about 40 nm to about 100 nm, about 40 nm to about 80 nm, about 40 nm to about 60 nm, from about 40 nm to about 50 nm, from about 50 nm to about 200 nm, from about 50 nm to about 180 nm, from about 50 nm to about 160 nm, from about 50 nm to about 140 nm, from about 50 nm to about 120 nm, from about 50 nm to about 100 nm, about 50 nm to about 80 nm, about 50 nm to about 60 nm, about 60 nm to about 200 nm, about 60 nm to about 180 nm, about 60 nm to about 160 nm, about 60 nm to about 140 nm, about 60 nm to about 120 nm, about 60 nm to about 100 nm, or about 60 nm to about 80 nm.

In any of the preceding antibody conjugates, the antibody may be an antibody fragment that binds to VEGF, for example, a fragment of an anti-VEGF antibody described herein that binds to VEGF. In some cases, an anti-VEGF antibody is an anti-VEGF antibody constructed by introducing cysteine as described herein (see, for example, Section 1(8)(d) above). In some instances, the antibody fragment is selected from the group consisting of Fab, Fab', Fab-C, Fab'-SH, Fv, scFv, and (Fab') ₂ fragments. In special cases, the antibody fragment is a Fab, Fab', or Fab-C. In some cases, the antibody fragment is Fab-C.

Any of the prior antibody conjugates may have an ocular half-life that is increased relative to a reference antibody that is not covalently attached to a polymer (eg, a hydrophilic polymer). In some cases, the elimination half-life of the eye is increased by at least about 2 times (e.g., about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times , about 10 times, about 12 times, about 14 times, about 16 times, about 18 times, about 20 times or more) relative to the original antibody. In some cases, the elimination half-life from the eye is increased by at least about 4 times relative to the reference antibody. In some cases, the ocular half-life is the vitreous elimination period. In some cases, the reference antibody is identical to the antibody of the antibody conjugate. In some cases, the reference antibody is not identical to the antibody of the antibody conjugate.

Any of the prior antibody conjugates may have a clearance that is reduced compared to a reference antibody that is not covalently attached to a polymer (eg, a hydrophilic polymer). In some cases, the clearance is reduced by at least about 2 times (for example, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 12 times, about 14 times, about 16 times, about 18 times, about 20 times or more) relative to the original antibody. In some cases, the clearance is reduced by at least about 4 times relative to the reference antibody. In some cases, the clearance is the clearance from the vitreous. In some cases, the reference antibody is identical to the antibody of the antibody conjugate. In some cases, the reference antibody is not identical to the antibody of the antibody conjugate.

In some cases, the time period between two intraocular injections (eg, intravitreal injections) of any of the prior antibody conjugates (eg, HA conjugates) is at least 1 month, such as at least 1 month, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks at least 15 weeks at least 16 weeks at least 20 weeks at least 24 weeks at least 28 weeks at least 32 weeks at least 36 weeks at least 40 weeks at least 44 weeks, at least 48 weeks, at least 52 weeks or more. In some cases, the maximum period between two intraocular injections is no more than four years, such as no more than three years, no more than two years, or no more than one year. The antibody conjugate can be administered, for example, every two to twelve months, for example, every four to ten months. In some cases, the antibody conjugate is administered every six months.

This invention also provides compositions (eg, pharmaceutical compositions) that contain any of the antibody conjugates described above. In some embodiments, the implementation of the composition contains one or more additional compounds. In certain embodiments, the additional compound binds to a second biological molecule selected from the group consisting of: IL-1β; IL-6; IL-6R; TCFR; angiopoietin; angiopoietin 2; Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin; apelina/APJ; erythropoietin; complement factor D; TNF-α; HtrA1; VEGF receptor; ST-2 receptor; and proteins genetically associated with the risk of age-related macular degeneration (AMD), such as components of the C2 complement pathway, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; interleukin-8 (IL-8); CX3CR1; TLR3; TLR4; SETP; LIPC; COL10A1; and TNFRSF10A. In some embodiments, the additional compound is an antibody, or an antigen-binding fragment thereof. For example, in some instances, the additional compound is a bispecific antibody (e.g., an anti-VEGF/anti-Ang2 bispecific antibody such as -Ang2 bispecific antibody as disclosed in WO 2010/069532 or WO 2016/073157, or a variant thereof In another example, in some instances, the additional compound is an anti-IL-6 antibody, such as EBI-031 (Eleven Biotereutics; See e.g. , WO 2016/073890), siltuximab (SYLVANT®), olokizumab, clasakizumab, sirukumab, elsilimoab, gerilimzumab, OPR-003, MEDI-5117, PF-04236921, or a variant thereof. anti-IL-eR antibody, eg tocilizumab (ACTEMRA®) (see eg WO 1992/019579), sarilumab, vobarilizumab (ALX-0061), SA-237, or a variant thereof.

In addition, the invention provides compositions (eg, pharmaceutical compositions) that contain any of the antibody conjugates described above and an additional VEGF antagonist.

2. Devices

Any of the antibodies (eg, cysteine-engineered anti-VEGF antibodies) or antibody conjugates (eg, monodisperse HA conjugates) described herein can be administered to the eye using an implantable delivery device. An implantable delivery device is an implantable, reusable device that can release a therapeutic agent (e.g., an anti-VEGF antibody conjugate) for several months (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12 or more months). Exemplary implantable delivery devices that may be used include those from ForSight Labs, LLC and/or ForSight VISION4, for example, as described in International Patent Application Publication Nos. WO 2010/088548, WO 2015/085234, WO 2013/116061, WO 2012/019176, WO 2013/040247, WO 2012/019047 and which are incorporated herein by reference in their entirety.

For example, the invention provides an implantable delivery device that includes reservoirs containing any of the antibodies or antibody conjugates described herein. The implantable delivery device may further comprise a proximal region, a tubular body connected to the proximal region in fluid communication with the reservoir, and one or more points in fluid communication with the reservoir and configured to release the composition into the eye. The tubular body may have an outer diameter capable of being inserted through a cut or hole in the eye of about 0.5 mm or less. The device may be from about 1 mm to about 15 mm in length (e.g., about 1 mm, about 2 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 9 mm, about 11 mm, about 13 mm, or about 15 mm long). The reservoir may be of any suitable volume. In some cases, the reservoir has a volume of from about 1 µl to about 100 µl (eg, about 1 µl, about 5 µl, about 10 µl, about 20 µl, about 50 µl, about 75 µl, or about 100 µl). The device or its components can be made from any suitable material, such as polyimide.

In some cases, the implantable delivery device contains a reservoir containing any of the antibodies or antibody conjugates described herein, and one or more additional compounds. In certain embodiments, the additional compound binds to a second biological molecule selected from the group consisting of IL-1β; IL-6; IL-6R; TCFR; angiopoietin; angiopoietin 2; Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin; apelina/APJ; erythropoietin; complement factor D; TNF-α; HtrA1; VEGF receptor; ST-2 receptor; and proteins genetically associated with the risk of AMD, such as components of the C2 complement pathway, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; interleukin (IL-8); CX3CR1; TLR3; TLR4; SETP; LIPC; COL10A1; and TNFRSF10A. In some embodiments, the additional compound is an antibody or an antigen-binding fragment thereof. For example, in some instances, the additional compound is a bispecific antibody (e.g., an anti-VEGF/anti-Ang2 bispecific antibody such as RG-7716 or any anti-VEGF/anti-Ang2 bispecific antibody disclosed in WO 2010/069532 or WO 2016 /073157, or a variant thereof In another example, in some cases, the additional compound is an anti-IL-6 antibody, e.g. EBI-031 (Eleven Biotereutics; See e.g. , clazakizumab, sirukumab, elsilimomab, gerilimzumab, OPR-003, MEDI-5117, PF-04236921, or a variant thereof In yet another example, in some instances, the additional compound is an anti-IL-6R antibody, such as tocilizumab (ACTEMRA®) ( see, for example, WO 1992/019579), sarilumab, vobarilizumab (ALX-0061), SA-237, or a variant thereof.

In some cases, the implantable delivery device contains any of the antibodies or antibody conjugates described herein and an additional VEGF antagonist.

III. EXAMPLES

The following are examples of methods and compositions according to this invention. It should be understood that given the general description provided above, various other embodiments of the present invention may be carried out.

Example 1: Linear hyaluronic acid (HA)-antibody conjugates derived from monodisperse HA to improve stability

Conjugation of Fab to a hyaluronic acid (HA) biopolymer can significantly improve the retention time of Fab in the eye, for example by slowing Fab diffusion and thus clearance from the vitreous. To obtain the HA-Fab conjugate, a two-stage method was used: first, commercial HA was modified with maleimide groups (HA-maleimide), and then, Fab-C (Fab with a free thiol on cysteine) was subjected to interaction with HA-maleimide to form covalent HA conjugates -Fab.

When produced by typical means, HA-protein conjugates may have two orthogonal components of variability. The first source of variability is polydispersity, which was provided by the polydispersity of the HA framework (Fig. 1A). The second source is the heterogeneity provided by differences in the number of Fab molecules attached to a given HA chain. This second source of variability is dictated by the stochastic nature of the maleimide modification of HA chains at the first stage of the HA conjugation process. On FIG. 1B shows the results of a Monte Carlo simulation in which each acid group of a 200 kDa HA chain was given a 5% chance of being reacted with a maleimide-containing linker, repeating per 1000 independent HA chains. The results indicate that while the average expected number of maleimides per HA chain in the simulation was 24.7, the absolute range was 11 to 42.

It may be desirable to maintain the physical and colloidal stability of antibody conjugates (e.g., antibody conjugates that contain linear HA and anti-VEGF antibody G6.31AARR; referred to herein as HA-G6.31.AARR antibody conjugates) in aqueous phase and vitreous. . Based in part on the simulations described above, it is believed that the molecular weight of the HA backbone and Fab loading level may be important parameters for physical stability. The Fab loading level refers to the average amount of an antibody molecule (eg, G6.31.AARR) attached to each HA chain, and is expressed in terms of the percentage of acidic groups on the HA backbone that are covalently modified with a Fab fragment (each repeating HA fragment contains one variable group of acids on the saccharide of glucuronic acid). While other parameters may contribute to conjugation stability (eg, Fab specific properties including net charge, surface charge distribution, hydrophobicity), they are generally not controlled within that particular molecule.

In order to evaluate the effect of MW HA and Fab loading on the physical stability of HA-G6.31.AARR, conjugates were made with three different HAs starting at MW (approximately 40 kDa, 200 kDa and 600 kDa) and different Fab loading levels. tested under physiological conditions and monitored for physical stability over several months to mimic biological effects.

(A) Materials and methods

(i) Materials

Sodium hyaluronate (HA, Lifecore Biomedical, Chaska, MN) was used in this study with three different molecular weights. Their properties, evaluated using size exclusion chromatography with included refractive index and multi-angle laser light scattering detectors (SEC-RI-MALS), are summarized in Table 4. Mn denotes the number average molecular weight; Mw is the weight average molecular weight; PDI stands for polydispersity index; a R _H indicates the hydrodynamic radius. Table 5 shows the data for Mn, Mw, and PDI of various polydisperse HA samples compared to a monodisperse sample, determined using SEC-RI-MALS. Data from Tables 4 and 5 are from two different batches of HA200K that had different polydispersities.

(ii) Synthesis of maleimide-functionalized HA (HA-mal)

HA was modified with maleimide groups using an aqueous reaction with the coupling agent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (DMTMM) and the linker N-(2-aminoethyl)maleimide trifluoroacetate salt (AEM). HA was dissolved in 100 mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.5) at 2.5 mg/mL, and DMTMM and AEM were added to this solution with stirring. The amounts of DMTMM and AEM added varied and were chosen for different target levels of maleimide functionalization ranging from 2 to 10%. The reaction mixture was heated to 70°C for 2 hours.

™ (GE Life Sciences) и уравновешивали 10 мМ ацетата натрия (рН 4,0) 150 мМ NaCl. Реакционную смесь впрыскивали в чистом виде в колонку, а пик ГК-мал собирали в соответствии с оптической плотностью при 302 нм и концентрировали до более чем 5 мг/мл с использованием центробежного устройства ультрафильтрации. Концентрация малеимида в ГК-мал исходного раствора измеряли по оптической плотности при 302 нм с помощью УФ-видимого спектрофотометра, и молярное отношение групп малеимида на цепь ГК оценивали с помощью эксклюзионной хроматографии с разными углами рассеяния света (SEC-MALS).Excess AET and DMTMM were removed from the reaction mixture using a desalting procedure. Desalting column HIPREP™ 26/10 was mounted on the purification system

™ (GE Life Sciences) and balanced 10 mm sodium acetate (pH 4.0) 150 mm NaCl. The reaction mixture was injected neat into the column, and the HA-mal peak was collected according to the optical density at 302 nm and concentrated to more than 5 mg/ml using a centrifugal ultrafiltration device. The concentration of maleimide in the HA-mal stock solution was measured by optical density at 302 nm using a UV-visible spectrophotometer, and the molar ratio of maleimide groups per HA chain was estimated using size exclusion chromatography with different light scattering angles (SEC-MALS).

(iii) Fab-C conjugation with GK-mal

The Fab-C solution was adjusted to pH 6.5 with 1M phosphate (pH 6.5) to a final phosphate concentration of 50 mM, and ethylenediaminetetraacetic acid (EDTA) was added to a final concentration of 2.5 mM. The Fab-C solution was stirred and HA-mal diluted in a reaction buffer consisting of 10 mM phosphate (pH 6.5), 150 mM NaCl, and 2.5 mM EDTA was added to it. The stoichiometry was set at 1.2 moles of Fab-C per mole of maleimide in the final reaction and the volume was set to give a final protein concentration of 1 mg/mL. The conjugation reaction was carried out at room temperature and stirring. After 3 hours, mercaptoethanol was added at a ratio of 2 moles per mole of maleimide to bind the unreacted maleimide groups. After 30 minutes, the reaction mixture was diluted with 10 mM phosphate (pH 6.5) to less than 50 mM NaCl.

Purification was performed using size exclusion chromatography (SEC) to separate free Fab-C and Fab dimer from the conjugate. A HILOAD® 26/600 SUPERDEX® 200 pg column (GE Healthcare) was equilibrated with 10 mM HisHCl (pH 5.5), 150 mM NaCl and the reaction mixture was injected neat. The peaks associated with the conjugate, Fab dimer and Fab monomer were eluted separately and the conjugate peak was collected.

(iv) Analysis of HA-Fab conjugates by SEC-RI-MALS

Residue-free Fab content, conjugate total molar mass, and protein mass fraction were assessed by SEC-RI-MALS-QELS (combination of size exclusion chromatography (SEC), refractive index (RI) of multi-angle light scattering (MALS) and quasi-elastic light scattering (QELS) )) on an Agilent 1200 HPLC with a Wyatt OPTILAB® T-rEX™ Refractive Index (RI) detector and a Wyatt HELEOS™-II Multi-View Light Scattering (MALS) detector. Two columns were run sequentially for SEC: ACCLAIM™ 7.8×150 mm pore size 1000 A followed by ACCLAIM™ 7.8×150 mm pore size 300 A with phosphate buffered saline (PBS) (pH 7.4) as running buffer. A bovine serum albumin (BSA) control was used to normalize the MALS detectors and correct for band broadening between the detectors. Free Fab content was measured by integrating the UV A ₂₈₀ peaks corresponding to Fab and HA-Fab conjugate. The molar mass of the conjugate was taken as the weight average molecular weight (Mw) of the conjugate peak. Protein weight fraction was calculated using conjugate protein analysis using differential refractive index (dRI) and UV A ₂₈₀ signals.

(v) Physiological stress and GK-G6.31.AARR conjugate analysis

Purified conjugates were dissolved in PBS buffer supplemented with 2 mM sodium azide and 0.01% polysorbate 20 (PBSTN). Final concentrations were approximately 5 mg/ml based on Fab. The samples were sealed and incubated at 37°C. This condition serves as a vitreous substitute (mimicking the pH, temperature, and ionic strength of the vitreous). The concentration of 5 mg/ml represents a "stress" concentration for the assessment of the concentration of precipitation in the vitreous body (2 mg dose in 4 ml of human vitreous body is equivalent to 0.5 mg/ml Fab). At regular intervals, samples were taken, diluted to 1 mg/mL in 10 mM HisHCl (pH 5.5) with 0.01% polysorbate 20 and 10% (w/v) trehalose, and analyzed for soluble protein concentration (A ₂₈₀ ), turbidity (A ₄₅₀ ), SEC retention time, and molecular weight (Mn and Mw via SEC-RI-MALS as above).

(B) Results

(i) Characterization of materials

Table 6 shows the SEC-RI-MALS characterization results for the nine conjugates used in this study. The conjugates differ both in the MW of the HA framework (40 kDa, 200 kDa, or 600 kDa) and in the level of Fab loading. Sample names are given as MW scaffolds (cDA) followed by Fab loading (%).

The polydispersity of the G6.31.AARR conjugates was experimentally evaluated using SEC-RI-MALS (Fig. 1C). The polydispersity index was 1.58, 1.78, and 1.41 for the GK40K, GK200K, and GK600K conjugates, respectively.

(ii) Conjugate Stability Under Physiological Stress Under conditions of physiological stress, some GK-G6.31.AARR conjugates showed significant physical changes during prolonged incubation in PBSTN. This change is most clearly indicative of shifts in average molecular weight (Mw - weight average molecular weight) over time, as shown in FIG. 2. While the Mw of some samples did not change during the 12-week study, those that did, all showed a decrease in Mw over time. The degree of reduction in Mw depends on both the molecular weight of the HA framework and the level of Fab loading. In general, precipitation was found to occur in samples in which the HA molecular weight and Fab loading were higher.

In order to understand this Mw shift, an analysis of the SEC retention profiles of the incubated GK-G6.31.AARR conjugates was performed. The retention time of SEC shifted to a later time with extended incubation, indicating that the average amount of conjugate became progressively smaller over time (FIG. 3). Since the SEC retention time is determined solely by the molecular weight of the HA backbone (and not by the Fab loading), this observation indicated that either: (a) the entire population of conjugates becomes smaller over time, or (b) the higher molecular weight subpopulation was precipitated from solution, resulting in a visible shift to smaller conjugates. These data are also shown in Table 7, which shows the difference between the retention time at baseline (T0) and week 12.

These changes measured by SEC and MALS were run in parallel with visual observations that samples with higher HA backbone molecular weight and higher Fab loading contained visible precipitate at later time points. This observation supports the latter explanation for the observed shift in SEC retention profiles, i.e. that higher HA molecular weight subpopulations precipitated and resulted in a shift in the average population remaining in solution.

Further, in support of this hypothesis, a re-analysis of the SEC-RI-MALS data was performed, examining the distribution of the HA-G6.31.AARR conjugate with respect to molecular weight, as shown in FIG. 4. For the GK40K scaffold sample (left panel), the overall distribution of conjugate molecules relative to molecular weight did not noticeably change during long-term exposure to physiological conditions. For the GK600K backbone sample (right), a continuous shift of all population fractions to lower molecular weights was observed, indicating a broad population of conjugate molecules that precipitated from solution. For the GK200K backbone sample (central panel), the shift in population fractions occurred mainly in higher molecular weight conjugate molecules. This indicates that there was a mixed population of relatively stable and unstable conjugate molecules in the GK200K backbone sample. Over time, high molecular weight (relatively unstable) conjugate molecules precipitate out of solution, leaving only a relatively stable subpopulation remaining in solution.

(iii) Monodisperse HA conjugates

Based on the understanding that in a polydisperse population of HA200K-G6.31.AARR, only the higher MW subpopulation is the driving force behind physical instability and precipitation, we hypothesized that switching from a polydisperse HA backbone to a monodisperse HA backbone could result in a more stable conjugate. antibodies.

Standard commercially available HA is produced by a multi-step process in which: (a) HA is synthesized via bacterial fermentation, providing HA with extremely high MW (MW 1-4 MDa), (b) HA is purified from cell culture, and (c) HA is chemically cleaved in a controlled way providing a random rupture of HA molecules. This final step of the process is critical for obtaining lower molecular weight HA, which would otherwise not be possible with fermentation alone. However, the random process of cutting degradation also leads to a wide (i.e., polydisperse) distribution of HA molecular weights.

HA can also be synthesized on an industrial scale using a synchronized chemo-enzymatic process (see Jing et al. J, Biol, Chem. 279:42345-42349, 2004; Jing et al. Anal. Blochem. 355:183-188, 2006 and US patent No. 8088604, which are incorporated into this description by reference in its entirety). In this method, the purified hyaluronan synthase enzyme is added to a mixture of small HA oligosaccharides (usually HA tetramer, HA ₄ ) and uridine diphosphate (UDP) saccharides UDP-glucuronic acid and UDP-N-acetylglucosamine. The enzyme hyaluronan synthase increases oligosaccharide fragments by using available sugar in a variable manner, resulting in synchronous HA polymerization. The molecular weight of the resulting HA polymer can be changed by the ratio of HA substrate (eg HA ₄ ) to sugars in the initial reaction. The result of this process is highly monodisperse HA polymers.

For comparison, the SEC-RI-MALS performance of polydisperse and monodisperse commercial HAs of similar size were compared in FIG. 5. Most noticeable is the difference in the polydispersity index, which was 1.779 for polydisperse HA200K and 1.001 for monodisperse HA150K. This lower level of polydispersity for monodisperse HA is believed to provide several key advantages in terms of HA-protein conjugates: (a) simplification of analytical characterization, since the elimination of HA polydispersity leaves differences in protein loading levels as the only source of heterogeneity in the sample; (b) elimination of low and higher molecular weight HA backbones in the general population, which can eliminate the heterogeneity and precipitation observed at higher molecular weight HA backbones within the subpopulation of HA-G6.31.AARR conjugates, and (c) potential reduction in viscosity formed GK-antibody conjugate.

In the context of using polydisperse and monodisperse HA starting materials for the preparation of G6.31.AARR conjugates, the same difference in polydispersity was observed: polydisperse HA200K-G6.31.AARR had a polydispersity index of 1.228, while monodisperse HA150K-G6.31 .AARR had a polydispersity index of 1.003 (FIG. 6). This monodispersity is expected to improve the stability of HA-G6.31.AARR conjugates due to the absence of a higher molecular weight subpopulation of the HA backbone that is thought to be responsible for the instabilities and precipitation observed under physiological stress of polydisperse HA-G6.31.AARR conjugates. .

Example 2 Optimized Fab Loading and Cysteine Engineered Sites for Antibody Linear GK Conjugates

Covalent conjugation of monoclonal antibodies (mAbs) or antibody fragments (Fabs) to a polymer backbone can be carried out through a wide range of chemicals, ranging from amine chemistry (direct amidation through solvent-accessible lysine residues) to chemoenzymatic conjugations using substrate-recognizing enzymes such as transglutaminase. Recently, thiol-maleimide chemical conjugation has gained considerable interest because it provides several key advantages over other approaches: (a) it is site-selective, reacting only to reduced, solvent-accessible cysteine residues, (b) it is an extremely fast reaction, (c) maleimide-containing linkers are readily available and generally readily available synthetically, (d) cysteine residues can generally be easily incorporated into the protein structure, and (e) thiol-maleimide conjugation can be performed near neutral pH in aqueous conditions.

In order to generate a Fab that is amenable to maleimide conjugation, one strategy was used to generate Fab-C in which the heavy chain sequence was extended by a hinge peptide from the first or second position of the hinge cysteine disulfide and truncated at that cysteine residue (Fig. 7) . In practice, this approach can potentially introduce a number of complications that affect conjugation to the polymer backbone. The primary consequence of the spatial proximity of this free cysteine residue to the interchain disulfide bond is a thiol rearrangement whereby these three nearby cysteine residues can form three possible disulfide configurations. These three possible disulfide configurations are shown graphically in FIG. 8A-8C. In each configuration, a different cysteine residue is reduced, making it a potential site for conjugation with the maleimide-containing backbone of the polymer. The consequence of this is heterogeneity at the site of attachment between the Fab and the HA scaffold, which may have consequences in terms of product quality, Fab stability, or safety of the conjugated material.

We hypothesized that movement in a free cysteine residue from a flexible, spatially proximal hinge sequence to a further location on the Fab surface might reduce or eliminate these conjugation variants. This approach is similar in nature to that which uses THIOMAB™ cysteine engineered monoclonal antibodies in which surface residues are mutated into cysteines for subsequent conjugation. In Fab format, we refer to Fabs containing surface-mutated cysteine as "ThioFab".

Protein-polymer conjugates made using thiol-maleimide chemistry can also suffer from deconjugation of the protein from the polymer backbone via a reverse Michael addition reaction. This can lead to a slow release of the free protein from the polymer backbone in a pH- and temperature-dependent manner. This behavior also depends on the local chemical environment around the cysteine residue and the structure of the maleimide-containing linker (eg, the presence of electron withdrawing groups or amines). Based on the results on the Michael back reaction sensitivity of various cysteine sites in THIOMAB™ development (see Shen et al. Nat, Biotechnol, 30:184-189, 2012), we also predicted that the movement of free cysteine from the hinge peptide to the surface site would result in to a decrease in the release rate of free Fab from the polymer backbone in the reverse Michael reaction. We also investigated the rate of deconjugation in the reverse Michael reaction of a model polymer polyethylene glycol (PEG)-maleimide on Fab-C and ThioFab format molecules under physiological conditions.

(A) Materials and methods

(i) Materials

Lys-C enzyme was purchased from Promega (Catalog #V1671, Madison, WI), hyaluronidase enzyme (recombinant human PH20, also referred to as TKase) was purchased from Halozyme (San Diego, CA), N-ethylmaleimide (NEM) was purchased at Sigma Aldrich (St. Louis, Missouri). The defined methoxy polyethylene glycol maleimide (d-mPEG4-Mal, Lot #10745) was purchased from Quanta Biodesign (Plain City, Ohio).

(ii) Lys-C restricted cleavage of G6.31.AARR.Fab-C

Limited digestion with Lys-C was performed on G6.31.AARR.Fab-C samples. Compared to more traditional Lys-C protein cleavage, "limited" Lys-C cleavage is performed with a reduced amount of the Lys-C enzyme and under non-denaturing conditions. This results in selective cleavage of the hinge peptide portion of the G6.31.AARR.Fab-C (KTHTC (SEQ ID NO: 61)) molecule, which is cleaved after the lysine residue. Four samples of G6.31.AARR.Fab-C were tested under different conditions: (a) +NEM, -cleavage, (b) +NEM +cleavage, (c) -NEM, +cleavage, and (d) -NEM, - split.

For each sample, up to 500 μg Fab-C in 500 μl 10 mM histidine acetate + 150 mM sodium chloride was added 50 μl 1 M Tris + 10 mM NEM (pH 5.5) (pH 7.5); NEM was not added for -NEM samples. Samples were incubated for 30 min at 37°C to bind any free thiols. The Lys-C enzyme was then added to the +Lys-C samples at a weight ratio of 1:500 Lys-C:Fab-C. Samples were incubated for 30 min at 37°C. After hydrolysis, the samples were frozen to stop the reaction and analyzed using RP-UPLC-TOF.

(iii) Lys-C restricted cleavage of GK200K-G6.31.AARR

To 500 μg (protein-based) GK200K-G6.31.AARR in 500 μl of 10 mM histidine acetate + 150 mM sodium chloride buffer (pH 5.5), 50 μl of 1 M Tris (pH 7.5) was added. Lys-C enzyme was added in a weight ratio of 1:500 Lys-C:Fab-C. Samples were incubated overnight at 37°C. After hydrolysis, the samples were frozen to stop the reaction and analyzed using RP-UPLC-TOF.

(iv) Cleavage of GK200K-G6.31.AARR with hyaluronidase

500 μg (protein based) GK200K-G6.31.AARR was diluted in 500 μl 10 mM histidine acetate + 150 mM sodium chloride (pH 5.5). Hyaluronidase was added in the amount of 10 units (U) per 1 µg of HA in the conjugate. The reaction mixture was incubated at 37°C for 4 hours. After hydrolysis, the samples were frozen to stop the reaction, and analyzed using RP-UPLC-TOF.

(v) Reverse Michael deconjugation of G6.31.AARR-PEG model conjugates

For G6.31. AARR. Fab-C, G6.31.AARR.A140C, G6.31.AARR.L174C and G6.31.AARR.K149C buffered with 10 mM phosphate (pH 6.5), 150 mM NaCl, 2.5 mM EDTA in concentrations from 0.2 to 0.5 mg/ml, and d-mPEG4-Mal was added in a 20-fold molar excess. The reaction mixture was incubated at room temperature for 2 hours followed by purification by desalting on PD-10 columns (GE Healthcare, Pittsburgh, PA) in PBS (pH 7.4). The conjugates were then concentrated to 1 mg/ml by centrifugal ultrafiltration and supplemented with oxidized glutathione (GSSG) to a final concentration of 2 mM. The constructs were incubated at 37°C and periodically sampled for analysis using RP-UPLC-TOF.

(vi) Reversed Phase Ultra High Performance Liquid Chromatography/Time-of-Flight (RP-UPLC-TOF) Mass Spectrometry (MS) analysis

Intact masses of samples were obtained by liquid chromatography and tandem mass spectrometry (LC/MS) analysis using an Agilent 6230 Time-of-Flight (TOF) mass spectrometer with electrospray ionization (ESI) Agilent 1290 Ultra High Performance Liquid Chromatography (UPLC) system. Approx. 2.5 μg of protein was injected per sample and desalted using reverse phase ultra high performance liquid chromatography (RP-UPLC) for direct online MS analysis. The resulting spectra were deconvoluted to zero charge using a MassHunter Workstation/Qualitative Analysis software workstation (Agilent Technologies Inc., Santa Clara, CA).

(B) Results

(iii) Disulfide status in G6.31.AARR.Fab-C

To confirm the presence of the three disulfide states shown in FIG. 8A-8C existing in G6.31.AARR. Fab-C, and maintained in dynamic thermodynamic equilibrium, we performed a series of experiments using maleimide capping using NEM and limited Lys-C cleavages to investigate both the instantaneous status of the three cysteine residues and their ability to dynamically rearrange.

In the first experiment, G6.31.AARR.Fab-C was capped using NEM (fixing the disulfide status by eliminating any free thiols) and then subjected to limited Lys-C cleavage, cleaving the heavy chain hinge peptide sequence to the putative free cysteine residue ( Fig. 9, second chromatogram). After denaturing analysis on RP-UPLC-TOF, species associated with the three disulfide states shown in FIG. 8A-8C: (a) intact Fab-minus hinge peptide (related to Fig. 8A); (b) light chain plus NEM and cyclized heavy chain (related to Fig. 8B); and (c) light chain plus hinge peptide and heavy chain minus hinge peptide plus NEM (related to Fig. 8C). This experiment demonstrated that G6.31.AARR.Fab-C does not exist in a homogeneous state, but contains three different species with different disulfide configurations and hence reactive cysteine residues.

In the second experiment, G6.31.AARR.Fab-C was subjected to limited Lys-C cleavage without NEM capping, leaving the cysteines to dynamically rearrange (FIG. 9, third chromatogram). In this experiment, almost all proteins analyzed by RP-UPLC-TOF were in the same state: Fab minus the hinge peptide. Taking into account the results of the first experiment, this second data set indicates that the three free cysteines rearranged dynamically and on a relatively accelerated time scale (30 min experiment duration). Upon cleavage of the hinge peptide with Lys-C, the three possible disulfide states are dynamically "scrambled" between the three cysteine residues. If the correct interchain disulfide is formed at any point in time, the hinge peptide sequence is left to be released into solution as it has been previously cleaved with Lys-C. As a result of this rearrangement process, the sample moves towards a fully properly formed interchain disulfide.

These two experiments confirmed that G6.31.AARR.Fab-C can exist in three different states with respect to the three proximal cysteine residues, and that their relative abundance is determined by dynamic thermodynamic equilibrium. This latter circumstance is important as it indicates that no further processing step can completely eliminate the two mis-disulfide configurations, since given any short time period, the three cysteines are re-scrambled to form the three variants observed in the first experiment. .

(ii) Conjugation variants in G6.31.AARR.Fab-C conjugates

While these three disulfide variants are present in G6.31.AARR.Fab-C, it remains to be shown that they also exist as variants in G6.31.AARR conjugates. In order to elucidate the exact location of the conjugation of individual G6.31.AARR.Fab-C molecules with HA-maleimide, we used two enzymatic digestion procedures. First, by limited Lys-C cleavage, G6.31.AARR was cleaved at the hinge peptide preceding the putative heavy chain free cysteine residue. In correctly conjugated G6.31.AARR molecules, this treatment should release free intact G6.31.AARR into solution. However, RP-UPLC-TOF analysis of digested samples under denaturing conditions identified additional species in solution: free light chain plus cleaved hinge peptide (FIG. 10A). The presence of this species confirms that the G6.31.AARR.Fab-C population was conjugated to HA via a heavy chain cysteine, usually occupied by an interchain disulfide presumably derived from the disulfide variant depicted in FIG. 8C.

The second enzymatic cleavage procedure kept all conjugations intact but completely cleaved the HA backbone with hyaluronidase (Gcase). This made it possible not only to analyze free molecules in solution, but also conjugated proteins with covalently bound small GA oligosaccharides. This method allowed direct observation of conjugated species at the individual level. In this experiment, G6.31.AARR molecules were observed correctly conjugated to HA through the putative heavy chain terminal cysteine; however, direct evidence of conjugation via the disulfide variant depicted in FIG. 8 B. This proposal was supported by two observations: (a) the presence of free cyclized heavy chain molecules in solution; and (b) the presence of HA oligosaccharides with a covalently linked maleimide linker plus a light chain (FIG. 10B). The presence of these species confirms that the G6.31.AARR.Fab-C population was conjugated to HA via a light chain cysteine, usually occupied by an interchain disulfide presumably derived from the disulfide variant depicted in FIG. 8B.

These two cleavage experiments confirmed that the disulfide rearrangement did lead to changes at the site of attachment of G6.31.AARR.Fab-C and HA-maleimide. This observation is expected to apply to any maleimide-containing polymer backbone used for conjugation and any Fab-C containing this configuration of three cysteine residues in close proximity.

(iii) Construction of a Fab containing free cysteine for more uniform conjugation

The data described above demonstrate that three conjugation options exist when a Fab-C molecule is attached to a maleimide-containing polymer backbone. This heterogeneity of conjugation is due to the presence of three different disulfide configurations in thermodynamic equilibrium, with each configuration leaving a different cysteine residue available for conjugation. That these three disulfide configurations are possible most likely depends on several factors, including the proximity of the putative free hinge cysteine residue to the interchain disulfide and the flexibility of the sequence of the hinge peptide to which the free cysteine is attached.

In order to avoid scrambling of the free cysteine with the interchain disulfide, we provided for moving the free cysteine away from the interchain disulfide. Thus, we truncated the hinge sequence, as is typical for a standard Fab-format molecule, and replaced Fab surface residues with cysteine residues. These sites were chosen to be far enough from the HVR so as not to adversely affect antigen binding and far enough from the interstrand disulfide to prevent scrambling.

Based on previous reports from the THIOMAB™ development, three sites were selected for the initial screening study to meet these criteria: LC-K149 (EU numbering), HC-A140 (EU numbering) and HC-L174 (EU numbering). Each site was replaced with a cysteine residue and the hinge peptide was terminated just before the first hinge disulfide cysteine (ie, KTNT; SEQ ID NO: 87).

In order to confirm that these cysteine sites are still reactive towards HA-maleimide, we conducted pilot pairings under standard conditions (10 mM phosphate (pH 6.5), 150 mM NaCl and 2.5 mM EDTA) and the products were evaluated by SEC-RI-MALS after being incubated overnight. Coupling proceeded normally with the generation of G6.31.AARR conjugates with the correct retention time compared to the G6.31.AARR.Fab-C conjugation reaction control (FIG. 11). The only significant difference noted for the thio-Fab samples was the lower Fab conversion to conjugate. This is likely caused by the relatively low pH of the conjugation reaction (pH 6.5), which may be better suited for the pKa of the cysteine hinge compared to such engineered cysteine residues of the thio-Fab. Increasing the pH of the conjugation reaction is expected to increase the yield of thio-Fab conjugations.

(iv) Sensitivity of the back reaction of Michael addition of Fab-C and muo-Fab formats

G6.31.AARR in Fab-C and thio-Fab formats were conjugated to PEG-maleimide model constructs and incubated in PBS at 37°C with GSSG to act as a trap for free thiols (i.e., preventing reverse addition reaction Michael released free thiol with PEG-maleimide) and then periodic analysis using RP-UPLC-TOF. In the Fab-C-PEG constructs, significant reverse Michael deconjugation was observed during the first seven days, resulting in a loss of 9.8% of the intact conjugate (FIG. 12). Thio-Fab conjugates showed different levels of improvement over Fab-C with A140C variant showing almost no deconjugation up to 14 days, L174C variant showing 5.9% deconjugation, and K149C variant showing 7.1% deconjugation in the same time . These data indicate that cysteine mutation variants may be protective against protein deconjugation mediated by reverse Michael reaction and may provide a significant advantage over the Fab-C format in maintaining intact protein-polymer conjugates.

Example 3 Exemplary Anti-VEGF Antibodies for Use in Antibody Conjugates of the Invention

Any of the anti-VEGF antibodies described herein can be used to prepare antibody conjugates as described in Examples 1 and 2. For example, any anti-VEGF antibody described in International Patent Application No. PCT/US2016/053454 can be used. Table 8 describes exemplary anti-VEGF antibodies that may be used, as well as amino acid sequences from the VH and VL domains for each antibody. Table 9 describes the HVR VL amino acid sequences for the anti-VEGF antibodies described in Table 8. Table 10 describes the HVR VH amino acid sequences for the anti-VEGF antibodies described in Table 8. In specific embodiments, anti-VEGF antibody G6 is used. AARR (also referred to in this document as "G6.31.AARR").

The upper hinge region of the Fab heavy chain of any of the antibodies listed above, eg G6.31 AARR, can be mutated to remove the anti-IgG1 hinge autoantibody reactivity reported in the literature. See, for example, Brezski et al., J. Immunol. 181:3183-3192, 2008 and Brezski et al., mAbs 2:3, 212-220, 2010. Thus, the C-terminal G6.31 AARR heavy chain amino acid can be either T (wild type version (WT)) or L (variant version, which lacks reactivity to Fab against human IgG). The full length amino acid sequence of the G6.31 wild-type AARR heavy chain is SEQ ID NO:48. for G6.31 AARR, and the version variant that lacks anti-human IgG Fab reactivity is SEQ ID NO: 50.

The light chain and heavy chain amino acid sequences of the cysteine engineered variant G6.31.AARR.LC-K149C are shown below (LC-C149 is in bold and underlined).

Light chain (LC):

Heavy chain (HC):

The light chain and heavy chain amino acid sequences of the cysteine engineered variant G6.31AARR.HC-A140C are shown below (HC-C140 in bold and underlined).

LC:

NS:

The light chain and heavy chain amino acid sequences of the cysteine engineered variant G6.31AARR.HC-L174C are shown below (HC-C174 in bold and underlined).

LC:

NS:

Example 4 HA-G6.31.AARR Conjugates Derived from Monodisperse HA Demonstrate Improved Physical Stability Under Physiological Stress Compared to HA-G6.31.AARR Conjugates Derived from Polydisperse HA

(A) Materials and methods

Monodisperse HA was synthesized by a chemo-enzymatic process using Pasteurella multocida hyaluronan synthase (PmHAS) obtained from Hyalose (Oklahoma City, Oklahoma). All other chemicals and reagents were from Sigma Aldrich (St. Louis, MO).

Monodisperse HA (monoHA, Mw 137 kDa, r _h 21.2 nm) was modified with N-(2-aminoethylmaleimide) as described above to contain 4.0% maleimide groups. G6.31.AARR.Fab-C then conjugated to backbone monoHA-maleimide as described above and purified by SEC on a HILOAD® SUPERDEX® 200 pg column with PBS, pH 7.4 running buffer. Conjugated fractions were pooled and confirmed using SEC-RI-MALS.

Purified monoHA-G6.31.AARR was concentrated by ultrafiltration to 5 mg/ml (Fab based) in PBS, supplemented with sodium azide and polysorbate 20 (PS20) to a final concentration of 2 mM and 0.01%, respectively, and incubated at 37°C. Samples were taken at specific time points and diluted to 1 mg/mL in buffer containing 10 mM His-HCl (pH 5.5), 150 mM NaCl, 10% (w/v) trehalose, and 0.01% polysorbate 20, and then analyzed for soluble protein concentration (A ₂₈₀ ), turbidity (A ₄₅₀ ), SEC retention time and molecular weight (Mn and Mw according to SEC-RI-MALS).

(B) Results

G6.31.AARR conjugates made using monodisperse HA showed significantly improved physical stability for up to four weeks compared to G6.31.AARR conjugate made with similarly sized polydisperse HA at the same Fab loading level (Fig. 14 ). The molecular weight (Mw) of monoHA-G6.31.AARR decreased slightly from week to week 2 and week 4, but this loss could be fully explained by the loss of free Fab due to deconjugation in the reverse Michael reaction, as is usually observed for conjugated with thiol-maleimide proteins. There was also no noticeable shift in SEC retention time for monoHA-G6.31.AARR, indicating that the physical size of the conjugate did not change over time.

These results clearly support the hypothesis that in the population of HA-G6.31.AARR conjugates derived from the starting material of polydisperse HAs, the high molecular weight HA-based subpopulation is responsible for the physical instability observed after physiological stress. Removing this subpopulation by homogenizing the parent HA material (ie, starting from monodisperse HA) results in a more stable conjugate molecule that is less prone to precipitation under physiological stress. These data further support the use of HA-G6.31.AARR conjugates derived from monodisperse HA material as therapeutic agents for in vivo use.

Although the foregoing invention has been described in some detail by way of illustration and examples for purposes of unambiguous understanding, the description and examples should not be taken as limiting the scope of the invention. The contents of all patents and scientific literature referenced in this document are expressly and in their entirety incorporated by reference.

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SEQUENCE LIST

<110> Genentech, Inc.

F. Hoffmann-La Roche AG

<120> OPTIMIZED ANTIBODY COMPOSITIONS FOR THE TREATMENT OF EYE DISEASE

<130> 50474-160WO3

<150> US 62/475163

<151> 2017-03-22

<160> 94

<170> PatentIn version 3.5

<210> 1

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 1

Asp Tyr Trp Ile His

fifteen

<210> 2

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> Xaa represents Ile or His

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> Xaa represents Ala or Arg

<220>

<221> MISC_FEATURE

<222>(8)..(8)

<223> Xaa is Tyr or Lys

<220>

<221> MISC_FEATURE

<222>(9)..(9)

<223> Xaa is Thr or Glu

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> Xaa is Arg, Tyr, Gln, or Glu

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> Xaa is Ala or Glu

<220>

<221> MISC_FEATURE

<222> (16)..(16)

<223> Xaa is Lys or Glu

<220>

<221> MISC_FEATURE

<222> (17)..(17)

<223> Xaa is Gly or Glu

<400> 2

Gly Xaa Thr Pro Xaa Gly Gly Xaa Xaa Xaa Tyr Xaa Asp Ser Val Xaa

1 5 10 15

xaa

<210> 3

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 3

Phe Val Phe Phe Leu Pro Tyr Ala Met Asp Tyr

1 5 10

<210> 4

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> Xaa is Asp or Arg

<400> 4

Arg Ala Ser Gln Xaa Val Ser Thr Ala Val Ala

1 5 10

<210> 5

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Xaa is Ser or Met

<400> 5

Xaa Ala Ser Phe Leu Tyr Ser

fifteen

<210> 6

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Xaa is Gln, Asn, or Thr

<220>

<221> MISC_FEATURE

<222>(6)..(6)

<223> Xaa is Ala, Asn, Gln, or Arg

<400> 6

Xaa Gln Gly Tyr Gly Xaa Pro Phe Thr

fifteen

<210> 7

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 7

Gly Ile Thr Pro Ala Gly Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 8

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 8

Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala

1 5 10

<210> 9

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 9

Ser Ala Ser Phe Leu Tyr Ser

fifteen

<210> 10

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 10

Gln Gln Gly Tyr Gly Ala Pro Phe Thr

fifteen

<210> 11

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 11

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Thr Pro Ala Gly Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Arg Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Val Phe Phe Leu Pro Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 12

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 12

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Gly Ala Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 13

<211> 30

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 13

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser

20 25 30

<210> 14

<211> 14

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 14

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

1 5 10

<210> 15

<211> 32

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 15

Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln

1 5 10 15

Met Arg Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 16

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 16

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 17

<211> 23

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 17

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys

twenty

<210> 18

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 18

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

1 5 10 15

<210> 19

<211> 32

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 19

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ala Ala Thr Tyr Tyr Cys

20 25 30

<210> 20

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 20

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

1 5 10

<210> 21

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 21

Gly Ile Thr Pro Ala Gly Gly Tyr Glu Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 22

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 22

Gly Ile Thr Pro Ala Gly Gly Tyr Glu Tyr Tyr Ala Asp Ser Val Glu

1 5 10 15

gly

<210> 23

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 23

Gln Gln Gly Tyr Gly Asn Pro Phe Thr

fifteen

<210> 24

<211> 32

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 24

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 25

<211> 23

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 25

Asp Ile Gln Met Thr Gln Ser Pro Glu Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Glu Val Thr Ile Thr Cys

twenty

<210> 26

<211> 23

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 26

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Glu Val Thr Ile Thr Cys

twenty

<210> 27

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 27

Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Lys Leu Leu Ile Tyr

1 5 10 15

<210> 28

<211> 32

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 28

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Glu Ser Leu Gln Pro Glu Asp Ala Ala Thr Tyr Tyr Cys

20 25 30

<210> 29

<211> 30

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 29

Glu Glu Gln Leu Val Glu Glu Gly Gly Gly Leu Val Gln Pro Gly Glu

1 5 10 15

Ser Leu Glu Leu Ser Cys Ala Ala Ser Gly Phe Glu Ile Ser

20 25 30

<210> 30

<211> 14

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 30

Trp Val Arg Gln Glu Pro Gly Glu Gly Leu Glu Trp Val Ala

1 5 10

<210> 31

<211> 32

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 31

Arg Phe Thr Ile Ser Ala Asp Thr Ser Glu Asn Thr Ala Tyr Leu Gln

1 5 10 15

Met Asn Glu Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 32

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 32

Trp Gly Gln Gly Glu Leu Val Thr Val Ser Ser

1 5 10

<210> 33

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 33

Glu Glu Gln Leu Val Glu Glu Gly Gly Gly Leu Val Gln Pro Gly Glu

1 5 10 15

Ser Leu Glu Leu Ser Cys Ala Ala Ser Gly Phe Glu Ile Ser Asp Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Glu Pro Gly Glu Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Thr Pro Ala Gly Gly Tyr Glu Tyr Tyr Ala Asp Ser Val

50 55 60

Glu Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Glu Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Glu Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Val Phe Phe Leu Pro Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Glu Leu Val Thr Val Ser Ser

115 120

<210> 34

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 34

Asp Ile Gln Met Thr Gln Ser Pro Glu Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Glu Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Gly Ala Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 35

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 35

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Glu Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Glu Ser Leu Gln Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Gly Ala Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 36

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 36

Asp Ile Gln Met Thr Gln Ser Pro Glu Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Glu Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Gly Ala Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 37

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 37

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Glu Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Gly Ala Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 38

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 38

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Gly Asn Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 39

<211> 14

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 39

Trp Val Arg Gln Glu Pro Gly Lys Gly Leu Glu Trp Val Ala

1 5 10

<210> 40

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 40

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Glu Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Thr Pro Ala Gly Gly Tyr Glu Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Val Phe Phe Leu Pro Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 41

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 41

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Gly Ala Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 42

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 42

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Thr Pro Ala Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Val Phe Phe Leu Pro Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 43

<211> 227

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 43

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Thr Pro Ala Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Val Phe Phe Leu Pro Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His

225

<210> 44

<211> 32

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 44

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ser Ala Thr Tyr Tyr Cys

20 25 30

<210> 45

<211> 23

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 45

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Asp Cys

twenty

<210> 46

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 46

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Asp Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Gly Ala Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 47

<211> 232

<212> Protein

<213> Homo sapiens

<400> 47

Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu

1 5 10 15

Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly

20 25 30

Gly Gly Gln Asn His Glu Val Val Lys Phe Met Asp Val Tyr Gln

35 40 45

Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu

50 55 60

Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu

65 70 75 80

Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro

85 90 95

Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His

100 105 110

Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys

115 120 125

Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Lys Lys Ser Val

130 135 140

Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg Lys Lys Ser Arg Tyr

145 150 155 160

Lys Ser Trp Ser Val Tyr Val Gly Ala Arg Cys Cys Leu Met Pro Trp

165 170 175

Ser Leu Pro Gly Pro His Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys

180 185 190

His Leu Phe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn

195 200 205

Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr

210 215 220

Cys Arg Cys Asp Lys Pro Arg Arg

225 230

<210> 48

<211> 228

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 48

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Thr Pro Ala Gly Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Arg Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Val Phe Phe Leu Pro Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr

225

<210> 49

<211> 228

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 49

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Thr Pro Ala Gly Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Arg Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Val Phe Phe Leu Pro Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Leu

225

<210> 50

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 50

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Gly Ala Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 51

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 51

Glu Glu Gln Leu Val Glu Glu Gly Gly Gly Leu Val Gln Pro Gly Glu

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Glu Ile Ser Asp Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Glu Pro Gly Glu Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Thr Pro Ala Gly Gly Tyr Glu Tyr Tyr Ala Asp Ser Val

50 55 60

Glu Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Glu Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Glu Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Val Phe Phe Leu Pro Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Glu Leu Val Thr Val Ser Ser

115 120

<210> 52

<211> 30

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 52

Glu Glu Gln Leu Val Glu Glu Gly Gly Gly Leu Val Gln Pro Gly Glu

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Glu Ile Ser

20 25 30

<210> 53

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 53

Gly Ile Thr Pro Ala Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 54

<211> 32

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 54

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys

20 25 30

<210> 55

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 55

Phe Gly Gln Gly Thr Lys Val Glu Val Lys

1 5 10

<210> 56

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 56

catcagatgg cgggaagatg aagacagatg gtgc 34

<210> 57

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 57

gccatccaga tgacccagtc tcc 23

<210> 58

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 58

ggctgcacca tctgtcttc 19

<210> 59

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 59

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Gly Asn Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 60

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 60

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Gly Ala Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 61

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 61

Lys Thr His Thr Cys

fifteen

<210> 62

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 62

Gly Tyr Tyr Met His

fifteen

<210> 63

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 63

Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

gly

<210> 64

<211> 20

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 64

Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr Pro Gly

1 5 10 15

Ala Phe Asp Ile

twenty

<210> 65

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 65

Gly Gly Asn Asn Ile Gly Ser Lys Ser Val His

1 5 10

<210> 66

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 66

Asp Asp Ser Asp Arg Pro Ser

fifteen

<210> 67

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 67

Gln Val Trp Asp Ser Ser Ser Asp His Trp Val

1 5 10

<210> 68

<211> 129

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 68

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr

100 105 110

Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser

115 120 125

Ser

<210> 69

<211> 110

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 69

Ser Tyr Val Leu Thr Gln Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

<210> 70

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 70

Gly Thr Lys Val Glu Cys Lys Arg Thr Val Ala

1 5 10

<210> 71

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 71

Lys Val Glu Ile Lys Cys Thr Val Ala Ala Pro

1 5 10

<210> 72

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 72

Asn Asn Phe Tyr Pro Cys Glu Ala Lys Val Gln

1 5 10

<210> 73

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 73

Ala Lys Val Gln Trp Cys Val Asp Asn Ala Leu

1 5 10

<210> 74

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 74

Gln Gly Thr Leu Val Cys Val Ser Ser Ala Ser

1 5 10

<210> 75

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 75

Thr Ser Gly Gly Thr Cys Ala Leu Gly Cys Leu

1 5 10

<210> 76

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 76

Thr Phe Pro Ala Val Cys Gln Ser Ser Gly Leu

1 5 10

<210> 77

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 77

Leu Gln Ser Ser Gly Cys Tyr Ser Leu Ser Ser

1 5 10

<210> 78

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 78

Leu Ser Ser Val Val Cys Val Pro Ser Ser Ser

1 5 10

<210> 79

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 79

His Lys Pro Ser Asn Cys Lys Val Asp Lys Lys

1 5 10

<210> 80

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 80

Pro Glu Val Thr Cys Cys Val Val Asp Val Ser

1 5 10

<210> 81

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 81

Thr Cys Leu Val Lys Cys Phe Tyr Pro Ser Asp

1 5 10

<210> 82

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 82

Leu Val Lys Gly Phe Cys Pro Ser Asp Ile Ala

1 5 10

<210> 83

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 83

Ile Ala Val Glu Trp Cys Ser Asn Gly Gln Pro

1 5 10

<210> 84

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 84

Gln Gly Asn Val Phe Cys Cys Ser Val Met His

1 5 10

<210> 85

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 85

His Glu Ala Leu His Cys His Tyr Thr Gln Lys

1 5 10

<210> 86

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 86

His Asn His Tyr Thr Cys Lys Ser Leu Ser Leu

1 5 10

<210> 87

<211> 4

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 87

Lys Thr His Thr

one

<210> 88

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 88

Lys Thr His Thr Ser Pro Pro Cys

fifteen

<210> 89

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 89

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Gly Ala Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Cys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 90

<211> 228

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 90

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Thr Pro Ala Gly Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Arg Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Val Phe Phe Leu Pro Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr

225

<210> 91

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 91

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Gly Ala Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 92

<211> 228

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 92

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Thr Pro Ala Gly Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Arg Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Val Phe Phe Leu Pro Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Cys Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr

225

<210> 93

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 93

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Gly Ala Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 94

<211> 228

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct

<400> 94

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Thr Pro Ala Gly Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Arg Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Val Phe Phe Leu Pro Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Cys Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr

225

<---

Claims (310)

1. An antibody conjugate for treating an eye disease in a subject or for reducing or inhibiting angiogenesis in a subject having an eye disease, comprising (i) an antibody that specifically binds to vascular endothelial growth factor (VEGF), and (ii) a hyaluronic acid (HA) polymer covalently attached to the antibody, wherein the HA polymer has a polydispersity index (PDI) of 1.1 or lower. 2. An antibody conjugate according to claim 1, characterized in that the HA polymer has a PDI of 1.0 to 1.1. 3. The antibody conjugate of claim 2, wherein the HA polymer has a PDI of 1.0 to about 1.07. 4. The antibody conjugate according to any one of paragraphs. 1-3, characterized in that the HA polymer has a PDI from about 1.0001 to about 1.06. 5. An antibody conjugate according to claim 4, characterized in that the HA polymer has a PDI of about 1.05. 6. The antibody conjugate according to any one of paragraphs. 1-5, characterized in that the HA polymer has a molecular weight of about 1 megadalton (MDa) or less. 7. An antibody conjugate according to claim 6, wherein the HA polymer has a molecular weight of from about 25 kDa to about 500 kDa. 8. An antibody conjugate according to claim 7, wherein the HA polymer has a molecular weight of from about 100 kDa to about 250 kDa. 9. An antibody conjugate according to claim 8, wherein the HA polymer has a molecular weight of about 150 kDa to about 200 kDa. 10. The antibody conjugate according to any one of paragraphs. 1-9, characterized in that the HA polymer is a linear HA polymer. 11. The antibody conjugate according to any one of paragraphs. 1-10, characterized in that the antibody conjugate has a hydrodynamic radius from about 10 nm to about 60 nm. 12. The antibody conjugate of claim 11, wherein the antibody conjugate has a hydrodynamic radius of about 25 nm to about 35 nm. 13. An antibody conjugate according to claim 12, characterized in that the hydrodynamic radius is from about 20 nm to about 30 nm. 14. The antibody conjugate according to any one of paragraphs. 1-13, characterized in that the antibody conjugate has an ocular half-life that is increased compared to a reference antibody that is not covalently attached to the HA polymer. 15. An antibody conjugate according to claim 14, characterized in that the half-life from the eye is increased by at least about 2 times relative to the reference antibody. 16. An antibody conjugate according to claim 15, characterized in that the elimination half-life from the eye is increased by at least about 4 times compared to the reference antibody. 17. The antibody conjugate according to any one of paragraphs. 14-16, characterized in that the half-life from the eye is the period of elimination from the vitreous body. 18. The antibody conjugate according to any one of paragraphs. 14-17, characterized in that the reference antibody is identical to the antibody of the antibody conjugate. 19. The antibody conjugate according to any one of paragraphs. 1-18, characterized in that the antibody contains the following six hypervariable regions (HVR): (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GX ₁ TPX ₂ GGX ₃ X ₄ X ₅ YX ₆ DSVX ₇ X ₈ (SEQ ID NO: 2), where X ₁ is Ile or His, X ₂ is Ala or Arg, X ₃ is Tyr or Lys, X ₄ is Thr or Glu, X ₅ is Arg, Tyr, Gln or Glu, X ₆ is Ala or Glu, X ₇ is Lys or Glu and X ₈ is Gly or Glu; (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQX ₁ VSTAVA (SEQ ID NO: 4), where X ₁ represents Asp or Arg; (e) HVR-L2 containing the amino acid sequence X ₁ ASFLYS (SEQ ID NO: 5), where X ₁ represents Ser or Met; and (f) HVR-L3 containing the amino acid sequence X ₁ QGYGX ₂ PFT (SEQ ID NO: 6), where X ₁ is Gln, Asn or Thr and X ₂ is Ala, Asn, Gln or Arg. 20. An antibody conjugate according to claim 19, characterized in that the antibody contains the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYTRYADSVKG (SEQ ID NO: 7), GITPAGGYEYYADSVKG (SEQ ID NO: 21) or GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGAPFT (SEQ ID NO: 10) or QQGYGNPFT (SEQ ID NO: 23). 21. An antibody conjugate according to claim 19 or 20, characterized in that the antibody contains the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYTRYADSVKG (SEQ ID NO: 7); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGAPFT (SEQ ID NO: 10). 22. The antibody conjugate according to any one of paragraphs. 19-21, characterized in that the antibody additionally contains the following framework regions (FR) of the heavy chain variable domain (VH): (a) FR-H1 containing the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTIS (SEQ ID NO: 13); (b) FR-H2 containing the amino acid sequence WVRQAPGKGLEWVA (SEQ ID NO: 14); (c) FR-H3 containing the amino acid sequence RFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO: 15); and (d) FR-H4 containing the amino acid sequence WGQGTLVTVSS (SEQ ID NO: 16). 23. The antibody conjugate according to any one of paragraphs. 19-22, characterized in that the antibody additionally contains the following light chain variable domain (VL) FRs: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20). 24. An antibody conjugate according to claim 19 or 20, characterized in that the antibody contains the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGNPFT (SEQ ID NO: 23). 25. An antibody conjugate according to claim 24, characterized in that the antibody additionally contains the following VL domain FRs: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 24); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20). 26. An antibody conjugate according to claim 19 or 20, characterized in that the antibody contains the following six HVRs: (a) HVR-H1 containing the amino acid sequence DYWIH (SEQ ID NO: 1); (b) HVR-H2 containing the amino acid sequence GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) HVR-H3 containing the amino acid sequence FVFFLPYAMDY (SEQ ID NO: 3); (d) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 8); (e) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 9); and (f) HVR-L3 containing the amino acid sequence QQGYGAPFT (SEQ ID NO: 10). 27. An antibody conjugate according to claim 26, characterized in that the antibody additionally contains the following VL domain FRs: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17), DIQMTQSPESLSASVGDEVTITC (SEQ ID NO: 25) or DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18) or WYQQKPGEAPKLLIY (SEQ ID NO: 27); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19) or GVPSRFSGSGSGTDFTLTIESLQPEDAATYYC (SEQ ID NO: 28); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20). 28. The antibody conjugate according to any one of paragraphs. 24-27, characterized in that the antibody additionally contains the following VH domain FRs: (a) FR-H1 containing the amino acid sequence EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29) or EEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 52); (b) FR-H2 containing the amino acid sequence WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) FR-H3 containing the amino acid sequence RFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) FR-H4 containing the amino acid sequence WGQGELVTVSS (SEQ ID NO: 32). 29. The antibody conjugate according to any one of paragraphs. 1-18, characterized in that the antibody contains (a) a VH domain containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 11, 40 or 42; (b) a VL domain containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 12, 41 or 46; or (c) a VH domain as in (a) and a VL domain as in (b). 30. An antibody conjugate according to claim 29, characterized in that the VH domain additionally contains the following FRs: (a) FR-H1 containing the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTIS (SEQ ID NO: 13); (b) FR-H2 containing the amino acid sequence WVRQAPGKGLEWVA (SEQ ID NO: 14) or WVRQEPGKGLEWVA (SEQ ID NO: 39); (c) FR-H3 containing the amino acid sequence RFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO: 15); and (d) FR-H4 containing the amino acid sequence WGQGTLVTVSS (SEQ ID NO: 16). 31. An antibody conjugate according to claim 30, characterized in that the VH domain contains the amino acid sequence of SEQ ID NO: 11. 32. The antibody conjugate according to any one of paragraphs. 29-31, characterized in that the VL domain additionally contains the following FRs: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17) or DIQMTQSPSSLSASVGDRVTIDC (SEQ ID NO: 45); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19), GVPSRFSGSGSGTDFTLTISSLQPEDSATYYC (SEQ ID NO: 44) or GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO: 54); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20) or FGQGTKVEVK (SEQ ID NO: 55). 33. An antibody conjugate according to claim 32, characterized in that the VL domain contains the amino acid sequence of SEQ ID NO: 12. 34. An antibody conjugate according to claim 29, characterized in that the antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 11, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 12. 35. An antibody conjugate according to claim 29, characterized in that the antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 40, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 12. 36. An antibody conjugate according to claim 29, characterized in that the antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 42, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 12. 37. An antibody conjugate according to claim 29, characterized in that the antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 42, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 41. 38. The antibody conjugate according to any one of paragraphs. 1-18, characterized in that the antibody contains (a) a VH domain containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 33 or 51; (b) a VL domain containing an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 12, 34, 35, 36, 37, or 38; or (c) a VH domain as in (a) and a VL domain as in (b). 39. An antibody conjugate according to claim 38, characterized in that the antibody additionally contains the following FRs: (a) FR-H1 containing the amino acid sequence EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29) or EEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 52); (b) FR-H2 containing the amino acid sequence WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) FR-H3 containing the amino acid sequence RFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) FR-H4 containing the amino acid sequence WGQGELVTVSS (SEQ ID NO: 32). 40. An antibody conjugate according to claim 39, characterized in that the VH domain contains the amino acid sequence of SEQ ID NO: 33. 41. An antibody conjugate according to claim 39, characterized in that the VH domain contains the amino acid sequence of SEQ ID NO: 51. 42. The antibody conjugate according to any one of paragraphs. 38-41, characterized in that the antibody additionally contains the following FRs: (a) FR-L1 containing the amino acid sequence DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17), DIQMTQSPESLSASVGDEVTITC (SEQ ID NO: 25) or DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) FR-L2 containing the amino acid sequence WYQQKPGKAPKLLIY (SEQ ID NO: 18) or WYQQKPGEAPKLLIY (SEQ ID NO: 27); (c) FR-L3 containing the amino acid sequence GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19), GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 24) or GVPSRFSGSGSGTDFTLTIESLQPEDAATYYC (SEQ ID NO: 28); and (d) FR-L4 containing the amino acid sequence FGQGTKVEIK (SEQ ID NO: 20). 43. An antibody conjugate according to claim 42, characterized in that the VL domain contains the amino acid sequence of SEQ ID NO: 34. 44. An antibody conjugate according to claim 42, characterized in that the VL domain contains the amino acid sequence of SEQ ID NO: 35. 45. An antibody conjugate according to claim 42, characterized in that the VL domain contains the amino acid sequence of SEQ ID NO: 36. 46. An antibody conjugate according to claim 42, characterized in that the VL domain contains the amino acid sequence of SEQ ID NO: 37. 47. An antibody conjugate according to claim 42, characterized in that the VL domain contains the amino acid sequence of SEQ ID NO: 12. 48. An antibody conjugate according to claim 42, characterized in that the VL domain contains the amino acid sequence of SEQ ID NO: 38. 49. An antibody conjugate according to claim 38, characterized in that the antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 33, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 38. 50. An antibody conjugate according to claim 38, characterized in that the antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 33, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 34. 51. An antibody conjugate according to claim 38, characterized in that the antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 33, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 35. 52. An antibody conjugate according to claim 38, characterized in that the antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 33, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 36. 53. An antibody conjugate according to claim 38, characterized in that the antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 33, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 37. 54. An antibody conjugate according to claim 38, characterized in that the antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 33, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 12. 55. An antibody conjugate according to claim 38, characterized in that the antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 51, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 38. 56. An antibody conjugate according to claim 38, characterized in that the antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 51, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 35. 57. An antibody conjugate according to claim 38, characterized in that the antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 51, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 37. 58. An antibody conjugate according to claim 38, characterized in that the antibody contains (a) a VH domain containing the amino acid sequence of SEQ ID NO: 51, and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 12. 59. An antibody conjugate according to claim 38, characterized in that the antibody contains (a) a heavy chain containing the amino acid sequence of SEQ ID NO: 48, and (b) a light chain containing the amino acid sequence of SEQ ID NO: 50. 60. An antibody conjugate according to claim 38, characterized in that the antibody contains (a) a heavy chain containing the amino acid sequence of SEQ ID NO: 49, and (b) a light chain containing the amino acid sequence of SEQ ID NO: 50. 61. An antibody conjugate according to any one of paragraphs. 1-60, characterized in that the antibody is capable of inhibiting the binding of VEGF to the VEGF receptor. 62. An antibody conjugate according to claim 61, wherein the VEGF receptor is VEGF receptor 1 (Flt-1). 63. The antibody conjugate of claim 61, wherein the VEGF receptor is the VEGF 2 receptor (KDR). 64. The antibody conjugate according to any one of paragraphs. 1-63, characterized in that the antibody binds to human VEGF (hVEGF) with a Kd of about 2 nM or less. 65. An antibody conjugate according to claim 64, wherein the antibody binds hVEGF with a Kd of about 75 pM to about 2 nM. 66. An antibody conjugate according to claim 65, wherein the antibody binds hVEGF with a Kd of about 75 pM to about 600 pM. 67. An antibody conjugate according to claim 66, wherein the antibody binds hVEGF with a Kd of about 75 pM to about 500 pM. 68. An antibody conjugate according to claim 67, characterized in that the antibody binds hVEGF with a Kd of about 60 pM. 69. The antibody conjugate according to any one of paragraphs. 19-23, 29-37 and 59-68, characterized in that the antibody has a melting point (Tm) of more than about 83.5°C. 70. An antibody conjugate according to claim 69, wherein said antibody has a Tm of about 85°C to about 91°C. 71. An antibody conjugate according to claim 70, characterized in that said antibody has a Tm of about 89°C. 72. An antibody conjugate according to any one of paragraphs. 19, 20, 24-28 and 38-58, characterized in that the antibody has an isoelectric point (pl) lower than 8. 73. The antibody conjugate of claim 72, wherein the antibody has a pl of about 5 to about 7. 74. An antibody conjugate according to claim 73, wherein the antibody has a pl of about 5 to about 6. 75. The antibody conjugate according to any one of paragraphs. 1-74, characterized in that the antibody is monoclonal, human, humanized or chimeric. 76. An antibody conjugate according to any one of claims 1-75, wherein the antibody is an antigen-binding fragment of an antibody. 77. An antibody conjugate according to claim 76, wherein the antibody fragment is selected from the group consisting of Fab, Fab-C, Fab'-SH, Fv, scFv and (Fab') ₂ fragments. 78. An antibody conjugate according to claim 77, characterized in that the antibody fragment is a Fab. 79. An antibody conjugate according to any one of claims 1-78, characterized in that the antibody is a monospecific antibody. 80. An antibody conjugate according to any one of claims 1-78, characterized in that the antibody is a multispecific antibody. 81. An antibody conjugate according to claim 80, wherein the multispecific antibody is a bispecific antibody. 82. An antibody conjugate according to claim 81, characterized in that the bispecific antibody binds VEGF and a second biological molecule selected from the group consisting of IL-1β; interleukin-6 (IL-6); interleukin-6 receptor (IL-6R); interleukin-13 (IL-13); IL-13 receptor (IL-13R); TCFR; angiopoietin; angiopoietin-2; Tie2; S1P; integrins αvβ3, αvβ5 and α5β1; betacellulin; apelina/APJ; erythropoietin; complement factor D; TNF-α; HtrA1; VEGF receptor; ST-2 receptor; and a protein genetically associated with the risk of age-related macular degeneration (AMD). 83. An antibody conjugate according to claim 82, wherein the VEGF receptor is VEGF1, VEGF2, VEGF3, the membrane-bound VEGF receptor (mbVEGF) or the soluble VEGF receptor (sVEGF). 84. An antibody conjugate according to claim 82, characterized in that the protein genetically associated with the risk of AMD is selected from the group consisting of components of the C2 complement pathway, factor B, factor H, CFHR3, C3b, C5, C5a and C3a; HtrA1; ARMS2; TIMP3; HLA; interleukin-8 (IL-8); CX3CR1; TLR3; TLR4; SETP; LIPC, COL10A1; and TNFRSF10A. 85. An antibody conjugate according to any one of paragraphs. 1-84, characterized in that the antibody is an antibody constructed by the introduction of cysteine. 86. The antibody conjugate of claim 85, wherein the cysteine-engineered antibody contains a heavy chain cysteine mutation selected from the group consisting of HC-A118C, HC-A140C, and HC-L174C (EU numbering), or a cysteine mutation in a light chain selected from the group consisting of LC-K149C and LC-V205C (Kabat numbering). 87. An antibody conjugate according to claim 86, wherein the cysteine mutation in the heavy chain is HC-A118C (EU numbering). 88. An antibody conjugate according to claim 86, wherein the cysteine mutation in the heavy chain is HC-A140C (EU numbering). 89. The antibody conjugate of claim 86 wherein the cysteine mutation in the heavy chain is HC-L174C (EU numbering). 90. An antibody conjugate according to claim 86, wherein the cysteine mutation in the light chain is LC-K149C (Kabat numbering). 91. An antibody conjugate according to claim 86, wherein the cysteine mutation in the light chain is LC-V205C (Kabat numbering). 92. The antibody conjugate according to any one of paragraphs. 86-91, characterized in that the HA polymer is covalently attached to the antibody at the site of the cysteine mutation. 93. An antibody conjugate for treating an eye disease in a subject or for reducing or inhibiting angiogenesis in a subject having an eye disease, comprising (i) an antibody that specifically binds to VEGF and (ii) a HA polymer covalently attached to the antibody, wherein the HA polymer has a PDI of 1.05, and wherein the antibody comprises (a) a VH domain containing the amino acid sequence of SEQ ID NO: 11 and (b) a VL domain containing the amino acid sequence of SEQ ID NO: 12. 94. An antibody conjugate for treating an eye disease in a subject or for reducing or inhibiting angiogenesis in a subject having an eye disease, comprising (i) an antibody that specifically binds to VEGF and (ii) a HA polymer covalently attached to the antibody, wherein the HA polymer has a PDI of 1.05, and where the antibody contains (a) a heavy chain containing the amino acid sequence of SEQ ID NO: 48 or SEQ ID NO: 49, and (b) a light chain containing the amino acid sequence of SEQ ID NO: 50. 95. The use of an antibody conjugate according to any one of paragraphs. 1-94 in the manufacture of a medicament for the treatment of an eye disease in a subject. 96. The use of an antibody conjugate according to any one of paragraphs. 1-94 in reducing or inhibiting angiogenesis in a subject having an eye disease. 97. The use of an antibody conjugate according to any one of paragraphs. 1-94 in the treatment of eye disease in a subject. 98. Application according to any one of paragraphs. 95-97, characterized in that the eye disease is selected from the group consisting of age-related macular degeneration (AMD), macular degeneration, macular edema, diabetic macular edema (DME), retinopathy, diabetic retinopathy (DR), other ischemia-related retinopathy, retinopathy prematurity (RN), retinal vein occlusion (RVO), CNV, corneal neovascularization, corneal neovascularization disease, retinal neovascularization, retinal neovascularization/choroidal neovascularization disease, pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, familial exudative vitreoretinopathy (VEVRP), Coats disease, Norrie disease, osteoporosis-pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, rubeosis, neovascular eye disease, neovascular glaucoma, retinitis pigmentosa (PR), hypertensive retinopathy, retinal angiomatous proliferation, macular telangiectasia, iris neovascularization, intraocular neovas retinal degeneration, cystic macular edema (CMO), vasculitis, papilledema, retinitis, conjunctivitis, Leber's congenital amaurosis, uveitis, choroiditis, ocular histoplasmosis, blepharitis, dry eye syndrome, traumatic eye injury, and Sjögren's syndrome. 99. The use of an antibody conjugate according to any one of paragraphs. 1-94 in the treatment of an ocular disease in a subject, wherein the ocular disease is AMD, DME, DR, or RVO. 100. Use according to claim 98 or 99, characterized in that the eye disease is AMD. 101. Application according to any one of paragraphs. 98-100, characterized in that AMD is wet AMD. 102. Use according to claim 98 or 99, characterized in that the eye disease is DME. 103. The use according to claim 98 or 99, characterized in that the DME is a focal DME outside the central zone or a diffuse DME involving the central zone of the retina. 104. Use according to claim 98 or 99, characterized in that the DR is proliferative DR (PDR), non-proliferative DR (NPDR), or high altitude DR. 105. Pharmaceutical composition for the treatment of an eye disease in a subject or for reducing or inhibiting angiogenesis in a subject having an eye disease, containing an antibody conjugate according to any one of paragraphs. 1-94 and a pharmaceutically acceptable carrier, excipient or diluent. 106. The pharmaceutical composition of claim 105 further comprising a second agent, the second agent being selected from the group consisting of an antibody, an anti-angiogenic agent, a cytokine, a cytokine antagonist, a corticosteroid, an analgesic, and a compound that binds to the second biological molecule. 107. The pharmaceutical composition according to claim 106, characterized in that the anti-angiogenic agent is a VEGF antagonist. 108. The pharmaceutical composition of claim 107, wherein the VEGF antagonist is an anti-VEGF antibody, an anti-VEGF receptor antibody, a soluble VEGF receptor fusion protein, an aptamer, an anti-VEGF DARPin®, or an inhibitor of VEGF tyrosine kinase (VEGF receptor). 109. The pharmaceutical composition of claim 108, wherein the anti-VEGF antibody is ranibizumab (LUCENTIS®), RTH-258, or a bispecific anti-VEGF antibody. 110. The pharmaceutical composition of claim 109, wherein the bispecific anti-VEGF antibody is an anti-VEGF/anti-Ang2 antibody. 111. Pharmaceutical composition according to claim 110, characterized in that the anti-VEGF/anti-Ang2 antibody is RG-7716. 112. The pharmaceutical composition of claim 108, wherein the soluble VEGF receptor fusion protein is aflibercept (EYLEA®). 113. The pharmaceutical composition of claim 108, wherein the aptamer is pegaptanib (MACUGEN®). 114. Pharmaceutical composition according to claim 108, characterized in that the anti-VEGF DARPin® is abicipar pegol. 115. Pharmaceutical composition according to claim 108, characterized in that the FESCR tyrosine kinase inhibitor is selected from the group consisting of 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline (ZD6474) SUTENT® (sunitinib). 116. Pharmaceutical composition according to p. 106, characterized in that the second biological molecule is selected from the group consisting of IL-1β; IL-6; IL-6R; IL-13; IL-13R; TCFR; angiopoietin; angiopoietin-2; Tie2; S1P; integrins αvβ3, αvβ5 and α5β1; betacellulin; apelina/APJ; erythropoietin; complement factor D; TNF-α; HTRA1; VEGF receptor; ST-2 receptor; and a protein genetically associated with the risk of AMD. 117. The pharmaceutical composition according to claim 116, characterized in that the VEGF receptor is VEGF1, VEGF2, VEGF3, mbVEGF, or sVEGF. 118. The pharmaceutical composition according to claim 116, characterized in that the protein genetically associated with the risk of AMD is selected from the group consisting of components of the C2 complement pathway, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; SETP; LIPC, COL10A1; and TNFRSF10A. 119. Pharmaceutical composition according to any one of paragraphs. 106 and 116-118, characterized in that the compound that binds the second biological molecule is an antibody or its antigen-binding fragment. 120. Pharmaceutical composition according to claim 119, characterized in that the antigen-binding antibody fragment is selected from the group consisting of Fab, Fab-C, Fab'-SH, Fv, scFv and (Fab') ₂ fragments. 121. The use of a pharmaceutical composition according to any one of paragraphs. 105-120 in the manufacture of a medicament for the treatment of an eye disease in a subject. 122. The use of a pharmaceutical composition according to any one of paragraphs. 105-120 in reducing or inhibiting angiogenesis in a subject having an eye disease. 123. The use of a pharmaceutical composition according to any one of paragraphs. 105-120 in the treatment of eye disease in a subject. 124. Application according to any one of paragraphs. 121-123, characterized in that the eye disease is selected from the group consisting of age-related macular degeneration (AMD), macular degeneration, macular edema, diabetic macular edema (DME), retinopathy, diabetic retinopathy (DR), other ischemia-related retinopathy, retinopathy prematurity (RN), retinal vein occlusion (RVO), CNV, corneal neovascularization, corneal neovascularization disease, retinal neovascularization, retinal neovascularization/choroidal neovascularization disease, pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, familial exudative vitreoretinopathy (VEVRP), Coats disease, Norrie disease, osteoporosis-pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, rubeosis, neovascular eye disease, neovascular glaucoma, retinitis pigmentosa (PR), hypertensive retinopathy, retinal angiomatous proliferation, macular telangiectasia, iris neovascularization, intraocular neov vascularization, retinal degeneration, cystic macular edema (CMO), vasculitis, papilledema, retinitis, conjunctivitis, Leber's congenital amaurosis, uveitis, choroiditis, ocular histoplasmosis, blepharitis, dry eye syndrome, traumatic eye injury, and Sjögren's syndrome. 125. The use of a pharmaceutical composition according to any one of paragraphs. 105-120 in the treatment of an eye disease in a subject, wherein the eye disease is AMD, DME, DR, or RVO. 126. Use according to claim 124 or 125, characterized in that the eye disease is AMD. 127. Application according to any one of paragraphs. 124-126, characterized in that AMD is wet AMD. 128. Use according to claim 124 or 125, characterized in that the eye disease is DME. 129. The use according to claim 124 or 125, characterized in that the DME is a focal DME outside the central zone or a diffuse DME involving the central zone of the retina. 130. Use according to claim 124 or 125, characterized in that the DR is proliferative DR (PDR), non-proliferative DR (NPDR), or high altitude DR. 131. A method of reducing or inhibiting angiogenesis in a subject having an eye disease, comprising administering to the subject an effective amount of an antibody conjugate according to any one of paragraphs. 1-94 or a pharmaceutical composition according to any one of paragraphs. 105-120, thereby reducing or inhibiting angiogenesis in the subject. 132. A method of treating an eye disease, comprising administering an effective amount of an antibody conjugate according to any one of paragraphs. 1-94 or a pharmaceutical composition according to any one of paragraphs. 105-120 to a subject in need of such treatment. 133. The method according to p. 131 or 132, characterized in that the eye disease is selected from the group consisting of age-related macular degeneration (AMD), macular degeneration, macular edema, diabetic macular edema (DME), retinopathy, diabetic retinopathy (DR), others ischemia-related retinopathy, retinopathy of prematurity (RP), retinal vein occlusion (RVO), CNV, corneal neovascularization, corneal neovascularization disease, retinal neovascularization, retinal neovascularization/choroidal neovascularization disease, pathological myopia, Hippel-Lindau disease , ocular histoplasmosis, familial exudative vitreoretinopathy (FEVR), Coats' disease, Norrie's disease, osteoporosis-pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, rubeosis, neovascular eye disease, neovascular glaucoma, retinitis pigmentosa (PR), hypertensive retinopathy, retinal angiomatous proliferation, macular telangiectasia, neovascularization of the iris retinitis, intraocular neovascularization, retinal degeneration, cystic macular edema (CMO), vasculitis, papilledema, retinitis, conjunctivitis, Leber's congenital amaurosis, uveitis, choroiditis, ocular histoplasmosis, blepharitis, dry eye syndrome, traumatic eye injury, and Sjögren's syndrome. 134. A method of treating an eye disease, comprising administering an effective amount of an antibody conjugate according to any one of paragraphs. 1-94 or a pharmaceutical composition according to any one of paragraphs. 105-120 to a subject in need of such treatment, characterized in that the eye disorder is AMD, DME, DR, or RVO. 135. The method according to p. 133 or 134, characterized in that the eye disease is AMD. 136. The method according to any one of paragraphs. 133-135, characterized in that AMD is wet AMD. 137. The method according to p. 133 or 134, characterized in that the eye disease is DME. 138. The method according to claim 133 or 134, characterized in that the DME is a focal DME outside the central zone or a diffuse DME involving the central zone of the retina. 139. The method according to p. 133 or 134, characterized in that the DR is a proliferative DR (PDR), non-proliferative DR (NPDR) or high altitude DR. 140. The method according to any one of paragraphs. 131-139 further comprising administering to the subject an effective amount of a second agent, the second agent being selected from the group consisting of an antibody, an antiangiogenic agent, a cytokine, a cytokine antagonist, a corticosteroid, an analgesic, and a compound that binds to the second biological molecule. 141. The method of claim 140, wherein the anti-angiogenic agent is a VEGF antagonist. 142. The method of claim 141, wherein the VEGF antagonist is an anti-VEGF antibody, an anti-VEGF receptor antibody, a soluble VEGF receptor fusion protein, an aptamer, an anti-VEGF DARPin®, or an inhibitor of VEGF tyrosine kinase (VEGF receptor). 143. The method of claim 142, wherein the anti-VEGF antibody is ranibizumab (LUCENTIS®), RTH-258, or a bispecific anti-VEGF antibody. 144. The method of claim 143, wherein the bispecific anti-VEGF antibody is an anti-VEGF/anti-Ang2 antibody. 145. The method of claim 144 wherein the anti-VEGF/anti-Ang2 antibody is RG-7716. 146. The method of claim 142 wherein the soluble VEGF receptor fusion protein is aflibercept (EYLEA®). 147. The method of claim 142 wherein the aptamer is pegaptanib (MACUGEN®). 148. The method of claim 142, wherein the anti-VEGF DARPin® is abicipar pegol. 149. The method according to p. 142, characterized in that the EGFR tyrosine kinase inhibitor is selected from the group consisting of 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline ( ZD6474), 4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline (AZD2171), vatalanib (PTK787), semaxaminib (SU5416), and SUTENT ® (sunitinib). 150. The method according to p. 140, characterized in that the second biological molecule is selected from the group consisting of IL-1β; IL-6; IL-6R; IL-13; IL-13R; TCFR; angiopoietin; angiopoietin-2; Tie2; S1P; integrins αvβ3, αvβ5 and α5β1; betacellulin; apelina/APJ; erythropoietin; complement factor D; TNF-α; HTRA1; VEGF receptor; ST-2 receptor; and a protein genetically associated with the risk of AMD. 151. The method of claim 150, wherein the VEGF receptor is VEGF1, VEGF2, VEGF3, mbVEGF, or sVEGF. 152. The method according to p. 151, characterized in that the protein genetically associated with the risk of AMD is selected from the group consisting of components of the C2 complement pathway, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; SETP; LIPC, COL10A1; and TNFRSF10A. 153. The method according to any one of paragraphs. 140 and 150-152, characterized in that the compound that binds the second biological molecule is an antibody or its antigen-binding fragment. 154. The method of claim 153, wherein the antigen-binding antibody fragment is selected from the group consisting of Fab, Fab-C, Fab'-SH, Fv, scFv, and (Fab') ₂ fragments. 155. The method according to any one of paragraphs. 131-154, characterized in that the antibody conjugate or the pharmaceutical composition is administered intravitreally, through the eye, intraocularly, into the periscleral region, into the sub-Tenon space, into the suprachoroidal space, locally, intravenously, intramuscularly, intradermally, transdermally, intraarterially, intraperitoneally, intrafocally, intracranially , intraarticular, inside the prostate, intrapleural, intratracheal, intrathecal, intranasal, intravaginal, rectal, topically, intratumoral, intraperitoneal, peritoneal, intraventricular, subcutaneous, subconjunctival, intravesicular, mucosal, intrapericardial, intraumbilical, intraorbital, oral, transdermal, by inhalation, by injections, in the form of eye drops, by implantation, by infusion, by continuous infusion, by localized perfusion directly through target cells, by catheter, by lavage, in creams or in lipid formulations. 156. The method according to p. 155, characterized in that the antibody conjugate or pharmaceutical composition is administered into the vitreous body, through the eye, intraocularly, into the periscleral region, into the sub-Tenon's space, into the suprachoroidal space, or locally. 157. The method according to p. 155 or 156, characterized in that the antibody conjugate or pharmaceutical composition is administered by injection into the vitreous body. 158. The method according to p. 155 or 156, characterized in that the antibody conjugate or pharmaceutical composition is administered topically in the form of eye drops or ointment. 159. The method according to p. 155 or 156, characterized in that the antibody conjugate or pharmaceutical composition is administered using an implantable delivery device. 160. The method according to any one of paragraphs. 131-159, characterized in that the subject is a human.

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