Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

• Sequence Listing XML is provided as a file entitled KDIAK190WOSeqList.xml, created January 30, 2024 which is 835,882 bytes in size.
• the information in the electronic format of the Sequence Listing XML is incorporated herein by reference in its entirety.
• the present disclosure relates to antibodies and conjugates thereof and methods of using and manufacturing said antibodies and conjugates thereof.
• High-temperature requirement Al is a secreted homo-oligomeric (trimer) serine protease. Each monomer contains an N-terminal insulin like growth factorbinding protein (IGFBP)/Kazal domain, a trypsin-like fold protease domain and a C-terminal PDZ domain. HTRA1 promiscuously cleaves a plethora of extracellular matrix (ECM) proteins and TGF-beta family members.
• IGFBP insulin like growth factorbinding protein
• ECM extracellular matrix
• an antibody conjugate comprising (1) an anti-HTRAl antibody and (2) a phosphorylcholine containing polymer, wherein the polymer is covalently bonded to the antibody at a non-native cysteine outside a variable region of the antibody.
• an antibody conjugate comprising an anti-HTRAl immunoglobulin G (IgG) bonded to a polymer, which polymer comprises MPC monomers
• the anti-HTRAl antibody heavy chain comprises: a heavy chain variable region (VH) comprising an amino acid sequence at least 80% identical to any one of SEQ ID NOs:725, 742, 744, 745, 746, C-terminally fused to a heavy chain constant region (HC) comprising an amino acid sequence of SEQ ID NO:536, wherein the polymer is bonded to C326 of SEQ ID NO:536, and wherein the anti-HTRAl antibody light chain comprises: a light chain variable region (VL) comprising an amino acid sequence at least 80% identical to SEQ ID NO:748, C-terminally fused to a light chain constant region (LC) comprising an amino acid sequence of SEQ ID NO:537.
• VH heavy chain variable region
• HC heavy chain constant region
• LC light chain constant region
• an antibody conjugate comprising an anti-HTRAl immunoglobulin G (IgG) covalently bonded to a polymer, which polymer comprises MPC monomers
• the anti-HTRAl antibody heavy chain comprises: a heavy chain variable region (VH) comprising an amino acid sequence comprising SEQ ID NOs:725, 742, 744, 745, 746, C-terminally fused to a heavy chain constant region (HC) comprising an amino acid sequence of SEQ ID NO:536, wherein the polymer is bonded to C326 of SEQ ID NO:536, and wherein the anti-HTRAl antibody light chain comprises: a light chain variable region (VL) comprising an amino acid sequence comprising SEQ ID NO:748, C-terminally fused to a light chain constant region (LC) comprising an amino acid sequence of SEQ ID NO: 537.
• VH heavy chain variable region
• HC heavy chain constant region
• LC light chain constant region
• an anti-HTRAl antibody which comprises a heavy chain and a light chain, wherein the heavy chain comprises: a CDRnl having an amino acid sequence of a CDRHI in any one of the VH sequences in Figs. 21, 22A, 23 A, and 24A, and Tables 1.2,
• a CDRu2 having an amino acid sequence of a CDRH2 in any one of the VH sequences in Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1,
• the anti-HTRAl antibody light chain comprises a light chain variable region (VL) comprising: a CDRLI having an amino acid sequence of a CDRLI in any one of the VL sequences in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1,
• a CDRL2 having an amino acid sequence of a CDRL2 in any one of the VL sequences in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and 8.2
• a CDRL3 having an amino acid sequence of a CDRL3 in any one of the VL sequences in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and 8.2.
• an anti-HTRAl antibody which comprises a heavy chain and a light chain, wherein the heavy chain comprises CDRHI : FYHVH (SEQ ID NO:830), CDRH2: SIYTSGYTEYASALES (SEQ ID NO:831), and CDRH3 : EGLQRVGVLDA (SEQ ID NO:834) or EGLQRVGVFDA (SEQ ID NO 832) or EGLQRVGVMDA (SEQ ID NO:833), and the light chain comprises CDRLI : RSSQSLLDEAGETYLA (SEQ ID NO:840), CDRL2: EVSLLES (SEQ ID NO: 841), and CDRL3 : QQATYFPYT (SEQ ID NO: 839), and the heavy chain isotype is IgGl, wherein the IgGl constant region comprises one or more of the following mutations to modulate effector function (EU numbering): L234A, L235A, and G237A.
• the heavy chain isotype is I
• an anti-HTRAl antibody which comprises a heavy chain and a light chain, wherein the heavy chain comprises CDRHI : GFSLTFYH (SEQ ID NO: SEQ ID NO 825), CDRH2: IYTSGYT (SEQ ID NO:826), and CDRH3 : AREGLQRVGVFDA (SEQ ID NO 827) or AREGLQRVGVMDA (SEQ ID NO:828) or AREGLQRVGVLDA (SEQ ID NO:829), and the anti-HTRAl antibody light chain variable region comprises CDRLI : QSLLDEAGETY (SEQ ID NO:837), CDRL2: EV (SEQ ID NO:838), and CDRL3 : QQATYFPYT (SEQ ID NO:839), and the heavy chain isotype is IgGl, wherein the IgGl constant region comprises one or more of the following mutations to modulate effector function (EU numbering): L234A, L235A, and G2
• an anti-HTRAl antibody which comprises a heavy chain and a light chain, wherein the heavy chain comprises CDRHI : GFSLTFY (SEQ ID NO: SEQ ID NO:835), CDRH2: YTSGY (SEQ ID NO:836), and CDRH3 : EGLQRVGVLDA (SEQ ID NO:834) or EGLQRVGVFDA (SEQ ID NO:832) or EGLQRVGVMDA (SEQ ID NO:833), and the anti-HTRAl antibody light chain variable region comprises CDRLI : RSSQSLLDEAGETYLA (SEQ ID NO:840), CDRL2: EVSLLES (SEQ ID NO:841), and CDR L 3 : QQATYFPYT (SEQ ID NO:839), wherein the IgGl constant region comprises one or more of the following mutations to modulate effector function (EU numbering): L234A, L235A, and G237A.
• the heavy chain comprises CDRHI :
• an antibody comprising: a heavy chain amino acid variable region that comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOs:725, 742, 744, 745, 746; and a light chain amino acid variable region that comprises an amino acid sequence at least 80% identical to SEQ ID NO:748.
• an antibody that binds to HTRA1 comprising: a CDRHI that is the CDRHI in any one of SEQ ID NOs:725, 742, 744, 745, 746; a CDRH2 that is the CDRH2 in any one of SEQ ID NOs:725, 742, 744, 745, 746; a CDRH3 that is the CDRH3 in any one of SEQ ID NOs:725, 742, 744, 745, 746; a CDRLI that is the CDRLI in SEQ ID NO:748; a CDRL2 that is the CDRL2 in SEQ ID NO:748; a CDRL3 that is the CDRL3 in SEQ ID NO:748; at least one of the following mutations (EU numbering): L234A, L235A, and G237A; and at least one of the following mutations(EU numbering): Q347C or L443C.
• composition that includes at least one of the antibody and/or antibody conjugate provided herein. Also provided is a method of treatment or prophylaxis of an ocular disease comprising administering the antibody conjugate and/or the antibody provided herein.
• an antibody conjugate comprising an anti-HTRAl antibody conjugated to a phosphorylcholine containing polymer
• the method comprising the step of: conjugating an anti-HTRAl antibody to a phosphorylcholine containing polymer, wherein the anti-HTRAl antibody comprises a non-native cysteine residue outside a variable region of the antibody, wherein the phosphorylcholine containing polymer comprises a sulfhydryl specific reacting group selected from the group consisting of a maleimide, a vinylsulfone, an orthopyridyl-disulfide, and an iodoacetamide, and wherein the sulfhydryl specific reacting group on the phosphorylcholine containing polymer reacts with the cysteine residue on the anti-HTRAl antibody to make the antibody conjugate
• an antibody that specifically binds to HTRA1 wherein the antibody inhibits a protease activity of HTRA1. Also provided is an antibody that specifically binds to HTRA1 , wherein the antibody binds to HTRA1 with a binding affinity (KD) of 1.0 x IO’ 10 M or less.
• KD binding affinity
• FIG. 1 is a bar graph showing antibody binding to an ELISA plate coated with Loop A peptide, according to some non-limiting embodiments of the present disclosure.
• FIG. 2 is a collection of Bar graphs showing the inhibitory activity profiling of various antibodies in a FRET -based (H2-Opt) blocking assay, according to some nonlimiting embodiments of the present disclosure.
• FIG. 4 is a collection of images showing SDS-PAGE monitoring of the amount of intact casein in the presence of HTRA1 and HTRA1 antibody mixtures, according to some non-limiting embodiments of the present disclosure.
• FIGS. 5A-5C are graphs showing size exclusion chromatography in line with SEC-MALS to assess antibody and HTRA1 complex molar mass, according to some nonlimiting embodiments of the present disclosure. Chromatograms plot UV traces indicating molar mass (g/mol) as function of time (minutes).
• FIG. 6B is a collection of graphs showing HTRA1 activity dose response curves as measured in H2-Opt assays, according to some non-limiting embodiments of the present disclosure.
• the graphs plot HTRA1 activity (RFU/s) as function of the antibody concentration (M).
• FIG. 7A is an image showing preparation of a vector backbone from pLL45 (Hu_aHTRAl_36_l-pCDisplay4) phagemid by Sa l and As/SI digestion.
• FIG. 7B is an image showing PCR amplification of VH and VL library, CL- pelB fragment.
• FIG. 8 is a graph of polyclonal phage ELISA showing the enrichment of phage pools in library Genl panning, according to some non-limiting embodiments of the present disclosure.
• the phage pools of library Genl, 5 rounds of panning outputs and parental pLL44 were tested for binding to HTRA1 (white bars), HTRA3 (gray bars) and uncoated surface (black bars).
• FIGS. 9C and 9D are collection of graphs showing BIAcore analysis of HTRA1 binding to aHTRAl_36-Fab captured on anti-HuIgG chip (FIG. 9C) and anti-Fab- VHH Chip (FIG. 9D), according to some non-limiting embodiments of the present disclosure. Binding data in the sensorgrams are presented in gray and kinetic fitting curves are in black.
• FIG. 9E is a table showing the kinetic parameters of aHTRAl_36 in Fab and IgG format on different capture surfaces, according to some non-limiting embodiments of the present disclosure.
• FIGS. 10A and 10B are a collection of graphs showing H2-Opt based HTRA1 inhibition with IgG variants from library Genl, according to some non-limiting embodiments of the present disclosure.
• FIG. 11A is a graph showing polyclonal phage ELISA of library Gen2 panning, according to some non-limiting embodiments of the present disclosure.
• the phage pools of library Gen2, 4 rounds of panning outputs and GEN1-P1D6 were tested for binding to HTRA1 (white bars), HTRA3 (gray bars) and uncoated surface (black bars).
• FIG. 1 IB is a graph showing polyclonal phage ELISA of optimized R3 phage outputs in comparison to Lib Gen2, phage outputs from R1-R3 and parental pLL44, according to some non-limiting embodiments of the present disclosure.
• FIGS. 12A-12C are a collection of BIAcore sensorgrams showing the kinetics of HTRA1 binding to parental pLL44 (FIG. 12A), GEN1-P1D6 (FIG. 12B) and Gen2- P1D1 (FIG. 12C) from PPE preps, according to some non-limiting embodiments of the present disclosure.
• the equilibrium dissociation constant (KD) is shown.
• Gen2-P1D1 and GEN1- P1D6 are clones from phage display screening and have highly improved affinity in comparison to parental pLL44.
• FIGS. 13A-13B are a collection of graphs showing BIAcore analysis of HTRA1 monomeric mutant and HTRA1 trimeric wild type binding to aHTRAl_36 and selected IgG variants from library Genl (HC5/LC4, HC6/LC4, HC7/LC4) and Gen2 (HC13/LC11, HC13/LC8), according to some non-limiting embodiments of the present disclosure. Binding data in the sensorgrams are presented in gray and kinetic fitting curves are in black.
• FIGS. 15A and 15B are a graph and a table showing polyreactivity profile of molecules HC17/LC21 and HC41/LC21 on selected general antigens (cardiolipin, KLH, LPS, ssDNA, dsDNA, and insulin), according to some non-limiting embodiments of the present disclosure.
• OG1950 and A_S574 serve as negative and positive controls.
• ELISA scores are shown in FIG. 15B.
• FIGS. 15C and 15D are a graph and a table showing polyreactivity profile of molecules HC17/LC21 and HC41/LC21 on baculovirus particle (BVP), according to some non-limiting embodiments of the present disclosure.
• OG1950 and A_S574 serve as negative and positive controls.
• BVP scores are shown in FIG. 15D.
• FIGS. 16A-16C are a collection of graphs and tables showing the potency of selected molecules before and after conjugation to the biopolymer OG1802.
• HC17/LC21 (FIG. 16A), HC41/LC21 (FIG. 16B) and HC40/LC21 (FIG. 16C) were tested by H2-Opt assay, according to some non-limiting embodiments of the present disclosure.
• the half maximal inhibitory concentration (IC50) is shown in the tables.
• FIGS. 17A and 17B are a collection of plots showing SEC-MALS chromatograms, according to some non-limiting embodiments of the present disclosure.
• the black curves display the UV profile of antibody elution.
• the gray curves show the elution profile of antibody and HTRA1 (S328 A) complex.
• the dash lines indicate the calculated molar mass.
• FIG. 17A represents HC17/LC21 and complex.
• FIG. 17B represents HC17/LC21 conjugated with OG1802 only and in complex with HTRA1 (S328A).
• FIGS. 18D and 18E are graphs showing mass distribution of antibody and HTRA1 mixtures with mass photometry, according to some non-limiting embodiments of the present disclosure.
• Both HC40/LC21 (FIG. 18D) and HC41/LC21 (FIG. 18E) binding complexes present two major peaks corresponding to free antibody and free HTRA1 and antibody HTRA1 complexes. The mean peak value, width of the fitted peak and counts are shown in the graphs.
• FIG. 19 shows amino acid sequences for human HTRA1, HTRA2, HTRA3 and HTRA4 proteins.
• FIGS. 20A and 20B show sequence alignments for heavy chain and light chain variable region amino acid sequences, respectively, of non-limiting embodiments of the anti-HTRAl antibodies. CDRs are indicated within boxes.
• FIG. 21 shows non-limiting examples of heavy chain and light chain variable region sequences of the present disclosure.
• FIGs. 22A and 22B show non-limiting examples of heavy chain and light chain variable region sequences of the present disclosure. CDRs are underlined.
• FIGs. 23 A and 23B show non-limiting examples of heavy chain and light chain variable region sequences of the present disclosure. CDRs are underlined.
• FIGs. 24A and 24B show non-limiting examples of heavy chain and light chain variable region sequences of the present disclosure. CDRs are underlined.
• FIGs. 25A and 25B show non-limiting examples of heavy chain and light chain sequences of the present disclosure. CDRs are underlined and constant regions are italicized.
• FIG. 26A is a graph showing molecules HC17/LC21, HC37/LC21, HC41/LC21 and aHTRAl_36 binding to the Loop A peptide, according to some non-limiting embodiments of the present disclosure.
• aHTRAl_37 serves as a positive control.
• FIG. 26B is a graph showing molecules HC17/LC21, HC37/LC21, HC41/LC21, aHTRAl_36 and aHTRAl_37 binding to HTRA1, according to some nonlimiting embodiments of the present disclosure.
• FIG. 27A shows OG1786.
• FIG. 27B shows OG1801.
• FIG. 27C shows OG1802.
• FIG. 28 A is a graph showing sequence alignment of HTRA1 protease domain (PD) in different species. The differential residues between HTRA1 in human and in other species were highlighted in grey.
• FIGS. 28B and 28C are a collection of graphs showing BIAcore kinetic analysis of molecules HC41/LC21 and HC17/LC21 binding to human, mouse, dog and rat HTRA1 PD domains, according to some non-limiting embodiments of the present disclosure. Binding data in the sensorgrams are presented in gray and kinetic fitting curves are in black.
• FIG. 29 shows amino acid sequences for human, mouse, dog and rat HTRA1 protease domain (PD).
• antibodies that bind to HTRA1, e.g., human HTRA1.
• conjugates of the antibodies that include an anti-HTRAl antibody covalently bonded to a polymer, such as a phosphorylcholine containing polymer.
• the antibodies and conjugates thereof of the present disclosure find use in treating an ocular disease, e.g., age-related macular degeneration (AMD).
• AMD age-related macular degeneration
• GWAS Genome-wide association studies
• HTRA1 has been hypothesized that cleavage of certain ECM proteins contribute to extracellular debris accumulation that in turn sensitizes cells to inflammatory stimuli (Lin et al., 2018)(Beguier et al., 2020). Without being bound to theory, this mechanism may explain how genetic variations that increase expression of HTRA1 increase the risk of developing the dry form of the disease. HTRA1 has also been shown to induce expression of inflammatory cytokines and VEGF(Lu et al., 2019), and overexpression of HTRA1 in the retina of mouse models recapitulates key physiological markers of AMD (Jones et al., 2011); (Vierkotten, Muether, & Fauser, 2011). These data corroborate the observation that HTRA1 risk alleles show strong and nearly equal association with wet and dry forms of AMD, and therefore indicate that HTRA1 might be successfully targeted in both forms of the disease.
• HTRA1 refers to a HtrA serine protease.
• HTRA1 can refer to, without limitation, human HTRA1 (Gene ID: 5654), mouse HTRA1 (Gene ID: 56213), rat HTRA1 (Gene ID: 65164), canine HTRA1 (Gene ID: 477852), chicken HTRA1 (Gene ID: 100857572), bovine HTRA1 (Gene ID: 282326).
• HTRA1 is human HTRA1 (“HuHTRAl”).
• a “neovascular disorder” is a disorder or disease state characterized by altered, dysregulated or unregulated angiogenesis.
• neovascular disorders include neoplastic transformation (e.g. cancer) and ocular neovascular disorders including diabetic retinopathy and age-related macular degeneration.
• An “ocular neovascular” disorder is a disorder characterized by altered, dysregulated or unregulated angiogenesis in the eye of a patient.
• Such disorders include optic disc neovascularization, iris neovascularization, retinal neovascularization, choroidal neovascul arization, corneal neovascularization, vitreal neovascularization, glaucoma, pannus, pterygium, macular edema, diabetic retinopathy, diabetic macular edema, vascular retinopathy, retinal degeneration, uveitis, inflammatory diseases of the retina, and proliferative vitreoretinopathy .
• HTRA1 activity e.g., HTRA1 proteolytic activity
• antibody includes intact antibodies and binding fragments thereof.
• a binding fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of binding fragments include Fv, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e g. scFv); and multispecific antibodies formed from antibody fragments. scFv antibodies are described in Houston JS. 1991. Methods in Enzymol. 203:46-96.
• antibody fragments comprise single chain polypeptides having the characteristics of a VH domain, namely being able to assemble together with a VL domain, or of a VL domain, namely being able to assemble together with a VH domain to a functional antigen binding site and thereby providing the antigen binding property of full length antibodies.
• Specific binding of an antibody to its target antigen(s) means an affinity of at least 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 M’ 1 . Specific binding is detectab ly higher in magnitude and distinguishable from non-specific binding occurring to at least one unrelated target. Specific binding can be the result of formation of bonds between particular functional groups or particular spatial fit (e.g., lock and key type) whereas nonspecific binding is usually the result of van der Waals forces. Specific binding does not however necessarily imply that an antibody or fusion protein binds one and only one target.
• a basic antibody structural unit is a tetramer of subunits.
• Each tetramer includes two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy" chain (about 50-70 kDa).
• the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. This variable region is initially expressed linked to a cleavable signal peptide.
• the variable region without the signal peptide is sometimes referred to as a mature variable region.
• a light chain mature variable region means a light chain variable region without the light chain signal peptide.
• the hinge region provides flexibility between the binding and effector regions of an antibody and also provides sites for intermolecular disulfide bonding between the two heavy chain constant regions in a tetramer subunit.
• the CH2 and CH3 regions are the primary site of effector functions and FcR binding.
• Light chains are classified as either kappa or lambda.
• Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively.
• the variable and constant regions are joined by a "J" segment of about 12 or more amino acids, with the heavy chain also including a "D” segment of about 10 or more amino acids.
• the mature variable regions of each light/heavy chain pair form the antibody binding site.
• an intact antibody has two binding sites, i.e., is divalent. In natural antibodies, the binding sites are the same.
• bispecific antibodies can be made in which the two binding sites are different (see, e.g., Songsivilai S, Lachmann PC. 1990. Bispecific antibody: a tool for diagnosis and treatment of disease. Clin Exp Immunol. 79:315- 321; Kostelny SA, Cole MS, Tso JY. 1992. Formation of bispecific antibody by the use of leucine zippers. J Immunol. 148: 1547-1553).
• epitope refers to a site on an antigen to which an antibody or extracellular trap segment binds.
• An epitope on a protein can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost on treatment with denaturing solvents.
• An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
• Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).
• Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay showing the ability of one antibody to compete with the binding of another antibody to a target antigen.
• the epitope of an antibody can also be defined by X-ray crystallography of the antibody (or Fab fragment) bound to its antigen to identify contact residues.
• two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
• Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
• Competition between antibodies is determined by an assay in which an antibody under test inhibits specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al., Cancer Res. 50: 1495, 1990).
• a test antibody competes with a reference antibody if an excess of a test antibody (e.g., at least 2x, 5x, lOx, 20x or lOOx) inhibits binding of the reference antibody by at least 50%.
• the test antibody inhibits binding of the reference antibody by 75%, 90%, or 99% as measured in a competitive binding assay.
• Antibodies identified by competition assay include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur.
• patient includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.
• Percentage sequence identities are determined with antibody sequences maximally aligned by the Kabat numbering convention for a variable region or EU numbering for a constant region. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage.
• a subject antibody region e.g., the entire mature variable region of a heavy or light chain
• Sequence identities of other sequences can be determined by aligning sequences using algorithms, such as BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI, using default gap parameters, or by inspection, and the best alignment (i.e., resulting in the highest percentage of sequence similarity over a comparison window). Percentage of sequence identity is calculated by comparing two optimally aligned sequences over a window of comparison, determining the number of positions at which the identical residues occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
• algorithms such as BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI, using default gap parameters, or by inspection, and the best alignment (i.e., resulting in
• compositions or methods "comprising" one or more recited elements may include other elements not specifically recited.
• a composition that comprises antibody may contain the antibody alone or in combination with other ingredients.
• ADCC antibody-dependent cellular cytotoxicity
• target cells i.e., cells with bound antibody
• immune cells possessing lytic activity also referred to as effector cells.
• effector cells include natural killer cells, monocytes/macrophages and neutrophils.
• ADCC is triggered by interactions between the Fc region of an antibody bound to a cell and Fey receptors, particularly FcyRI and FcyRIII, on immune effector cells such as neutrophils, macrophages and natural killer cells.
• the target cell is eliminated by phagocytosis or lysis, depending on the type of mediating effector cell. Death of the antibody-coated target cell occurs as a result of effector cell activity.
• opsonization also known as "antibody-dependent cellular phagocytosis", or ADCP, refers to the process by which antibody-coated cells are internalized, either in whole or in part, by phagocytic immune cells (e.g., macrophages, neutrophils and dendritic cells) that bind to an immunoglobulin Fc region.
• phagocytic immune cells e.g., macrophages, neutrophils and dendritic cells
• complement-dependent cytotoxicity refers to a mechanism for inducing cell death in which an Fc effector domain(s) of a target-bound antibody activates a series of enzymatic reactions culminating in the formation of holes in the target cell membrane.
• antigen-antibody complexes such as those on antibody- coated target cells bind and activate complement component Clq which in turn activates the complement cascade leading to target cell death.
• Activation of complement may also result in deposition of complement components on the target cell surface that facilitate ADCC by binding complement receptors (e.g., CR3) on leukocytes.
• complement receptors e.g., CR3
• a humanized antibody is a genetically engineered antibody in which the CDRs from a non-human "donor” antibody are grafted into human "acceptor” antibody sequences (see, e.g., Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539, Carter, US 6,407,213, Adair, US 5,859,205 6,881,557, Foote, US 6,881,557).
• the acceptor antibody sequences can be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a germLine region sequence.
• a humanized antibody is an antibody having some or all CDRs entirely or substantially from a donor antibody and variable region framework sequences and constant regions, if present, entirely or substantially from human antibody sequences.
• a humanized heavy chain has at least one, two and usually all three CDRs entirely or substantially from a donor antibody heavy chain, and a heavy chain variable region framework sequence and heavy chain constant region, if present, substantially from human heavy chain variable region framework and constant region sequences.
• a humanized light chain has at least one, two and usually all three CDRs entirely or substantially from a donor antibody light chain, and a light chain variable region framework sequence and light chain constant region, if present, substantially from human light chain variable region framework and constant region sequences.
• a humanized antibody comprises a humanized heavy chain and a humanized light chain.
• a CDR in a humanized antibody is substantially from a corresponding CDR in a non-human antibody when at least 85%, 90%, 95% or 100% of corresponding residues (as defined by Kabat) are identical between the respective CDRs.
• the variable region framework sequences of an antibody chain or the constant region of an antibody chain are substantially from a human variable region framework sequence or human constant region respectively when at least 85, 90, 95 or 100% of corresponding residues defined by Kabat are identical.
• humanized antibodies often incorporate all six CDRs (which can be as defined by Kabat) from a mouse antibody, they can also be made with less than all CDRs (e.g., at least 3, 4, or 5 CDRs from a mouse antibody) (e.g., De Pascalis R, Iwahashi M, Tamura M, et al. 2002. Grafting “Abbreviated” Complementary-Determining Regions Containing Specificity-Determining Residues Essential for Ligand Contact to Engineer a Less Immunogenic Humanized Monoclonal Antibody. J Immunol. 169:3076-3084; Vajdos FF, Adams CW, Breece TN, Presta LG, de Vos AM, Sidhu, SS.
• CDRs which can be as defined by Kabat
• a chimeric antibody is an antibody in which the mature variable regions of light and heavy chains of a non-human antibody (e.g., a mouse) are combined with human light and heavy chain constant regions. Such antibodies substantially or entirely retain the binding specificity of the mouse antibody, and are about two-thirds human sequence.
• a veneered antibody is a type of humanized antibody that retains some and usually all of the CDRs and some of the non-human variable region framework residues of a non-human antibody but replaces other variable region framework residues that may contribute to B- or T-cell epitopes, for example exposed residues (Padlan EA. 1991. A possible procedure for reducing the immunogenicity of antibody variable regions while preserving their ligandbinding properties. Mol Immunol. 28:489-98) with residues from the corresponding positions of a human antibody sequence. The result is an antibody in which the CDRs are entirely or substantially from a non-human antibody and the variable region frameworks of the non- human antibody are made more human-like by the substitutions.
• a human antibody can be isolated from a human, or otherwise result from expression of human immunoglobulin genes (e.g., in a transgenic mouse, in vitro or by phage display).
• Methods for producing human antibodies include the trioma method of Ostberg L, Pursch E. 1983. Human x (mouse x human) hybridomas stably producing human antibodies. Hybridoma 2:361-367; Ostberg, U.S. Patent No.
• “naturally occurring amino acids” found in proteins and polypeptides are L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamine, L-glutamic acid, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and or L-valine.
• “Non-naturally occurring amino acids” found in proteins are any amino acid other than those recited as naturally occurring amino acids.
• Non-naturally occurring amino acids include, without limitation, the D isomers of the naturally occurring amino acids, and mixtures of D and L isomers of the naturally occurring amino acids.
• Other amino acids such as N-alpha- methyl amino acids (e.g. sarcosine), 4-hydroxyproline, desmosine, isodesmosine, 5-hydroxylysine, epsilon-N-methyllysine, 3-methylhistidine, although found in naturally occurring proteins, are considered to be non-naturally occurring amino acids found in proteins for the purpose of this disclosure as they are generally introduced by means other than ribosomal translation of mRNA.
• “Pharmaceutically acceptable excipient” refers to an excipient that can be included in compositions and that causes no significant adverse toxicological effect on the patient and is approved or approvable by the FDA for therapeutic use, particularly in humans.
• Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose and the like.
• Therapeutic proteins are administered in an effective regime meaning a dosage, route of administration and frequency of administration that delays the onset, reduces the severity, inhibits further deterioration, and/or ameliorates at least one sign or symptom of a disorder. If a patient is already suffering from a disorder, the regime can be referred to as a therapeutically effective regime. If the patient is at elevated risk of the disorder relative to the general population but is not yet experiencing symptoms, the regime can be referred to as a prophylactically effective regime. In some instances, therapeutic or prophylactic efficacy can be observed in an individual patient relative to historical controls or past experience in the same patient. In other instances, therapeutic or prophylactic efficacy can be demonstrated in a preclinical or clinical trial in a population of treated patients relative to a control population of untreated patients.
• the “biological half-life” of a substance is a pharmacokinetic parameter which specifies the time required for one half of the substance to be removed from a tissue or an organism following introduction of the substance.
• BCVA denotes Best Corrected Visual Acuity.
• OCT-A denotes OCT-Angiography
• SD-OCT denotes Spectral Domain Optical Coherence Tomography.
• a “polymer” is a molecule composed of many repeating subunits.
• the subunits also sometimes referred to as “monomers” can be the same or different.
• DNA, protein and complex carbohydrates are examples of natural polymers.
• Poly-styrene and poly-acrylamide are examples of synthetic polymers.
• a polymer composed of repeating units of a single monomer is called a homopolymer.
• a polymer composed of two or more monomers is called a copolymer or sometimes a heteropolymer.
• a copolymer in which certain monomer types are clustered together are sometimes called block copolymers.
• Polymers can be linear or branched.
• polymer When the polymer is branched, polymer chains having a common origin are sometimes referred to as a polymer arm(s).
• biopolymer denotes that a polymer has been linked to the protein of interest. The term can also be described as the “conjugated” form of the protein.
• An “initiator” is a compound capable of serving as a substrate on which one or more polymerizations can take place using monomers or comonomers as described herein.
• the polymerization can be a conventional free radical polymerization or preferably a controlled/”living” radical polymerization, such as Atom Transfer Radical Polymerization (ATRP), Reversible Addition-Fragmentation-Termination (RAFT) polymerization or nitroxide mediated polymerization (NMP).
• the polymerization can be a “pseudo” controlled polymerization, such as degenerative transfer.
• Initiators suitable for ATRP contain one or more labile bonds which can be homolytically cleaved to form an initiator fragment, I, being a radical capable of initiating a radical polymerization, and a radical scavenger, I’, which reacts with the radical of the growing polymer chain to reversibly terminate the polymerization.
• the radical scavenger F is typically a halogen, but can also be an organic moiety, such as a nitrile.
• the initiator contains one or more 2- bromoisobutyrate groups as sites for polymerization via ATRP.
• a “chemical linker” refers to a chemical moiety that links two groups together, such as a half-life extending moiety and a protein.
• the linker can be cleavable or non-cleavable.
• Cleavable linkers can be hydrolysable, enzymatically cleavable, pH sensitive, photolabile, or disulfide linkers, among others.
• Other linkers include homobifunctional and heterobifunctional linkers.
• a “linking group” is a functional group capable of forming a covalent linkage consisting of one or more bonds to a bioactive agent. Non-limiting examples include those illustrated in Table 1 of WO2013059137 (incorporated by reference).
• reactive group refers to a group that is capable of reacting with another chemical group to form a covalent bond, i.e. is covalently reactive under suitable reaction conditions, and generally represents a point of attachment for another substance.
• the reactive group is a moiety, such as maleimide or succinimidyl ester, is capable of chemically reacting with a functional group on a different moiety to form a covalent linkage.
• Reactive groups generally include nucleophiles, electrophiles and photoactivatable groups.
• phosphorylcholine also denoted as “PC,” refers to the following: where * denotes the point of attachment.
• the phosphoryl choline is a zwitterionic group and includes salts (such as inner salts), and protonated and deprotonated forms thereof.
• phosphorylcholine-based polymer is a polymer that contains phosphorylcholine.
• Zwitterion containing polymer refers to a polymer that contains a zwitterion.
• Poly(acryloyloxyethyl phosphorylcholine) containing polymer refers to a polymer containing 2-(acryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate as monomer.
• Poly(methacryloyloxyethyl phosphoryl choline) containing polymer refers to a polymer containing 2-(methacryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate as monomer.
• “molecular weight” in the context of the polymer can be expressed as either a number average molecular weight, or a weight average molecular weight or a peak molecular weight. Unless otherwise indicated, all references to molecular weight herein refer to the peak molecular weight. These molecular weight determinations, number average (Mn), weight average (Mw) and peak (Mp), can be measured using size exclusion chromatography or other liquid chromatography techniques.
• Alkyl can include any number of carbons, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6 and 5-6 carbons.
• carboxyalkyl means an alkyl group (as defined herein) substituted with a carboxy group.
• carboxycycloalkyl means an cycloalkyl group (as defined herein) substituted with a carboxy group.
• alkoxyalkyl means an alkyl group (as defined herein) substituted with an alkoxy group.
• carboxy employed herein refers to carboxylic acids and their esters.
• endocyclic refers to an atom or group of atoms which comprise part of a cyclic ring structure.
• heterocycloalkyl refers to a ring system having from 3 ring members to about 20 ring members and from 1 to about 5 heteroatoms such as N, O and S. Additional heteroatoms can also be useful, including, but not limited to, B, Al, Si and P. The heteroatoms can also be oxidized, such as, but not limited to, -S(O)- and -S(O)2-.
• aryl refers to a monocyclic or multicyclic (e.g., fused bicyclic, tricyclic or greater) aromatic ring assembly containing 6 to 16 carbon atoms.
• aryl may be phenyl, benzyl or naphthyl, preferably phenyl.
• substituted phenyl groups as R are, e.g. 4-chlorophen-l-yl, 3,4-dichlorophen-l-yl, 4-methoxyphen-l-yl, 4-methylphen-l-yl, 4-aminomethylphen-l-yl, 4-methoxy ethylaminomethylphen- 1 -yl, 4-hydroxy ethylaminomethylphen- 1 -yl, 4-hydroxyethyl-(methyl)-aminomethylphen-l-yl, 3-aminomethylphen-l-yl,
• heteroaryl refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, where from 1 to 4 of the ring atoms are a heteroatom each N, O or S.
• heteroaryl is pyridyl, indolyl, quinolinyl, pyrrolyl, thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl, benzothienyl, oxazolyl, indazolyl, or any of the radicals substituted, especially mono- or di-substituted.
• nucleophile refers to an ion or atom or collection of atoms, which may be ionic, having a nucleophilic center, i.e., a center that is seeking an electrophilic center or capable of reacting with an electrophile.
• a nucleophile or nucleophilic reagent is a reagent that forms a bond to its reaction partner (the electrophile) by donating both bonding electrons.
• a “nucleophilic group” refers to a nucleophile after it has reacted with a reactive group. Non limiting examples include amino, hydroxyl, alkoxy, haloalkoxy and the like.
• Size exclusion chromatography is a chromatography technique in which molecules in solution are separated by their size. Typically an aqueous solution is applied to transport the sample through the column which is packed with resins of various pore sizes. The resin is expected to be inert to the analyte when passing through the column and the analytes separate from each other based on their unique size and the pore size characteristics of the selected column.
• Coupling the SEC with MALS or SEC/MALS provides accurate distribution of molar mass and size (root mean square radius) as opposed to relying on a set of SEC calibration standards.
• This type of arrangement has many advantages over traditional column calibration methods. Since the light scattering and concentration are measured for each eluting fraction, the molar mass and size can be determined independently of the elution position. This is particularly relevant for species with non-globular shaped macromolecules such as the biopolymers (e.g., OG1802) or bioconjugates (e.g., a conjugate of OG1802 with an anti-HTRAl antibody); such species typically do not elute in a manner that might be described by a set of column calibration standards.
• the biopolymers e.g., OG1802
• bioconjugates e.g., a conjugate of OG1802 with an anti-HTRAl antibody
• a SEC/MALS analysis includes a Waters HPLC system with Alliance 2695 solvent delivery module and Waters 2996 Photodiole Array Detector equipped with a Shodex SEC -HPLC column (7.8x300mm). This is connected online with a Wyatt miniDawn TREOS and Wyatt Optilab T-rEX differential refractometer.
• the Empower software from Waters can be used to control the Waters HPLC system and the ASTRA V 6.1 .7.16 software from Wyatt can be used to acquire the MALS data from the Wyatt miniDawn TREOS, dn/dc data from the T-rEX detector and the mass recovery data using the A280 absorbance signal from the Waters 2996 Photodiole Array detector.
• SEC can be carried out at ImL/min in IxPBS pH 7.4, upon sample injection, the MALS and RI (or UV) signals can be analyzed by the ASTRA software for determination of absolute molar mass (Mp, Mw, Mn) and polydisperse index (PDI).
• the calculation also involves the input dn/dc values for polymer and protein as 0.142 and 0.183, respectively.
• the dn/dc is calculated based on the weighted MW of the polymer and the protein to be about 0.148 using the formula below:
• Conjugate dn/dc 0.142 x [ MW polymer /(MWpolymer+MWprotein)]+ 0.183 x [MW/rate/// /(MWpolymer+MWprotein)] where MW polymer for OG1802 is 800 kDa and the MW protein for HC41/LC21 is 146 kDa.
• aHTRAl and “anti-HTRAl antibody” are used interchangeably herein to refer to an antibody that binds to HTRA1, as provided herein.
• a or “an” entity refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound.
• a compound refers to one or more compounds or at least one compound.
• the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.
• an anti- HTRAl antibody includes a heavy chain (and/or heavy chain variable region (VH)) and a light chain (and/or light chain variable region (VL)), wherein the heavy chain (or VH) has: a heavy chain complementarity determining region 1 (CDRHI) having an amino acid sequence of a CDRHI in any one of the heavy chain variable region (VH) sequences in Figs.
• CDRHI heavy chain complementarity determining region 1
• the light chain (or VL) has: a light chain complementarity determining region 1 (CDRLI) having an amino acid sequence of a CDRi.l in any one of the light chain variable region (VL) sequences in Figs.
• CDRLI light chain complementarity determining region 1
• the 3 VH CDR sequences are paired with the 3 VL CDR sequences according to any one of the paired arrangements of VH and VL sequences provided in FIG. 21 and Tables 0.4, 0.5, 0.7, 0.10, 0.11, 0.12, 1.2, 6.3, and 7.1.
• the antibody (or conjugate thereof) includes a heavy chain variable region having 3 CDR sequences that each have an amino acid sequence based on a consensus sequence of the corresponding CDR from a sequence alignment of the VH sequences in one or more of Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7, 7.12, 7.17, and 8.1, where each variable position is defined as one of the options in the alignment, and a light chain variable region having 3 CDR sequences that each have an amino acid sequence based on a consensus sequence of the corresponding CDR from a sequence alignment of the VL sequences in one or more of Figs.
• each CDR can be specified according to the Kabat, Chothia or IMGT definition.
• the anti-HTRAl antibody light chain variable region includes at least one of the CDRHI, CDRH2, and CDRH3 sequences in any one of the VH sequences set forth in Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7, 7.12, 7.17, and 8.1.
• the anti-HTRAl antibody heavy chain variable region includes a CDRHI, CDRH2, and CDRH3 sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to an amino acid sequence of a corresponding CDRHI, CDRH2, and CDRH3 set forth in any one of the VH sequences in Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7, 7.12, 7.17, and 8.1.
• the anti-HTRAl antibody heavy chain variable region includes a CDRHI, CDRH2, and CDRH3 sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%
• HTRAl antibody light chain variable region includes at least one of the CDRLI, CDRL2, and CDRL3 sequences in any one of the VL sequences set forth in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and 8.2.
• the anti-semiconductor anti-semiconductor anti-semiconductor anti-s includes at least one of the CDRLI, CDRL2, and CDRL3 sequences in any one of the VL sequences set forth in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and 8.2.
• HTRAl antibody light chain variable region includes a CDRLI, CDRL2, and CDRL3 sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to an amino acid sequence of a corresponding CDRHI, CDRL2, and CDRL3 set forth in any one of the VL sequences in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and 8.2.
• the 3 CDRH sequences are paired with the 3 CDRL sequences according to at least one of the paired arrangements of VH and VL sequences provided in FIG. 21 and Tables 0.4, 0.5, 0.7, 0.10, 0.11, 0.12, 1.2, 6.3, and 7.1.
• each CDR can be specified according to the Kabat, Chothia or IMGT definition.
• an anti-HTRAl antibody (or conjugate thereof) includes a heavy chain (and/or heavy chain variable region (VH)) and a light chain (and/or light chain variable region (VL)), wherein the heavy chain (or VH) has: a heavy chain complementarity determining region 1 (CDRHI) having an amino acid sequence of a CDRHI in any one of the heavy chain variable region (VH) sequences in Table 0.1, or with a variation (e.g., substitution, deletion) at no more than 5 (e.g., no more than 4, no more than 3, no more than 2, no more than 1) residues thereof; a CDRH2 having an amino acid sequence of a CDRH2 in any one of the VH sequences in Table 0.1, or with a variation (e.g., substitution, deletion) at no more than 5 (e.g., no more than 4, no more than 3, no more than 2, no more than 1) residues thereof; and a CDRH3 having an amino acid sequence of
• the 3 CDRH sequences are paired with the 3 CDRL sequences according to any one of the paired arrangements of VH and VL sequences provided in Table 0.1.
• the antibody (or conjugate thereof) includes a heavy chain variable region having 3 CDR sequences that each have an amino acid sequence based on a consensus sequence of the corresponding CDR from a sequence alignment of the VH sequences in one or more of Table 0.1, where each variable position is defined as one of the options in the alignment, and a light chain variable region having 3 CDR sequences that each have an amino acid sequence based on a consensus sequence of the corresponding CDR from a sequence alignment of the VL sequences in one or more of Table 0.1, where each variable position is defined as one of the options in the alignment.
• each CDR can be specified according to the Kabat, Chothia or IMGT definition.
• the anti-HTRAl antibody (or conjugate thereof) having the 3 CDRs in each of the VH and VL sequence of any one of the VH/VL pairings as shown in Table 0.1 is an antagonistic anti-HTRAl antibody (or conjugate thereof) that inhibits HTRA1 function (e.g., protease activity).
• an anti-HTRAl antibody (or conjugate thereof) derived from (e.g., through one or more rounds of affinity maturation) an anti-HTRAl antibody having the 3 CDRs in each of the VH and VL sequence of any one of the VH/VL pairings as shown in Table 0.1 is an antagonistic anti-HTRAl antibody (or conjugate thereof) that inhibits HTRA1 function (e.g., protease activity).
• the anti-HTRAl antibody heavy chain variable region includes any of the CDRul, CDRH2, and CDRH3 sequences in any one of the VH sequences set forth in Table 0.1.
• the anti-HTRAl antibody heavy chain variable region includes a CDRul, CDRu2, and CDRu3 sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80- 100%, 85-99%, 90-97%, 95-100%, etc ), identical to an amino acid sequence of a corresponding CDRHI, CDRH2, and CDRH3 set forth in any one of the VH sequences in Table 0.1.
• the anti-HTRAl antibody light chain variable region includes any of the CDRLI, CDRL2, and CDRL3 sequences in any one of the VL sequences set forth in Table 0.1.
• the anti-HTRAl antibody light chain variable region includes a CDRLI , CDRL2, and CDRL3 sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90- 97%, 95-100%, etc ), identical to an amino acid sequence of a corresponding CDRul, CDRL2, and CDRL3 set forth in any one of the VL sequences in Table 0.1.
• the 3 CDRH sequences are paired with the 3 CDRL sequences according to any one of the paired arrangements of VH and VL sequences provided in Table 0.1.
• each CDR can be specified according to the Kabat, Chothia or IMGT definition.
• an anti-HTRAl antibody (or conjugate thereof) includes a heavy chain (and/or heavy chain variable region (VH)) and a light chain (and/or light chain variable region (VL)), wherein the heavy chain (or VH) has: a heavy chain complementarity determining region 1 (CDRHI ) having an amino acid sequence of a CDRHI in the heavy chain variable region (VH) sequence in aHTRAl_36 (SEQ ID NO: 43), or with a variation (e.g., substitution, deletion) at no more than 5 (e.g., no more than 4, no more than 3, no more than 2, no more than 1) residues thereof; a CDRu2 having an amino acid sequence of a CDRH2 in the VH sequence in aHTRAl_36 (SEQ ID NO: 43), or with a variation (e.g., substitution, deletion) at no more than 5 (e.g., no more than 4, no more than 3, no more than 2, no more than 1) residues thereof;
• CDRu2 having an amino
• each CDR can be specified according to the Kabat, Chothia or IMGT definition.
• the anti-HTRAl antibody (or conjugate thereof) is an antagonistic anti-HTRAl antibody (or conjugate thereof) that inhibits HTRA1 function (e.g., protease activity).
• the anti-HTRAl antibody heavy chain variable region includes the CDRul, CDRu2, and CDRH3 sequences in the VH sequence of aHTRAl_36 (SEQ ID NO: 43).
• the anti-HTRAl antibody heavy chain variable region includes a CDRHI, CDRH2, and CDRH3 sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80- 100%, 85-99%, 90-97%, 95-100%, etc.), identical to an amino acid sequence of a corresponding CDRul, CDRu2, and CDRu3 in the VH sequence of aHTRAl_36 (SEQ ID NO: 43).
• the anti-HTRAl antibody light chain variable region includes the CDRLI , CDRL2, and CDRL3 in the VL sequence of aHTRAl_36 (SEQ ID NO: 307).
• the anti-HTRAl antibody light chain variable region includes a CDRLI, CDRL2, and CDRL3 sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to an amino acid sequence of a corresponding CDRHI, CDRL2, and CDRL3 in the VL sequence of aHTRAl 36 (SEQ ID NO: 307).
• each CDR can be specified according to the Kabat, Chothia or IMGT definition.
• the antibody (or conjugate thereof) includes a heavy chain variable region having 3 CDR sequences that each have an amino acid sequence based on a consensus sequence of the corresponding CDR from a sequence alignment of the VH sequences as shown in Fig. 20B, and a light chain variable region having 3 CDR sequences that each have an amino acid sequence based on a consensus sequence of the corresponding CDR from a sequence alignment of the VL sequences as shown in Fig. 20A.
• each CDR can be specified according to the Kabat, Chothia or IMGT definition.
• the anti-HTRAl antibody heavy chain variable region includes any of the CDRHI, CDRH2, and CDRH3 sequences set forth in Table 0.2. In some embodiments, the anti-HTRAl antibody heavy chain variable region includes a CDRHI, CDRH2, and CDRH3 sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to a corresponding CDRnl, CDRn2, and CDRH3 sequence set forth in Table 0.2.
• the anti-HTRAl antibody heavy chain variable region includes a CDRHI, CDRH2, and CDRH3 sequence of a corresponding CDRHI, CDRH2, and CDRH3 sequence set forth in Table 0.2 with a variation (e.g., substitution, deletion) at no more than 5 (e.g., no more than 4, no more than 3, no more than 2, no more than 1) residues in each of the CDRHI, CDRH2, and CDRH3 sequences.
• the CDR sequences are as defined by Kabat, Chothia or IMGT.
• the anti-HTRAl antibody light chain variable region includes any of the CDRLI, CDRL2, and CDRL3 sequences set forth in Table 0.3.
• the anti-HTRAl antibody light chain variable region includes a CDRLI, CDRL2, and CDRL3 sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to a corresponding CDRHI, CDRL2, and CDRL3 sequence set forth in Table 0.3.
• the anti-HTRAl antibody light chain variable region includes a CDRLI , CDRL2, and CDRL3 sequence of a corresponding CDRLI, CDRL2, and CDRL3 sequence set forth in Table 0.3 with a variation (e.g., substitution, deletion) at no more than 5 (e.g., no more than 4, no more than 3, no more than 2, no more than 1) residues in each of the CDRLI, CDRL2, and CDRL3 sequences.
• the CDR sequences are as defined by Kabat, Chothia or IMGT. Table 0.3
• the anti-HTRAl antibody heavy chain variable region includes any of the CDRHI, CDRH2, and CDRH3 sequences set forth in Table 0.2, and the anti-HTRAl antibody light chain variable region includes any of the CDRLI, CDRL2, and CDRL3 sequences set forth in Table 0.3.
• the anti-HTRAl antibody heavy chain variable region includes a CDRHI, CDRH2, and CDRH3 sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to a corresponding CDRHI , CDRH2, and CDRH3 sequence set forth in Table 0.2, and the anti-HTRAl antibody light chain variable region includes a CDR sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95- 100%, etc.), identical to a corresponding CDRLI, CDRL2, and
• the anti-HTRAl antibody heavy chain variable region includes a CDRHI, CDRH2, and CDRH3 sequence of a corresponding CDRHI, CDRH2, and CDRH3 sequence set forth in Table 0.2 with a variation (e.g., substitution, deletion) at no more than 5 (e.g., no more than 4, no more than 3, no more than 2, no more than 1) residues in each of the CDRHI, CDRH2, and CDRH3 sequences
• the anti-HTRAl antibody light chain variable region includes a CDRLI, CDRL2, and CDRL3 sequence of a corresponding CDRLI , CDRL2, and CDRL3 sequence set forth in Table 0.3 with a variation (e.g., substitution, deletion) at no more than 5 (e.g., no more than 4, no more than 3, no more than 2, no more than 1) residues in each of the CDRLI , CDRL2, and CDRL3 sequences.
• the CDRHI, CDRH2, CDRH3, CDRLI, CDRL2, and CDRL3 sequences are as defined by Kabat, Chothia or IMGT.
• an anti-HTRAl antibody (or conjugate thereof) having a heavy chain variable region that includes any of the CDRHI , CDRH2, and CDRH3 sequences set forth in Table 0.2 (or variants thereof), and a light chain variable region that includes any of the CDRLI, CDRL2, and CDRL3 sequences set forth in Table 0.3 (or variants thereof) is an antagonistic anti-HTRAl antibody (or conjugate thereof) that inhibits HTRA1 function (e.g., protease activity).
• the anti-HTRAl antibody heavy chain variable region includes CDRHI : FYHVH (SEQ ID NO:830), CDRH2: SIYTSGYTEYASALES (SEQ ID NO:831), and CDRH3 : EGLQRVGVLDA (SEQ ID NO:834) or EGLQRVGVFDA (SEQ ID NO:832) or EGLQRVGVMDA (SEQ ID NO:833), and the anti-HTRAl antibody light chain variable region includes CDRLI : RSSQSLLDEAGETYLA (SEQ ID NO:840), CDRL2: EVSLLES (SEQ ID NO: 841), and CDRL3 : QQATYFPYT (SEQ ID NO: 839).
• the anti-HTRAl antibody heavy chain variable region includes CDRHI : GFSLTFYH (SEQ ID NO: SEQ ID NO:825), CDRH2: IYTSGYT (SEQ ID NO:826), and CDRH3: AREGLQRVGVFDA (SEQ ID NO: 827) or AREGLQRVGVMDA (SEQ ID NO:828) or AREGLQRVGVLDA (SEQ ID NO:829), and the anti-HTRAl antibody light chain variable region comprises CDRLI : QSLLDEAGETY (SEQ ID NO:837), CDRL2: EV (SEQ ID NO:838), and CDRL3 : QQATYFPYT (SEQ ID NO:839).
• the anti-HTRAl antibody heavy chain variable region includes CDRHI : GFSLTFY (SEQ ID NO: SEQ ID NO: 835), CDRH2: YTSGY (SEQ ID NO: 836), and CDRH3 : EGLQRVGVLDA (SEQ ID NO:834) or EGLQRVGVFDA (SEQ ID NO 832) or EGLQRVGVMDA (SEQ ID NO:833), and the anti-HTRAl antibody light chain variable region comprises CDRLI : RSSQSLLDEAGETYLA (SEQ ID NO:840), CDRL2: EVSLLES (SEQ ID NO:841), and CDRL3 : QQATYFPYT (SEQ ID NO:839).
• the anti-HTRAl antibody heavy chain variable region includes CDRHI : FYHVH (SEQ ID NO: 830), CDRH2: SIYTSGYTEYASALES (SEQ ID NO:831), and CDRH3 : EGLQRVGVFDA (SEQ ID NO:832), and the anti-HTRAl antibody light chain variable region includes CDRLI : RSSQSLLDEAGETYLA (SEQ ID NO:840), CDRL2: EVSLLES (SEQ ID NO:841), and CDRL3 : QQATYFPYT (SEQ ID NO:839).
• the anti-HTRAl antibody heavy chain variable region includes CDRHI : FYHVH (SEQ ID NO:830), CDRH2: SIYTSGYTEYASALES (SEQ ID NO:831), and CDRH3 : EGLQRVGVLDA (SEQ ID NO:834), and the anti-HTRAl antibody light chain variable region includes CDRLI : RSSQSLLDEAGETYLA (SEQ ID NO:840), CDRL2: EVSLLES (SEQ ID NO:841), and CDRL3 : QQATYFPYT (SEQ ID NO:839).
• the anti-HTRAl antibody heavy chain variable region includes CDRHI : FYHVH (SEQ ID NO:830), CDRH2: SIYTSGYTEYASALES (SEQ ID NO:831), and CDR H 3: EGLQRVGVMDA (SEQ ID NO:833), and the anti-HTRAl antibody light chain variable region includes CDRLI : RSSQSLLDEAGETYLA (SEQ ID NO:840), CDRi 2: EVSLLES (SEQ ID NO:841), and CDRL3 : QQATYFPYT (SEQ ID NO:839).
• the anti-HTRAl antibody heavy chain variable region includes CDRHI : GFSLTFYH (SEQ ID NO:825), CDRH2: IYTSGYT (SEQ ID NO:826), and CDRH3 : AREGLQRVGVFDA (SEQ ID NO:827), and the anti-HTRAl antibody light chain variable region comprises CDRLI : QSLLDEAGETY (SEQ ID NO:837), CDRL2: EV (SEQ ID NO:838), and CDRL3 : QQATYFPYT (SEQ ID NO:839).
• the anti-HTRAl antibody heavy chain variable region includes CDRHI : GFSLTFYH (SEQ ID NO:825), CDRH2: IYTSGYT (SEQ ID NO 826), and CDRH3 : AREGLQRVGVMDA (SEQ ID NO: 828), and the anti-HTRAl antibody light chain variable region comprises CDRLI : QSLLDEAGETY (SEQ ID NO:837), CDRL2: EV (SEQ ID NO:838), and CDRL3 : QQATYFPYT (SEQ ID NO:839).
• the anti- HTRAl antibody heavy chain variable region includes CDRHI : GFSLTFYH (SEQ ID NO 825), CDRH2: IYTSGYT (SEQ ID NO: 826), and CDRH3 : AREGLQRVGVLDA (SEQ ID NO:829), and the anti-HTRAl antibody light chain variable region comprises CDRLI : QSLLDEAGETY (SEQ ID NO:837), CDRL2: EV (SEQ ID NO:838), and CDRL3 : QQATYFPYT (SEQ ID NO:839).
• the anti-HTRAl antibody heavy chain variable region includes CDRHI : GFSLTFY (SEQ ID NO:835), CDRH2: YTSGY (SEQ ID NO:836), and CDRH3 : EGLQRVGVLDA (SEQ ID NO:834), and the anti-HTRAl antibody light chain variable region comprises CDRLI : RSSQSLLDEAGETYLA (SEQ ID NO:840), CDRL2: EVSLLES (SEQ ID NO:841), and CDRL3: QQATYFPYT (SEQ ID NO:839).
• the anti-HTRAl antibody heavy chain variable region includes CDRHI : GFSLTFY (SEQ ID NO:835), CDRH2: YTSGY (SEQ ID NO:836), and CDRH3: EGLQRVGVFDA (SEQ ID NO:832), and the anti-HTRAl antibody light chain variable region comprises CDRLI : RSSQSLLDEAGETYLA (SEQ ID NO:840), CDR L 2: EVSLLES (SEQ ID NO: 841), and CDRL3 : QQATYFPYT (SEQ ID NO: 839).
• the anti-HTRAl antibody heavy chain variable region includes CDRHI : GFSLTFY (SEQ ID NO: 835), CDRH2: YTSGY (SEQ ID NO: 836), and CDRH3 : EGLQRVGVMDA (SEQ ID NO:833), and the anti-HTRAl antibody light chain variable region comprises CDRLI : RSSQSLLDEAGETYLA (SEQ ID NO:840), CDRL2: EVSLLES (SEQ ID NO:841), and CDRL3 : QQATYFPYT (SEQ ID NO:839).
• the anti-HTRAl antibody (or conjugate thereof) is an antagonistic anti-HTRAl antibody (or conjugate thereof) that inhibits HTRA1 function (e.g., protease activity).
• the anti-HTRAl antibody heavy chain variable region includes CDRHI : GFSLTSYHVH (SEQ ID NO:538), CDRH2: VIWTSGNTEYN SALES (SEQ ID NO:539), and CDRH3 : AREGLRRVGVMDA (SEQ ID NO:540), and the anti-HTRAl antibody light chain variable region includes CDRLI : RSSQSLLDSDGDTYLN (SEQ ID NO:541), CDRL2: SVSNLES (SEQ ID NO:542), and CDRL3 : MQATHAPYT (SEQ ID NO:543).
• the anti-HTRAl antibody (or conjugate thereof) is an antagonistic anti-HTRAl antibody (or conjugate thereof) that inhibits HTRA1 function (e.g., protease activity).
• an anti-HTRAl antibody (or conjugate thereof) of the present disclosure includes a VH having an amino acid sequence of at least one of the VH sequences provided in Figs. 21, 22A, 23A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7,
• an anti-HTRAl antibody of the present disclosure includes a VH having an amino acid sequence at least about 80% identical to any one of the VH sequences provided in Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7,
• an anti-HTRAl antibody of the present disclosure includes a VH having an amino acid sequence at least about 90% identical to any one of the VH sequences provided in Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7,
• an anti-HTRAl antibody of the present disclosure includes a VH having an amino acid sequence at least about 95% identical to any one of the VH sequences provided in Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7,
• an anti-HTRAl antibody of the present disclosure includes a VH having an amino acid sequence at least about 97% identical to any one of the VH sequences provided in Figs. 21, 22A, 23 A, and 24A, and Tables 1 .2, 6.3, 6.5, 7.1, 7.4, 7.7,
• any variation within the amino acid sequence of the VH of the anti-HTRAl antibody from any one of the VH sequences provided in Figs. 21, 22A, 23A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7,
• an anti-HTRAl antibody of the present disclosure includes a VH having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100% identical to any one of the VH sequences provided in Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7, 7.12, 7.17, and 8.1, where any variation (e.g., substitution, deletion) within the amino acid sequence of the VH of the anti-HTRAl antibody from any one of the VH sequences provided in Figs.
• an anti-HTRAl antibody of the present disclosure includes a VH having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100% identical to any one of the VH sequences provided in Figs.
• an anti-HTRAl antibody (or conjugate thereof) of the present disclosure includes a VL having an amino acid sequence of at least one of the VL sequences provided in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13,
• an anti-HTRAl antibody of the present disclosure includes a VL having an amino acid sequence at least about 80% identical to any one of the VL sequences provided in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8,
• an anti-HTRAl antibody of the present disclosure includes a VL having an amino acid sequence at least about 90% identical to any one of the VL sequences provided in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and 8.2.
• an anti-HTRAl antibody of the present disclosure includes a VL having an amino acid sequence at least about 95% identical to any one of the VL sequences provided in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8,
• an anti-HTRAl antibody of the present disclosure includes a VL having an amino acid sequence at least about 97% identical to any one of the VL sequences provided in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8,
• an anti-HTRAl antibody of the present disclosure includes a VL having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100% identical to any one of the VL sequences provided in Figs.
• an anti-HTRAl antibody of the present disclosure includes a VL having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100% identical to any one of the VL sequences provided in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and 8.2, where any sequence variation from the VL sequence provided in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and 8.2 is not within a CDR of the VL sequence.
• an anti-HTRAl antibody (or conjugate thereof) of the present disclosure includes a VH having an amino acid sequence of at least one of the VH sequences provided in Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7, 7.12, 7.17, and 8.1, and a VL having an amino acid sequence of at least one ofthe VL sequences provided in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and
• an anti-HTRAl antibody (or conjugate thereof) of the present disclosure includes a VH having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95- 100%, etc.), identical to any one of the VH sequences provided in Figs.
• VL having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the VL sequences provided in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and 8.2.
• an anti-HTRAl antibody (or conjugate thereof) of the present disclosure includes a VH having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90- 97%, 95-100%, etc.), identical to any one of the VH sequences provided in Figs.
• 6.3, 6.5, 7.1, 7.4, 7.7, 7.12, 7.17, and 8.1 is in no more than 5, 4, 3, 2, or 1 residue of a CDR of the VH sequence, and a VL having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95- 100%, etc.), identical to any one of the VL sequences provided in Figs.
• an anti-HTRAl antibody (or conjugate thereof) of the present disclosure includes a VH having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the VH sequences provided in Figs. 21, 22A, 23 A, and 24A, and Tables 1 .2, 6.3, 6.5, 7.1, 7.4, 7.7, 7.12, 7.17, and 8.1, where any sequence variation from the VH sequence provided in Figs.
• VH and VL sequences of the anti-HTRAl antibody are paired according to at least one of the paired arrangements provided in FIG. 21 and Tables 0.1, 0.4, 0.5, 0.7, 0.10, 0.11, 0.12, 1.2, 6.3, and 7.1.
• the anti-HTRAl antibody (or conjugate thereof) includes a heavy chain variable region having at least an amino acid sequence of a consensus sequence from a sequence alignment of the VH sequences in one or more of Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7, 7.12, 7.17, and 8.1, where each variable position is defined as one of the options in the alignment, and a light chain variable region having at least an amino acid sequence of a consensus sequence from a sequence alignment of the VL sequences in one or more of Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and 8.2, where each variable position is defined as one of the options in the alignment.
• the antibody (or conjugate thereof) includes at least one of the pairings of VH and VL as shown in Fig. 21. In some embodiments the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 80% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Fig. 21. In some embodiments the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 90% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Fig. 21.
• the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 95% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Fig. 21 . In some embodiments the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 97% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Fig. 21. In some embodiments, the antibody (or conjugate thereof) includes a VH and VL each having CDRs of the corresponding CDRs in at least one of the pairings of VH and VL as shown in Fig. 21.
• the antibody (or conjugate thereof) includes at least one of the pairings of VH and VL as shown in Table 0.1. In some embodiments the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 80% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Table 0.1. In some embodiments the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 90% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Table 0.1.
• the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 95% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Table 0.1. In some embodiments the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 97% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Table 0.1. In some embodiments, the antibody (or conjugate thereof) includes a VH and VL each having CDRs of the corresponding CDRs in at least one of the pairings of VH and VL as shown in Table 0.1.
• the anti-HTRAl antibody having a VH/VL pairing as shown in Table 0.1 is an antagonistic anti-HTRAl antibody that inhibits HTRA1 function (e.g., protease activity).
• the anti-HTRAl antibody (or conjugate thereof) having a VH/VL pairing as shown in Table 0.1 is an antagonistic anti- HTRAl antibody (or conjugate thereof) that inhibits HTRA1 function (e.g., protease activity).
• an anti-HTRAl antibody (or conjugate thereof) derived from (e.g., through one or more rounds of affinity maturation) an anti-HTRAl antibody having a VH/VL pairing as shown in Table 0.1 is an antagonistic anti-HTRAl antibody (or conjugate thereof) that inhibits HTRA1 function (e.g., protease activity).
• the antibody (or conjugate thereof) includes at least one of the pairings of VH and VL as shown in Table 0.4 (where the HC# corresponds to a VH sequence as provided in Fig. 22A or Table 6.5, and the LC# corresponds to a VL sequence as provided in Fig. 22B or Table 6.6). In some embodiments the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 80% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Table 0.4.
• the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 90% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Table 0.4. In some embodiments the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 95% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Table 0.4. In some embodiments the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 97% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Table 0.4. In some embodiments, the antibody (or conjugate thereof) includes a VH and VL each having CDRs of the corresponding CDRs in at least one of the pairings of VH and VL as shown in Table 0.4.
• the antibody (or conjugate thereof) includes at least one of the pairings of VH and VL as shown in Table 0.5. In some embodiments the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 80% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Table 0.5. In some embodiments the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 90% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Table 0.5.
• the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 95% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Table 0.5. In some embodiments the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 97% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Table 0.5. In some embodiments, the antibody (or conjugate thereof) includes a VH and VL each having CDRs of the corresponding CDRs in at least one of the pairings of VH and VL as shown in Table 0.5.
• the antibody includes at least one VH point mutation, one VL point mutation, or both as provided in Table 0.6. In some embodiments, the antibody includes at least two VH point mutations, at least one VL point mutation, or both as provided in Table 0.6. In some embodiments, the antibody includes at least one VH point mutation, at least two VL point mutations, or both as provided in Table 0.6. In some embodiments, the antibody includes at least two VH point mutations, at least two VL point mutations, or both as provided in Table 0.6. In some embodiments, the antibody includes 1, 2, 3, 4, 5, 6, or 7 VH point mutations, as provided in Table 0.6.
• the antibody includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 VL point mutations, as provided in Table 0.6. In some embodiments, the antibody includes 1, 2, 3, 4, 5, 6, or 7 VH point mutations, 1, 2, 3, 4, 5, 6, 7, 8, or 9 VL point mutations, or the respective number of point mutations in both VH and VL, as provided in Table 0.6. In some embodiments, the VH point mutations provided in Table 0.6 are relative to SEQ ID NO: 43. In some embodiments, the VL point mutations provided in Table 0.6 are relative to SEQ ID NO: 307.
• the antibody (or conjugate thereof) includes at least one of the pairings of VH and VL as shown in Table 0.7 (where the HC# corresponds to a VH sequence as provided in Fig. 22A or 23A, and the LC# corresponds to a VL sequence as provided in Fig. 22B or 23B). In some embodiments the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 80% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Table 0.7.
• the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 90% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Table 0.7. In some embodiments the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 95% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Table 0.7. In some embodiments the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 97% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Table 0.7. In some embodiments, the antibody (or conjugate thereof) includes a VH and VL each having CDRs of the corresponding CDRs in at least one of the pairings of VH and VL as shown in Table 0.7.
• the antibody (or conjugate thereof) of the present disclosure includes at least one, at least two, at least three, at least four, at least five, or any six of the CDRs shown in Tables 0.8 and 0.9. In some embodiments, the antibody (or conjugate thereof) includes at least one, at least two, at least three, at least four, at least five, or any six of the CDRs shown in Tables 0.8 and 0.9 with no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 point mutation per CDR.
• the antibody (or conjugate thereof) of the present disclosure includes a combination of CDRs selected from any combination of the CDRs shown in Table 0.8, with no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 point mutation per CDR, and includes a combination of CDRs selected from any combination of the CDRs shown in Table 0.9, with no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 point mutation per CDR.
• the antibody (or conjugate thereof) includes one or more CDRs that is a Kabat, Chothia, or IMGT option or subpart of the corresponding CDR of Tables 0.8 or 0.9.
• the antibody (or conjugate thereof) includes at least one of the pairings of VH and VL as shown in Table 0.10 (where the HC# corresponds to a VH sequence as provided in Fig. 22A, 23 A, or 24A, and the LC# corresponds to a VL sequence as provided in Fig. 22B, 23B, or 24B). In some embodiments the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 80% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Table 0.10.
• the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 90% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Table 0.10. In some embodiments the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 95% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Table 0.10. In some embodiments the antibody (or conjugate thereof) includes at least a VH/VL pairing where each VH and VL sequence is at least about 97% identical to a corresponding VH and VL sequence of one of the sequence pairs shown in Table 0.10. In some embodiments, the antibody (or conjugate thereof) includes a VH and VL each having CDRs of the corresponding CDRs in at least one of the pairings of VH and VL as shown in Table 0.10. Table 0.10
• an anti-HTRAl antibody (or conjugate thereof) of the present disclosure includes a VH having an amino acid sequence of SEQ ID NO:725, and a VL having an amino acid sequence of SEQ ID NO:748.
• the anti- HTRAl antibody (or conjugate thereof) includes a VH having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80- 100%, 85-99%, 90-97%, 95-100%, etc.), identical to SEQ ID NO:725, and a VL having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-10
• any variation within the amino acid sequence of the VH of the anti-HTRAl antibody from SEQ ID NO:725 is not within a CDR of the VH sequence. In some embodiments, any variation within the amino acid sequence of the VL of the anti-HTRAl antibody from SEQ ID NO:748 is not within a CDR of the VL sequence.
• an anti-HTRAl antibody (or conjugate thereof) of the present disclosure includes a VH having an amino acid sequence of SEQ ID NO:746, and a VL having an amino acid sequence of SEQ ID NO:748.
• the anti- HTRA1 antibody (or conjugate thereof) includes a VH having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80- 100%, 85-99%, 90-97%, 95-100%, etc.), identical to SEQ ID NO:746, and a VL having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%>, at least about 97%o, or about 100%, or a percentage in a range defined by any two of the preceding values, identical to SEQ ID NO: 748.
• any variation within the amino acid sequence of the VH of the anti-HTRAl antibody from SEQ ID NO:746 is not within a CDR of the VH sequence. In some embodiments, any variation within the amino acid sequence of the VL of the anti-HTRAl antibody from SEQ ID NO:748 is not within a CDR of the VL sequence.
• an anti-HTRAl antibody (or conjugate thereof) of the present disclosure includes a VH having an amino acid sequence of SEQ ID NO:745, and a VL having an amino acid sequence of SEQ ID NO:748.
• the anti- HTRAl antibody (or conjugate thereof) includes a VH having an amino acid sequence at least about 80%o, at least about 85%>, at least about 90%, at least about 95%, at least about 97%>, or about 100%o, or a percentage in a range defined by any two of the preceding values (e.g., 80- 100%, 85-99%o, 90-97%o, 95-100%), etc.), identical to SEQ ID NO:745, and a VL having an amino acid sequence at least about 80%, at least about 85%, at least about 90%>, at least about 95%o, at least about 97%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%), 85-
• any variation within the amino acid sequence of the VH of the anti-HTRAl antibody from SEQ ID NO:745 is not within a CDR of the VH sequence. In some embodiments, any variation within the amino acid sequence of the VL of the anti-HTRAl antibody from SEQ ID NO:748 is not within a CDR of the VL sequence.
• an anti-HTRAl antibody (or conjugate thereof) of the present disclosure includes a VH having an amino acid sequence of SEQ ID NO:742, and a VL having an amino acid sequence of SEQ ID NO:748.
• the anti- HTRAl antibody (or conjugate thereof) includes a VH having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%>, or about 100%, or a percentage in a range defined by any two of the preceding values (e g., 80- 100%, 85-99%, 90-97%, 95-100%, etc.), identical to SEQ ID NO:742, and a VL having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95
• any variation within the amino acid sequence of the VH of the anti-HTRAl antibody from SEQ ID NO:742 is not within a CDR of the VH sequence. In some embodiments, any variation within the amino acid sequence of the VL of the anti-HTRAl antibody from SEQ ID NO:748 is not within a CDR of the VL sequence.
• an anti-HTRAl antibody (or conjugate thereof) of the present disclosure includes a VH having an amino acid sequence of SEQ ID NO:744, and a VL having an amino acid sequence of SEQ ID NO:748.
• the anti- HTRAl antibody (or conjugate thereof) includes a VH having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80- 100%, 85-99%, 90-97%, 95-100%, etc.), identical to SEQ ID NO:744, and a VL having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-10
• any variation within the amino acid sequence of the VH of the anti-HTRAl antibody from SEQ ID NO:744 is not within a CDR of the VH sequence. In some embodiments, any variation within the amino acid sequence of the VL of the anti-HTRAl antibody from SEQ ID NO:748 is not within a CDR of the VL sequence.
• an anti-HTRAl antibody (or conjugate thereof) of the present disclosure includes a VH having an amino acid sequence of SEQ ID NO:563, and a VL having an amino acid sequence of SEQ ID NO:569.
• the anti- HTRAl antibody (or conjugate thereof) includes a VH having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80- 100%, 85-99%, 90-97%, 95-100%, etc.), identical to SEQ ID NO:563, and a VL having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%
• any variation within the amino acid sequence of the VH of the anti-HTRAl antibody from SEQ ID NO: 563 is not within a CDR of the VH sequence. In some embodiments, any variation within the amino acid sequence of the VL of the anti-HTRAl antibody from SEQ ID NO:569 is not within a CDR of the VL sequence.
• the anti-HTRAl antibody contemplates the antibody portion of a conjugate of the anti-HTRAl antibody, as provided herein.
• An anti-HTRAl antibody of the present disclosure (or a conjugate thereof) binds to HTRA1 from any suitable species. In some embodiments, the antibody (or conjugate thereof) binds to human HTRA1. In some embodiments, the antibody (or conjugate thereof) binds to human HTRA1 having the amino acid sequence of SEQ ID NO: 1, or a portion thereof.
• the anti-HTRAl antibody (or conjugate thereof) binds to the loop A peptide of HTRA1. In some embodiments, the anti-HTRAl antibody (or conjugate thereof) binds to a loop A peptide of HTRA1, where the loop A peptide includes the amino acid sequence of FRKLPFSKREVPV (SEQ ID NO:851). In some embodiments, the anti-HTRAl antibody (or conjugate thereof) binds to a loop A peptide of HTRA1, where the loop A peptide has the amino acid sequence of FRKLPFSKREVPV (SEQ ID NO:851). Binding to the loop A peptide (or lack thereof) can be determined using any suitable option, e.g., by ELISA.
• the anti-HTRAl antibody (or conjugate thereof) that binds to the loop A peptide of HTRA1 includes a heavy chain variable region having any of the CDRHI, CDRH2, and CDRH3 sequences in any one of the VH sequences set forth in Table 0. 11.
• the anti-HTRAl antibody heavy chain variable region includes a CDRHI, CDRH2, and CDRH3 sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95- 100%, etc.), identical to an amino acid sequence of a corresponding CDRHI, CDRH2, and CDRH3 set forth in any one of the VH sequences in Table 0.11.
• the anti-HTRAl antibody (or conjugate thereof) that binds to the loop A peptide of HTRA1 includes a light chain variable region having any of the CDRLI , CDRL2, and CDRL3 sequences in any one of the VL sequences set forth in Table 0.11.
• the anti-HTRAl antibody light chain variable region includes a CDRLI, CDRL2, and CDRL3 sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to an amino acid sequence of a corresponding CDRLI, CDRL2, and CDRL3 set forth in any one of the VL sequences in Table 0.11.
• the 3 CDRH sequences are paired with the 3 CDRL sequences according to at least one of the paired arrangements of VH and VL sequences provided in Table 0.11.
• each CDR can be specified according to the Kabat, Chothia or IMGT definition.
• the anti-HTRAl antibody (or conjugate thereof) that binds to the loop A peptide of HTRA1 includes a heavy chain variable region having at least one of the VH sequences set forth in Table 0.11.
• the anti-HTRAl antibody heavy chain variable region includes a VH sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the VH sequences in Table 0.11.
• any variation (e.g., substitution, deletion) within the amino acid sequence of the VH of the anti-HTRAl antibody from any one of the VH sequences provided in Table 0.11 is in no more than 5, 4, 3, 2, or 1 residue of a CDR of the VH sequence. In some embodiments, any variation within the amino acid sequence of the VH of the anti-HTRAl antibody from a VH sequence provided in Table 0.11 is not within a CDR of the VH sequence.
• the anti-HTRAl antibody heavy chain variable region includes a VH sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the VH sequences in Table 0.11, where any variation (e.g., substitution, deletion) within the amino acid sequence of the VH of the anti-HTRAl antibody from any one of the VH sequences provided in Table 0.11 is in no more than 5, 4, 3, 2, or 1 residue of a CDR of the VH sequence.
• a VH sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85
• the anti-HTRAl antibody heavy chain variable region includes a VH sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the VH sequences in Table 0.11, where any variation within the amino acid sequence of the VH of the anti-HTRAl antibody from a VH sequence provided in Table 0.11 is not within a CDR of the VH sequence.
• the anti-HTRAl antibody (or conjugate thereof) that binds to the loop A peptide of HTRA1 includes a light chain variable region having at least one of the VL sequences set forth in Table 0.11.
• the anti-HTRAl antibody light chain variable region includes a VL sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the VL sequences in Table 0.11.
• any variation (e.g., substitution, deletion) within the amino acid sequence of the VL of the anti-HTRAl antibody from any one of the VL sequences provided in Table 0.11 is in no more than 5, 4, 3, 2, or 1 residue of a CDR of the VL sequence. In some embodiments, any variation within the amino acid sequence of the VL of the anti-HTRAl antibody from a VL sequence provided in Table 0.11 is not within a CDR of the VL sequence.
• the anti-HTRAl antibody light chain variable region includes a VL sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the VL sequences in Table 0.11, where any variation (e.g., substitution, deletion) within the amino acid sequence of the VL of the anti-HTRAl antibody from any one of the VL sequences provided in Table 0.11 is in no more than 5, 4, 3, 2, or 1 residue of a CDR of the VL sequence.
• a VL sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85
• the anti-HTRAl antibody light chain variable region includes a VL sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the VL sequences in Table 0.11, where any variation within the amino acid sequence of the VL of the anti-HTRAl antibody from a VL sequence provided in Table 0.11 is not within a CDR of the VL sequence.
• the anti-HTRAl antibody (or conjugate thereof) that binds to the loop A peptide of HTRA1 includes a heavy chain variable region having at least one of the VH sequences set forth in Table 0.11, and a light chain variable region having at least one of the VL sequences set forth in Table 0.11.
• the anti-HTRAl antibody heavy chain variable region includes a VH sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the VH sequences in Table 0.11, and the anti-HTRAl antibody light chain variable region includes a VL sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the VL sequences in Table 0.11.
• the anti-HTRAl antibody heavy chain variable region includes a VH sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95- 100%, etc.), identical to any one of the VH sequences in Table 0.11, where any variation (e.g., substitution, deletion) within the amino acid sequence of the VH of the anti-HTRAl antibody from any one of the VH sequences provided in Table 0.11 is in no more than 5, 4, 3, 2, or 1 residue of, or is not within, a CDR of the VH sequence, and the anti-HTRAl antibody light chain variable region includes a VL sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a
• the anti-HTRAl antibody (or conjugate thereof) does not bind to the loop A peptide of HTRA1. In some embodiments, the anti-HTRAl antibody (or conjugate thereof) does not bind to the loop A peptide of HTRA1, where the loop A peptide includes the amino acid sequence of FRKLPFSKREVPV (SEQ ID NO:851). In some embodiments, the anti-HTRAl antibody (or conjugate thereof) does not bind to the loop A peptide of HTRA1, where the loop A peptide has the amino acid sequence of FRKLPFSKREVPV (SEQ ID NO:851).
• the anti-HTRAl antibody does not require the loop A peptide of HTRA1 or binding to HTRA1, where the loop A peptide has the amino acid sequence of FRKLPFSKREVPV (SEQ ID NO:851). Binding to the loop A peptide (or lack thereof) can be determined using any suitable option, e.g., by ELISA.
• the anti-HTRAl antibody (or conjugate thereof) that does not bind to the loop A peptide of HTRA1 includes a heavy chain variable region having any of the CDRHI , CDRH2, and CDRH3 sequences in any one of the VH sequences set forth in Table 0.12.
• the anti-HTRAl antibody heavy chain variable region includes a CDRHI, CDRH2, and CDRH3 sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to an amino acid sequence of a corresponding CDRHI, CDRH2, and CDRH3 set forth in any one of the VH sequences in Table 0.12.
• the anti-HTRAl antibody (or conjugate thereof) that does not bind to the loop A peptide of HTRA1 includes a light chain variable region having any of the CDRLI, CDRL2, and CDRL3 sequences in any one of the VL sequences set forth in Table 0.12.
• the anti-HTRAl antibody light chain variable region includes a CDRLI, CDRL2, and CDRL3 sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to an amino acid sequence of a corresponding CDRHI, CDRL2, and CDRL3 set forth in any one of the VL sequences in Table 0.12.
• the 3 CDRu sequences are paired with the 3 CDRL sequences according to any one of the paired arrangements of VH and VL sequences provided in Table 0 0.12.
• each CDR can be specified according to the Kabat, Chothia or IMGT definition.
• the anti-HTRAl antibody (or conjugate thereof) that does not bind to the loop A peptide of HTRA1 includes a heavy chain variable region having at least one of the VH sequences set forth in Table 0.12.
• the anti- HTRAl antibody heavy chain variable region includes a VH sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80- 100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the VH sequences in Table 0.12.
• any variation (e g., substitution, deletion) within the amino acid sequence of the VH of the anti-HTRAl antibody from any one of the VH sequences provided in Table 0 0.12 is in no more than 5, 4, 3, 2, or 1 residue of a CDR of the VH sequence. In some embodiments, any variation within the amino acid sequence of the VH of the anti- HTRAl antibody from a VH sequence provided in Table 0.12 is not within a CDR of the VH sequence.
• the anti-HTRAl antibody heavy chain variable region includes a VH sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the VH sequences in Table 0.12, where any variation (e.g., substitution, deletion) within the amino acid sequence of the VH of the anti-HTRAl antibody from any one of the VH sequences provided in Table 0.12 is in no more than 5, 4, 3, 2, or 1 residue of a CDR of the VH sequence.
• a VH sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85
• the anti-HTRAl antibody heavy chain variable region includes a VH sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the VH sequences in Table 0.12, where any variation within the amino acid sequence of the VH of the anti-HTRAl antibody from a VH sequence provided in Table 0.12 is not within a CDR of the VH sequence.
• the anti-HTRAl antibody (or conjugate thereof) that does not bind to the loop A peptide of HTRA1 includes a light chain variable region having at least one of the VL sequences set forth in Table 0.12.
• the anti-HTRAl antibody light chain variable region includes a VL sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the VL sequences in Table 0.12.
• any variation (e.g., substitution, deletion) within the amino acid sequence of the VL of the anti-HTRAl antibody from any one of the VL sequences provided in Table 0.12 is in no more than 5, 4, 3, 2, or 1 residue of a CDR of the VL sequence. In some embodiments, any variation within the amino acid sequence of the VL of the anti-HTRAl antibody from a VL sequence provided in Table 0.12 is not within a CDR of the VL sequence.
• the anti-HTRAl antibody light chain variable region includes a VL sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the VL sequences in Table 0.12, where any variation (e.g., substitution, deletion) within the amino acid sequence of the VL of the anti-HTRAl antibody from any one of the VL sequences provided in Table 0.12 is in no more than 5, 4, 3, 2, or 1 residue of a CDR of the VL sequence.
• the anti-HTRAl antibody light chain variable region includes a VL sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the VL sequences in Table 0.12, where any variation within the amino acid sequence of the VL of the anti-HTRAl antibody from a VL sequence provided in Table 0.12 is not within a CDR of the VL sequence.
• the anti-HTRAl antibody (or conjugate thereof) that does not bind to the loop A peptide of HTRA1 includes a heavy chain variable region having at least one of the VH sequences set forth in Table 0.12, and a light chain variable region having at least one of the VL sequences set forth in Table 0.12.
• the anti-HTRAl antibody heavy chain variable region includes a VH sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the VH sequences in Table 0.12, and the anti-HTRAl antibody light chain variable region includes a VL sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the VL sequences in Tables 0.12.
• the anti-HTRAl antibody heavy chain variable region includes a VH sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95- 100%, etc.), identical to any one of the VH sequences in Table 0.12, where any variation (e.g., substitution, deletion) within the amino acid sequence of the VH of the anti-HTRAl antibody from any one of the VH sequences provided in Table 0.12 is in no more than 5, 4, 3, 2, or 1 residue of, or is not within, a CDR of the VH sequence, and the anti-HTRAl antibody light chain variable region includes a VL sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a
• an anti-HTRAl antibody (or a conjugate thereof) of the present disclosure can bind to HTRA1 with a high affinity.
• the anti-HTRAl antibody binds to HTRA1 with a binding affinity (KD) of about 1.0 x 10' 9 M or less, about 1.0 x IO 10 M or less, about 5.0 x 10' 11 M or less, about 2.0 x 10' 11 M or less, about 1.0 x 10' 11 M or less, about 5.0 x IO’ 12 M or less, about 2.0 x 10' 12 M or less, or about 1.0 x 10' 12 M or less, or with a binding affinity in a range defined by any two of the preceding values (e.g., 1.0 x 10' 9 - 1.0 x IO’ 12 M, 1.0 x IO’ 10 - 1.0 x IO’ 12 M, 5.0 x 10’ 11 - 5.0 x IO’ 12 M, 1.0 x 10’ 11 - 1.0 1.0 1.0 IO
• the anti-HTRAl antibody (or conjugate thereof) binds to a HTRA1 monomer or a trimer. In some embodiment, the anti-HTRAl antibody weakly binds to a HTRA1 monomer. In some embodiments, the anti-HTRAl antibody (or conjugate thereof) selectively binds to a HTRA1 trimer over a HTRA1 monomer. In some embodiments, the anti-HTRAl antibody (or conjugate thereof) binds to a HTRA1 trimer with a higher affinity than to a HTRA1 monomer.
• the anti-HTRAl antibody binds to a HTRA1 trimer with an affinity that is two times or more, 3 times or more, 5 times or more, 10 times or more, 20 times or more, 50 times or more, 100 times or more, 1,000 times or more, 10,000 times or more, or 100,000 times or more, or a fold difference defined by any two of the preceding values (e.g., 2-100,000 times, 3-100,000 times, 10-10,000 times, 20-1,000 times, 2-10 times, 2-50 times, 5-100 times, etc.), stronger (e.g., with a lower KD) than to a HTRA1 monomer.
• the relative affinity can be determined using any suitable method, e.g., a kinetic analysis.
• an anti-HTRAl antibody (or a conjugate thereof) binds to HTRA1 with any suitable stoichiometry of antibody to HTRA1.
• the anti-HTRAl antibody (or conjugate thereof) binds to a HTRA1 trimer with a stoichiometry of at least about 0.3: 1, at least about 0.7: 1, at least about 0.9: 1, or at least about 1: 1 of anti-HTRAl antibody to HTRA1 trimer.
• the anti-HTRAl antibody (or conjugate thereof) binds to a HTRA1 trimer with a stoichiometry of at most about 6: 1, at most about 3: 1, at most about 1.5: 1, at most about 1.1 :1, or at most about 1 : 1 of anti-HTRAl antibody to HTRA1 trimer. In some embodiments, the anti-HTRAl antibody (or conjugate thereof) binds to a HTRA1 trimer with a stoichiometry in a range of about 0.3:1 to about 6: 1, about 0.7: 1 to about 3:1, about 0.7:1 to about 1.5:1, or about 0.9: 1 to about 1.1 :1, of anti-HTRAl antibody to HTRA1 trimer.
• the anti-HTRAl antibody (or conjugate thereof) binds to a HTRA1 trimer with a stoichiometry of about 1 :1.
• the stoichiometry of binding can be determined using any suitable option, e.g., size exclusion chromatography.
• An anti-HTRAl antibody (or a conjugate thereof) of the present disclosure can specifically bind HTRA1.
• the anti-HTRAl antibody (or conjugate thereof) does not bind to one or more of HTRA2, HTRA3, HTRA4, e.g., as determined by kinetic analysis.
• the anti-HTRAl antibody binds to HTRA1 with a higher affinity than to HTRA2, HTRA3, and/or HTRA4, e.g., as determined by kinetic analysis.
• the anti-HTRAl antibody binds uniquely to HTRA1 (e.g., binds to HTRA1 and does not bind to HTRA2, HTRA3 and HTRA4), e.g., as determined by kinetic analysis.
• HTRA1 has the amino acid sequence of SEQ ID NO: 1 or 756, or a fragment thereof, or the amino acid sequence of SEQ ID NO:756 without the signal peptide (e.g., as shown underlined in Fig 19) or fragment thereof.
• HTRA2 has the amino acid sequence of SEQ ID NO:757, or a fragment thereof, or the amino acid sequence of SEQ ID NO:757 without the signal peptide (e.g., as shown underlined in Fig. 19) or fragment thereof.
• HTRA3 has the amino acid sequence of SEQ ID NO:5 or 758, or a fragment thereof, or the amino acid sequence of SEQ ID NO:758 without the signal peptide (e.g., as shown underlined in Fig 19) or fragment thereof.
• HTRA4 has the amino acid sequence or a fragment of SEQ ID NO:759, or a fragment thereof, or the amino acid sequence of SEQ ID NO:759 without the signal peptide (e.g., as shown underlined in Fig. 19) or fragment thereof.
• the anti-HTRAl antibody e.g., antagonistic anti- HTRA1 antibody
• conjugate thereof inhibits an enzymatic activity, e.g., protease activity, of HTRA1 when the antibody is bound thereto.
• the anti-HTRAl antibody or conjugate thereof inhibits the protease activity of HTRA1.
• the anti-HTRAl antibody inhibits HTRA1 protease activity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or about 100%, or by a percentage within a range defined by any two of the preceding values (e.g., 10-100%, 20-100%, 30-80%, 40- 80%, 50-90%, 85-99%, etc.). In some embodiments, percentage inhibition of HTRA1 protease activity is relative to a suitable control (e.g., a blank solution, a non-specific antibody, etc.).
• a suitable control e.g., a blank solution, a non-specific antibody, etc.
• the anti-HTRAl antibody inhibits the protease activity of HTRA1 with an IC50 of 1 x 10’ 9 M or less, 5 x 10' 10 M or less, 2 x 10' 10 M or less, 1 x IO 10 M or less, 5 x 10 11 M or less, 2 x 10 11 M or less, 1 x 10 11 M or less, 5 x 10 12 M or less, 2 x IO’ 12 M or less, or 1 x 10' 12 M or less, or with an IC50 in a range defined by any two of the preceding values (e.g., 1 x 10’ 9 -1 x 10' 12 M, 5 x 10' 10 -1 x 10' 12 M, 2 x 10' 10 -2 x 10' 12 M, 1 x IO’ 10 -2 x IO’ 12 M, 1 x 10’ 11 -5 x IO’ 12 M, etc.).
• the anti-HTRAl antibody that inhibits or neutralizes HTRA1 function includes at least one of the VH/VL pairing as shown in Table 0.1.
• the anti-HTRAl antibody that inhibits or neutralizes HTRA1 function includes at least the 3 CDRs of each of the VH and VL sequences in any one of the VH/VL pairing as shown in Table 0.1.
• the anti-HTRAl antibody is derived from one or more antagonistic anti-HTRAl antibodies as provided herein (e.g., by affinity maturation).
• the anti-HTRAl antibody that inhibits or neutralizes HTRA1 function is derived from one or more parental antagonistic anti- HTRAl antibodies as provided herein (e.g., by affinity maturation).
• the anti-HTRAl antibody that inhibits or neutralizes HTRA1 function includes at least one of the following VH/VL pairing: HC4/LC1, HC1/LC1, HC7/LC1, HC4/LC3, HC4/LC4, HC1/LC3, HC7/LC4, HC17/LC21, HC37/LC21, HC39/LC21, HC40/LC21, HC41/LC21 (with reference to Figs. 22A, 22B, 24A, 24B).
• the anti-HTRAl antibody that inhibits or neutralizes HTRA1 function includes a VH and VL amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to the corresponding VH and VL sequences in any one of the following VH/VL pairing: HC4/LC1, HC1/LC1, HC7/LC1, HC4/LC3, HC4/LC4, HC1/LC3, HC7/LC4, HC17/LC21, HC37/ LC21, HC39/LC21, HC40/LC21, HC41/LC21 (with reference to Figs.
• the anti-HTRAl antibody that inhibits or neutralizes HTRA1 function includes at least the 3 CDRs of each of the VH and VL sequences in any one of the following VH/VL pairing: HC4/LC1, HC1/LC1, HC7/LC1, HC4/LC3, HC4/LC4, HC1/LC3, HC7/LC4, HC17/LC21, HC37/ LC21, HC39/LC21, HC40/LC21, HC41/LC21 (with reference to Figs. 22A, 22B, 24A, 24B).
• the anti-HTRAl antibody e.g., antagonistic anti- HTRAl antibody
• conjugate thereof neutralizes (or reduces or inhibits) HTRA1 cleavage activity.
• the anti-HTRAl antibody (or conjugate thereof) reduces HTRA1 cleavage activity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or about 100%, or by a percentage within a range defined by any two of the preceding values (e.g., 10-100%, 20- 100%, 30-80%, 40-80%, 50-90%, 85-99%, etc.).
• the percentage reduction in HTRA1 cleavage activity is relative to a suitable control (e.g., a blank solution, a non-specific antibody, etc.).
• a suitable control e.g., a blank solution, a non-specific antibody, etc.
• the anti-HTRAl antibody e.g., antagonistic anti-HTRAl antibody
• conjugate thereof neutralizes (or reduces or inhibits) HTRA1 serine protease activity.
• the anti-HTRAl antibody reduces HTRA1 serine protease activity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or about 100%, or by a percentage within a range defined by any two of the preceding values (e.g., 10-100%, 20-100%, 30-80%, 40-80%, 50-90%, 85-99%, etc.).
• the percentage reduction in HTRA1 serine protease activity is relative to a suitable control (e.g., a blank solution, a non-specific antibody, etc.).
• an anti-HTRAl antibody (or conjugate thereof) that inhibits or neutralizes HTRA1 function (e.g., antagonistic anti-HTRAl antibody), and does not bind to the loop
• a peptide of HTRA1 includes a heavy chain variable region having at least one of the VH sequences of HC17, HC39, HC41 (with reference to Fig. 24A), and a light chain variable region having the VL sequence of LC21 (with reference to Fig. 24B).
• the anti-HTRAl antibody heavy chain variable region includes a VH sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the VH sequences of HC17, HC39, HC41, and the anti-HTRAl antibody light chain variable region includes a VL sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to the VL sequences of LC21.
• the anti-HTRAl antibody heavy chain variable region includes a VH sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the VH sequences of HC17, HC39, HC41, where any variation (e.g., substitution, deletion) within the amino acid sequence of the VH of the anti-HTRAl antibody from any one of the VH sequences of HC17, HC39, HC41 is in no more than 5, 4, 3, 2, or 1 residue of, or is not within, a CDR of the VH sequence, and the anti- HTRAl antibody light chain variable region includes a VL sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least
• HTRA1 enzymatic activity e.g., serine protease activity
• cleavage activity can be measured using any suitable option.
• inhibition of HTRA1 enzymatic activity by an anti-HTRAl antibody or conjugate thereof of the present disclosure is measured using an H2-Opt assay, as provided herein.
• the substrate for the H2-Opt assay has the amino acid sequence Mca-Ile-Arg-Arg-VaLSer-Tyr- Ser-Phe-Lys(Dnp)-Lys-OH (SEQ ID NO:750), where Mca is 7-methoxycoumarin-4-acetic acid and Dnp is N-dinitrophenyldiaminopropionic acid.
• the anti- HTRAl antibody or conjugate thereof inhibits casein digestion by HTRA1 when the antibody is bound thereto.
• inhibition of HTRA1 enzymatic activity by an anti- HTRAl antibody or conjugate thereof of the present disclosure is measured using a casein digestion assay, as provided herein.
• the HTRA1 neutralizing activity of the antibody is not substantially affected by the presence of a polymer covalently bonded to the antibody, as provided herein.
• the antibody conjugate inhibits the enzymatic activity, e g., protease activity, of HTRA1 when the antibody conjugate is bound thereto as well as the antibody does when the unconjugated antibody is bound to HTRA1.
• the antibody conjugate retains at least 20% HTRA1 neutralizing activity relative to unconjugated antibody.
• the antibody conjugate retains at least 50% HTRA1 neutralizing activity relative to unconjugated antibody.
• the antibody conjugate retains at least 90% HTRA1 neutralizing activity relative to unconjugated antibody. In some embodiments, the antibody conjugate retains at least 95% HTRA1 neutralizing activity relative to unconjugated antibody. In some embodiments, the antibody conjugate retains about 100% HTRA1 neutralizing activity relative to unconjugated antibody.
• the HTRA1 neutralizing activity of the antibody is not substantially affected by exposure to changes in pH, e.g., low pH.
• the anti-HTRAl antibody substantially retains HTRA1 neutralizing activity after being treated with a lower pH, such as a pH of about 6.5 or lower, about 6.0 or lower, about 5.5 or lower, about 5.0 or lower, about 4.5 of lower, about 4.0 or lower, about 3.7 or lower, about 3.5 or lower, or about 3.4, and/or a pH of about 3.0 or higher, about 3.2 or higher, about 3.3 or higher, or a pH in a range defined by any two of the preceding values (e.g., pH 3.0-6.5, pH 3.0-5.5, pH 3.2-4.5, pH 3.0-4.0, pH 3.2-3.7).
• the anti-HTRAl antibody substantially retains HTRA1 neutralizing activity after being treated with a lower pH (e.g., about pH 3.4) for about 30 minute or more, about 1 hour or more, about 1.5 hours or more, about 2 hours or more, about 2.5 hours or more, about 3 hours of more, about 3.5 hours or more, about 4 hours or more, and/or for about 6 hours or less, about 5.5 hours or less, about 5 hours or less, or about 4.5 hours or less, or by time interval in a range defined by any two of the preceding values (e.g., 0.5-6 hours, 1-5.5 hours, 2-5 hours, 2.5-4.5 hours, 1.5-4.5 hours, etc ).
• a lower pH e.g., about pH 3.4
• At least about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95% or more, or a percentage in a range defined by any two of the preceding values (e.g., 50-95%, 60-90%, 70-85%, 70-95%, etc.) of the HTRA1 neutralizing activity of the anti-HTRAl antibody is retained after being treated with a lower pH (e.g., about pH 3.4).
• An anti-HTRAl antibody of the present disclosure can be generated using any suitable option.
• the antibody that binds to HTRA1, e.g., human HTRA1, of the present disclosure is generated by at least screening/selecting clones (e.g., clonal antibody-secreting cell lineages) from the immunized animal based on binding characteristics of the antibodies produced by the clones to a human HTRA1 N-terminal truncation (HTRA1 PD/PDZ), HTRA1 loop A, and/or HTRA3.
• HTRA1 PD/PDZ human HTRA1 N-terminal truncation
• HTRA1 loop A HTRA1 loop A
• an antibody that binds to HTRA1, e.g., human HTRA1, of the present disclosure is obtained by at least screening/selecting for a clone that binds to a human HTRA1 N-terminal truncation (HTRA1 PD/PDZ) and/or binds to or does not bind to a HTRA1 loop A and/or does not bind HTRA3.
• An anti-HTRAl antibody of the present disclosure can be obtained by at least screening/selecting antibodies produced by clones from the immunized animals based on one or more of the following properties: 1. Binding to human HTRA1 (e.g., a human HTRA1 N-terminal truncation (HTRA1 PD/PDZ));
• an anti-HTRAl antibody of the present disclosure is obtained by at least further carrying out affinity maturation of antibodies identified by screening or selecting for clones, as provided herein.
• the anti-HTRAl antibody is a humanized antibody. In some embodiments, the anti-HTRAl antibody includes at least a humanized antibody. In some embodiments, an antibody conjugate of the present disclosure includes an anti-HTRAl antibody that is humanized. In some embodiments, the anti-HTRAl antibody includes a heavy chain variable domain including a framework region (HFR) 1, HFR2, HFR3, and HFR4 each having an amino acid sequence as set forth in Table 0.13, or with a variation (e.g., substitution, deletion) at no more than 6 (e.g., no more than 5, no more than 4, no more than 3, no more than 2, no more than 1) residues in each of the HFR1, HFR2, HFR3, and HFR4.
• HFR framework region
• the anti-HTRAl antibody includes a light chain variable domain including a framework region (LFR) 1, LFR2, LFR3, and LFR4 each having an amino acid sequence as set forth in Table 0.13, with a variation (e.g., substitution, deletion) at no more than 6 (e.g., no more than 5, no more than 4, no more than 3, no more than 2, no more than 1) residues in each of the LFR1, LFR2, LFR3, and LFR4.
• LFR framework region
• LFR3 LFR4
• the anti-HTRAl antibody (or conjugate thereof) includes a light chain and a heavy chain where the heavy chain has an Fc region.
• the anti-HTRAl heavy chain isotype is IgGl.
• the IgGl constant region has one or more mutations relative to a native IgGl constant region to modulate effector function.
• the effector function mutations are one or more of the following: (EU numbering) E233X, L234X, L235X, G236X, G237X, A327X, A330X, and P33 IX wherein X is any natural or unnatural amino acid.
• the effector function mutations are one or more of the following: (EU numbering) L234X, L235X, and G237X, wherein X is any natural or unnatural amino acid.
• the mutations are selected from the group consisting of (EU numbering): E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331 S.
• the mutations are selected from the group consisting of (EU numbering): L234A, L235A, and G237A.
• antibody conjugate has the following mutations (EU numbering): L234A, L235A, and G237A.
• the IgG domain can be IgG2, IgG3 or IgG4 or a composite in which a constant regions is formed from more than one of these isotypes (e.g., CHI region from IgG2 or IgG4, hinge, CH2 and CH3 regions from IgGl).
• Such domains can contain mutations to reduce and/or modulate effector function at one or more of the EU position mentioned for IgGl .
• Human IgG2 and IgG4 have reduced effector functions relative to human IgGl and IgG3.
• the anti-HTRAl antibody (or conjugate thereof) includes a heavy chain variable region having framework regions (e.g., HFR1, HFR2, HFR3, HFR4) each independently having an amino acid sequence at least 80%, e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95- 100%, etc.), identical to the corresponding framework regions of SEQ ID NOs:544-547.
• framework regions e.g., HFR1, HFR2, HFR3, HFR4
• the anti-HTRAl antibody (or conjugate thereof) includes a heavy chain having constant regions (e.g., CHI , CH2, CH3, FC), each independently having an amino acid sequence at least 80%, e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.) identical to the corresponding constant regions of SEQ ID NO:536.
• constant regions e.g., CHI , CH2, CH3, FC
• the anti-HTRAl antibody (or conjugate thereof) includes a light chain variable region having framework regions (e.g., LFR1, LFR2, LFR3, LFR4) each independently having an amino acid sequence at least 80%, e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95- 100%, etc.) identical to the corresponding framework regions of SEQ ID NOs:548-551.
• framework regions e.g., LFR1, LFR2, LFR3, LFR4
• the anti-HTRAl antibody (or conjugate thereof) includes a light chain having a constant region having an amino acid sequence at least 80%, e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95- 100%, etc.) identical to the constant region of SEQ ID NO:537.
• the anti-HTRAl antibody (or conjugate thereof) includes a non-native cysteine residue outside a variable region of the antibody.
• the cysteine is in the Fc region and the anti-HTRAl antibody is an immunoglobulin G (IgG).
• the anti-HTRAl antibody has a cysteine outside a variable region of the antibody covalently bonded to a phosphorylcholine containing polymer, where the cysteine has been added via recombinant DNA technology (e.g., is nonnative).
• cysteine residue is in the anti-HTRAl heavy chain and is Q347C (EU numbering) or L443C (EU numbering).
• the cysteine residue is L443C (EU numbering).
• “added by recombinant DNA technology” means that the cysteine residue replaces a non-cysteine amino acid that occurs in the same position in a known or existing antibody or in a consensus antibody sequence or a native sequence.
• the antibody is an IgGl and the heavy chain possess a leucine at EU position 443, the leucine is replaced via recombinant DNA technology with a cysteine (L443C, EU numbering).
• the native IgGl sequence at EU position 347 is Q (glutamine) and the Q is replaced with cysteine via recombinant DNA technology to yield Q347C.
• the anti-HTRAl antibody (or conjugate thereof) includes a heavy chain constant region (HC) having an amino acid sequence of SEQ ID NO 536 (as provided in Table 0.14), and the anti-HTRAl antibody includes a light chain constant region (LC) having an amino acid sequence of SEQ ID NO:537 (as provided in Table 0.14).
• HC heavy chain constant region
• LC light chain constant region
• the anti-HTRAl antibody (or conjugate thereof) includes a heavy chain constant region (HC) having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100%, or a percentage in a range defined any two of the preceding values (e.g., 80-100%, 85-99%, 90- 97%, 95-100%, etc.), identical to SEQ ID NO: 536 (as provided in Table 0.14).
• HC heavy chain constant region
• the anti-HTRAl antibody includes a light chain constant region (LC) having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100%, or a percentage in a range defined any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to SEQ ID NO:537 (as provided in Table 0.14).
• LC light chain constant region
• the anti-HTRAl antibody (or conjugate thereof) includes a heavy chain constant region (HC) having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100%, or a percentage in a range defined any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to SEQ ID NO:536 (as provided in Table 0.14), and the anti-HTRAl antibody includes a light chain constant region (LC) having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100%, or a percentage in a range defined any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to SEQ ID NO:537 (as provided in Table 0.14).
• HC heavy chain constant region
• the anti-HTRAl antibody (or conjugate thereof) includes a heavy chain having an amino acid sequence as provided in any one of SEQ ID NOs: 819-823 (Fig. 25 A), and a light chain having an amino acid sequence as provided in SEQ ID NO:824 (Fig. 25B).
• the anti-HTRAl antibody (or conjugate thereof) includes a heavy chain having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100%, or a percentage in a range defined any two of the preceding values (e g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the heavy chain sequences as provided in any one of SEQ ID NOs:819-823 (Fig.
• a light chain having an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100%, or a percentage in a range defined any two of the preceding values (e.g., 80-100%, 85-99%, 90-97%, 95-100%, etc.), identical to any one of the heavy chain sequences as provided in SEQ ID NO: 824 (Fig. 25B).
• antibody conjugates that includes: any one of the anti- HTRAl antibodies as disclosed herein; and a polymer covalently bonded to the antibody at a non-native cysteine outside a variable region of the antibody.
• the polymer is a phosphorylcholine containing polymer.
• the polymer is bonded to a single cysteine in the antibody.
• the antibody is conjugated with a poly(acryloyloxyethyl phosphorylcholine) containing polymer, such as a polymer of acrylic acid containing at least one acryloyloxyethyl phosphorylcholine monomer such as 2- methacryloyloxyethyl phosphorylcholine (i.e., 2-methacryloyl-2'-trimethylammonium ethyl phosphate).
• a poly(acryloyloxyethyl phosphorylcholine) containing polymer such as a polymer of acrylic acid containing at least one acryloyloxyethyl phosphorylcholine monomer such as 2- methacryloyloxyethyl phosphorylcholine (i.e., 2-methacryloyl-2'-trimethylammonium ethyl phosphate).
• the polymer is a water-soluble polymer, which refers to a polymer that is soluble in water.
• a solution of a water-soluble polymer may transmit at least about 75%, more preferably at least about 95% of light, transmitted by the same solution after filtering.
• a water-soluble polymer or segment thereof may be at least about 35%, at least about 50%, about 70%, about 85%, about 95% or 100% (by weight of dry polymer) soluble in water.
• the polymer has at least 2 or 3 or more arms. In some embodiments, the polymer has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 arms. In some embodiments, the polymer has 3, 6 or 9 arms. In some embodiments, the polymer has 9 arms. In some embodiments, the polymer peak molecular weight is between 300,000 and 1,750,000 Da. In some embodiments, the polymer has a peak molecular weight between 500,000 and 1,000,000 Da. In some embodiments, the polymer has a peak molecular weight between 600,000 to 800,000 Da.
• the polymer has a peak molecular weight between 300,000 and 1,750,000 Daltons as measured by size exclusion chromatography - multi angle light scattering (hereinafter “SEC-MALS”). In some embodiments, the polymer has a peak molecular weight between 500,000 and 1,000,000 Daltons as measured by SEC-MALS. In some embodiments, the polymer has a peak molecular weight between 600,000 to 800,000 Daltons as measured by SEC-MALS.
• SEC-MALS size exclusion chromatography - multi angle light scattering
• zwitterionic polymer-trap-antibody fusion conjugates having one or more functional agents and one or more polymer arms wherein each of the polymer arms has one or more monomer units wherein at least one of the units has a zwitterion.
• such a method can have the steps of: providing an initiator having one or more sites for monomer polymerization and a first linker having an amine group wherein the initiator is a trifluoro acetic acid salt; providing one or more monomers suitable for polymerization wherein at least one of the monomers is zwitterionic; reacting the monomers with the initiator to form one or more polymer arms each corresponding to the sites for monomer polymerization to provide an initiator-polymer conjugate having the first linker with the amine group; providing a second linker having at least second and third reactive groups; coupling one of the second and third reactive groups of the second linker to the amine group of the first linker of the initiator-polymer conjugate to provide a linker-initiator-polymer conjugate having one or more reactive groups that were not used in the coupling step; and coupling one or more functional agents to one or more of the unreacted reactive groups of the 1 inker-ini ti at or- polymer moiety
• the conjugation group e.g. maleimide
• the conjugation group is added after polymer synthesis. This is sometimes referred to as a “snap-on strategy” or “universal polymer strategy”. See, e.g., U.S. Patent Application No. 14/916,180 (published as U.S. Patent Application Publication No. 20160199501), hereby incorporated by reference in its entirety.
• a single initiator moiety can be used for large scale polymer synthesis. Thus, conditions can be developed for scaled up optimal polymer synthesis. Such polymers can then be adapted to various types of functional agents by “snapping-on” various types of linkers.
• a longer linker sequence can be snapped on to the polymer.
• smaller functional agents may call for relatively shorter linker sequences.
• the initiator has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 sites for polymer initiation. In some embodiments, the initiator has about 3, about 6, or about 9 sites for polymer initiation.
• a second linker has second, third, fourth, fifth, and sixth reactive groups. More preferably, a second linker has just second and third reactive groups.
• each polymer arm has from about 20 to about 2000 monomer units. Preferably, each arm has from about 100 to 500 monomer units or from about 500 to 1000 monomer units or from about 1000 to 1500 monomer units or from about 1500 to 2000 monomer units.
• the peak molecular weight of the polymer-functional agent conjugate is about 100,000 to 1,500,000 Da.
• the peak molecular weight of the polymer-functional agent conjugate is about 200,000 to about 300,000 Da, about 400,000 to about 600,000 Da or about 650,000 to about 850,000 Da.
• the first linker is preferably alkyl, substituted alkyl, alkylene, alkoxy, carboxyalkyl, haloalkyl, cycloalkyl, cyclic alkyl ether, alkenyl, alkenylene, alkynyl, alkynylene, cycloalkylene, heterocycloalkyl, heterocycloalkylene, aryl, arylene, arylene-oxy, heteroaryl, amino, amido or any combination thereof. More preferably, the first linker has the formula:
• the first linker has the above formula (Formula (1)) and m is 4.
• the initiator preferably includes a structure selected from group consisting of
• Formula (4) wherein X is selected from the group consisting of NCS, F, Cl, Br and T. More preferably, X in Formula (2), Formula (3) and/or Formula (4) is Br.
• the monomer is selected from the group consisting of
• the monomer is selected from the group consisting of 2- (methacryloyloxyethyl)-2’ -(trimethylammonium ethyl) phosphate (HEMA-PC) and 2- (acryloyl oxy ethyl)-2’ -(trimethylammoniumethyl) phosphate.
• the monomer is 2-(methacryloyloxyethyl)-2’- (trimethylammoniumethyl) phosphate.
• the second linker moiety preferably comprises an activated ester having the structure
• the polymer has 9 arms, m is 2-4, R9 is
• the radically polymerizable monomer is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-(2-aminoethyl)-2-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
• R1 is H or Cl -6 alkyl
• R2, R3, R4 are the same or different and are H or Cl- 4alkyl
• X and Y are the same or different and are integers from 1-6.
• Rl, R2, R3 and R4 are each methyl and X and Y are each 2 in Formula (12).
• the radically polymerizable monomer is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-(2-aminoethyl)-2-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
• Rl is H or Cl-6alkyl
• R2 and R3 are the same or different and are H or Cl- 4alkyl
• R4 is PO4-, SO3- or CO2-
• X and Y are the same or different and are integers from 1-6.
• Rl , R2 and R3 are methyl
• R4 is PO4- and X and Y are each 2 in
• the monomer is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
• R1 is H or Cl-6alkyl
• R2, R3 and R4 are the same or different and are H or Cl-4alkyl
• R5 is PO4-, SO3- or CO2-
• X and Y are the same or different and are integers from 1-6.
• Rl, R2, R3 and R4 are methyl
• R5 is PO4- and X and Y are 2 in Formula (14).
• the polymer when a polymer is to be conjugated via a cysteine (or other specified residue), the polymer can be linked directly or indirectly to the residue (e.g., with an intervening initiator, and or spacer or the like).
• the phosphorylcholine containing polymer comprises 2-(methacryloyloxyethyl)-2'-(trimethylammonium)ethyl phosphate (MPC) monomers as set forth below:
• n is an integer from 1 to 3000 and the wavy lines indicate the points of attachment between monomer units in the polymer.
• the polymer has three or more arms, or is synthesized with an initiator comprising 3 or more polymer initiation sites.
• the polymer has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 arms, oris synthesized with an initiator comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 polymer initiation sites. More preferably, the polymer has 3, 6, or 9 arms, or is synthesized with an initiator comprising 3, 6, or 9 polymer initiation sites. In some embodiments, the polymer has 9 arms, or is synthesized with an initiator comprising 9 polymer initiation sites.
• the polymer of the antibody conjugate has a molecular weight between about 300,000 and about 1,750,000 Da (SEC-MALs). In some embodiments, the polymer has a molecular weight between about 500,000 and about 1,000,000 Da. In some embodiments, the polymer has a molecular weight of between about 600,000 to about 900,000 Da. In some embodiments, the polymer has a molecular weight of between about 750,000 to about 850,000 Da. In some embodiments, the polymer has a molecular weight of between about 800,000 to about 850,000 Da. In some embodiments, the polymer has a molecular weight of between about 750,000 to about 800,000 Da.
• any of the antibodies described herein can be further conjugated to a polymer to form a bioconjugate or antibody conjugate.
• the molecular weight of the bioconjugate or antibody conjugate (in total, SEC-MALs) can be between about 350,000 and 2,000,000 Daltons, for example, between about 450,000 and 1,900,000 Daltons, between about 550,000 and 1,800,000 Daltons, between about 650,000 and 1,700,000 Daltons, between about 750,000 and 1,600,000 Daltons, between about 850,000 and 1,500,000 Daltons, between about 900,000 and 1,400,000 Daltons, between about 950,000 and 1,300,000 Daltons, between about 900,000 and 1,000,000 Daltons, between about 1,000,000 and 1,300,000 Daltons, between about 850,000 and 1,300,000 Daltons, between about 850,000 and 1,000,000 Daltons, and between about 1,000,000 and 1,200,000 Daltons.
• the antibody conjugate is purified.
• the polymer is aspect of the antibody conjugate is polydisperse, i.e. the polymer PDI is not 1.0.
• the PDI is less than 1.5.
• the PDI is less than 1.4.
• the PDI is less than 1.3.
• the PDI is less than 1.2.
• the PDI is less than 1.1.
• an antibody conjugate comprising an anti-HTRAl immunoglobulin G (IgG) bonded to a polymer, which polymer comprises MPC monomers, wherein the anti-HTRAl antibody heavy chain comprises: a heavy chain variable region (VH) comprising an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100%, or a percentage in a range defined any two of the preceding values (e g., 80-100%, 85-99%, 90-97%, 95-100%, etc ), identical to any one of SEQ ID NOs: 725, 742, 744, 745, 746, C-terminally fused to a heavy chain constant region (HC) comprising an amino acid sequence of SEQ ID NO: 536, wherein the polymer is bonded to C326 of SEQ ID NO:536 (as provided in Table 0.14), and wherein the anti-HTRAl antibody light chain comprises: a light chain
• the antibody conjugate has an anti-HTRAl immunoglobulin G (IgG) bonded to a polymer, which polymer comprises MPC monomers, wherein the sequence of the anti-HTRAl heavy chain includes a VH sequence of at least one of the VH sequences provided in Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7, 7.12, 7.17, and 8.1, or a sequence at least 80% identical thereto, and the sequence of the anti-HTRAl light chain includes a VL sequence of at least one of the VL sequences provided in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and
• the antibody conjugate has an anti-HTRAl immunoglobulin G (IgG) bonded to a polymer, which polymer comprises MPC monomers, wherein the sequence of the anti-HTRAl heavy chain includes a VH sequence at least 85% to any one of the VH sequences provided in Figs. 21, 22A, 23 A, and 24A, and Tables
• the sequence of the anti-HTRAl light chain includes a VL sequence at least 85% to any one of the VL sequences provided in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and 8.2, and wherein the antibody is bonded only at C443 (EU numbering) to the polymer.
• the antibody conjugate has an anti-HTRAl immunoglobulin G (IgG) bonded to a polymer, which polymer comprises MPC monomers, wherein the sequence of the anti-HTRAl heavy chain includes a VH sequence at least 90% to any one of the VH sequences provided in Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7, 7.12, 7.17, and 8.1, and the sequence of the anti-HTRAl light chain includes a VL sequence at least 90% to any one of the VL sequences provided in Figs.
• IgG anti-HTRAl immunoglobulin G
• the antibody conjugate has an anti-HTRAl immunoglobulin G (IgG) bonded to a polymer, which polymer comprises MPC monomers, wherein the sequence of the anti-HTRAl heavy chain includes a VH sequence at least 95% to any one of the VH sequences provided in Figs.
• IgG anti-HTRAl immunoglobulin G
• the sequence of the anti-HTRAl light chain includes a VL sequence at least 95% to any one of the VL sequences provided in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and 8.2, and wherein the antibody is bonded only at C443 (EU numbering) to the polymer.
• the antibody conjugate has an anti-HTRAl immunoglobulin G (IgG) bonded to a polymer, which polymer comprises MPC monomers, wherein the sequence of the anti-HTRAl heavy chain includes a VH sequence at least 97% to any one of the VH sequences provided in Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7, 7.12, 7.17, and 8.1, and the sequence of the anti-HTRAl light chain includes a VL sequence at least 97% to any one of the VL sequences provided in Figs.
• IgG anti-HTRAl immunoglobulin G
• the VH and VL sequences of the antibody conjugate are paired according to any one of the paired arrangements provided in FIG. 21 and Tables 0.1, 0.4, 0.5, 0.7, 0.10, 0.11, 0.12, 1.2, 6.3, and 7.1.
• the polymer has 9 arms and has a molecular weight of between about 600,000 to about 1,000,000 Da.
• the antibody conjugate has an anti-HTRAl immunoglobulin G (IgG) bonded to a polymer, which polymer comprises MPC monomers, wherein the sequence of the anti-HTRAl heavy chain includes a VH sequence of at least one of the VH sequences provided in Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1,
• the sequence of the anti-HTRAl light chain includes a VL sequence of at least one of the VL sequences provided in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and 8.2, or a sequence at least 80% identical thereto, where any sequence variation from the VL sequence provided in Figs.
• the antibody conjugate has an anti-HTRAl immunoglobulin G (IgG) bonded to a polymer, which polymer comprises MPC monomers, wherein the sequence of the anti-HTRAl heavy chain includes a VH sequence at least 85% to any one of the VH sequences provided in Figs.
• IgG anti-HTRAl immunoglobulin G
• the antibody conjugate has an anti-HTRAl immunoglobulin G (IgG) bonded to a polymer, which polymer comprises MPC monomers, wherein the sequence of the anti-HTRAl heavy chain includes a VH sequence at least 90% to any one of the VH sequences provided in Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7, 7.12, 7.17, and
• the antibody conjugate has an anti- HTRAl immunoglobulin G (IgG) bonded to a polymer, which polymer comprises MPC monomers, wherein the sequence of the anti-HTRAl heavy chain includes a VH sequence at least 95% to any one of the VH sequences provided in Figs. 21, 22A, 23 A, and 24A, and Tables
• the sequence of the anti-HTRAl light chain includes a VL sequence at least 95% to any one of the VL sequences provided in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and 8.2, where any sequence variation from the VL sequence provided in Figs.
• the antibody conjugate has an anti-HTRAl immunoglobulin G (IgG) bonded to a polymer, which polymer comprises MPC monomers, wherein the sequence of the anti- HTRAl heavy chain includes a VH sequence at least 97% to any one of the VH sequences provided in Figs.
• IgG anti-HTRAl immunoglobulin G
• the VH and VL of the antibody conjugate include CDRs of the corresponding CDRs in are paired according to at least one of the paired arrangements provided in FIG.
• the polymer has 9 arms and has a molecular weight of between about 600,000 to about 1,000,000 Da.
• the antibody conjugate has the structure of Formula (I):
• each heavy chain of the anti-HTRAl antibody is denoted by the letter H, and each light chain of the anti-HTRAl antibody is denoted by the letter L; the polymer is bonded to the anti-HTRAl antibody through the sulfhydryl of C443 (EU numbering), which bond is depicted on one of the heavy chains; PC is, where the curvy line indicates the point of attachment to the rest of the polymer; wherein X is a) -OR where R is - H, Methyl, ethyl, propyl, isopropyl, b) H, or c) any halogen, including -Br, -Cl, or -I; and either i) nl, n2, n3, n4, n5, n6, n7, n8 and n9 are the same or different and are integers from 0 to 3000; or ii) nl, n2, n3, n4, n
• nl, n2, n3, n4, n5, n6, n7, n8 and n9 is about 1500 to about 3500 plus or minus about 10% to about 20%.
• X is -OR, where R is a sugar, an aminoalkyl, monosubstituted, poly-substituted or unsubstituted variants of the following residues: saturated Ci - C24 alkyl, unsaturated C2 -C24 alkenyl or C2 -C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxy carbonyl, alkoxy carbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy, alky
• the antibody conjugate has the structure of Formula
• n.” is an integer from 1 to 50 and “n.i” is an integer from 1 to 50; each heavy chain of the anti-HTRAl antibody is denoted by the letter H, and each light chain of the anti-HTRAl antibody is denoted by the letter L; the polymer is bonded to the anti- HTRAl antibody through the sulfhydryl of C443 (EU numbering), which bond is depicted on one of the heavy chains; PC is, where the curvy line indicates the point of attachment to the rest of the polymer; wherein X is a) -OR where R is -H, Methyl, ethyl, propyl, isopropyl, b) H, or c) any halogen, including -Br, -Cl, or -I; and either i) nl , n2, n3, n4, n5, n6, n7, n8 and n9 are the same or different and are integers from 0 to 3000; or
• the polymers disclosed herein can comprise one or more of the following: a zwitterion, a phosphoryl choline, or a PEG linker bridging a center of a polymer branching point to the maleimide functional group.
• any of the polymers provided herein can be added to a protein via the methods provided herein.
• the half-life of the anti-HTRAl antibodies is extended by attachment of a “half-life (“half life”) extending moieties” or “half-life (“half life”) extending groups”.
• Half-life extending moieties include peptides and proteins which can be expressed in frame with the biological drug of issue (or conjugated chemically depending on the situation) and various polymers which can be attached or conjugated to one or more amino acid side chain or end functionalities such as -SH, -OH, -COOH, -CONH2, -NH2, or one or more N- and/or O-glycan structures.
• Half-life extending moieties generally act to increase the in vivo circulatory half-life of biologic drugs.
• Suitable peptide/protein half-life extending moieties include, without limitation, Fc fusion, human serum albumin (HAS) fusion, carboxy terminal peptide (CTP) fusion, genetic fusion of non-exact repeat peptide sequence (XTEN) fusion, elastin like peptide (ELPylation) (MCpherson DT, Morrow C, Minehan DS, et al.
• G-(VPGVG)19-VPGV Production and purification of a recombinant elastomeric polypeptide, G-(VPGVG)19-VPGV, from Escheriachia coli, human transferrin fusion, proline-alanine-serine (PASylation), homo-amino acid polymer (HAPylation) and gelatin like protein (GLK) fusion.
• polymer half-life extending moieties include polyethylene glycol (PEG), branched PEG, PolyPEG® (Warwick Effect Polymers; Coventry, UK), polysialic acid (PSA), starch, hydroxylethyl starch (HES), hydroxyalkyl starch (HAS), carbohydrate, polysaccharides, pullulane, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, dextran, carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, polyethylene-co-maleic acid anyhydride, polystyrene-co-maleic acid anhydride, poly(
• a half-life extending moiety can be conjugated to an antibody via free amino groups of the protein using N-hydroxysuccinimide (NHS) esters.
• NHS N-hydroxysuccinimide
• Reagents targeting conjugation to amine groups can randomly react to c-amine group of lysines, a-amine group of N-terminal amino acids, and 5-amine group of histidines.
• a half-life extending moiety is coupled to one or more free SH groups using any appropriate thiol -reactive chemistry including, without limitation, maleimide chemistry, or the coupling of polymer hydrazides or polymer amines to carbohydrate moieties of the antibody after prior oxidation.
• maleimide coupling is used.
• coupling occurs at cysteines naturally present or introduced via genetic engineering.
• a polymer is covalently attached to a cysteine residue introduced into anti-HTRAl antibodies by site directed mutagenesis, or to a non-native cysteine.
• the cysteine residue is employed in the Fc portion of the antibody.
• the non-native cysteine residue is in the Fc portion of the antibody.
• the sites to introduce cysteine residues into an Fc region are provided in WO 2013/093809, US 7,521,541, WO 2008/020827, US 8,008,453, US 8,455,622 and US2012/0213705, incorporated herein by reference for all purposes.
• the cysteine mutations are Q347C (EU numbering) and L443C referring to the human IgG heavy chain by EU numbering.
• conjugates of antibody and high MW polymers serving as half-life extenders are provided.
• a conjugate comprises an antibody that is coupled to a zwitterionic polymer wherein the polymer is formed from one or more monomer units and wherein at least one monomer unit has a zwitterionic group is provided.
• the zwitterionic group is phosphorylcholine.
• one of the monomer units is HEMA-PC.
• a polymer is synthesized from a single monomer which is HEMA-PC.
• some antibody conjugates have 2, 3, or more polymer arms wherein the monomer is HEMA-PC.
• the conjugates have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 polymer arms wherein the monomer is HEMA-PC.
• the conjugates have 3, 6 or 9 arms. In some embodiments, the conjugate has 9 arms.
• polymer-antibody conjugates have a polymer portion with a molecular weight of between 100,000 and 1,500,000 Da. In some embodiments, the conjugate has a polymer portion with a molecular weight between 500,000 and 1,000,000 Da. In some embodiments, the conjugate has a polymer portion with a molecular weight between 600,000 to 800,000 Da. In some embodiments, the conjugate has a polymer portion with a molecular weight between 600,000 and 850,000 Da and has 9 arms. When a molecular weight is given for an antibody conjugated to a polymer, the molecular weight will be the addition of the molecular weight of the protein, including any carbohydrate moi eties associated therewith, and the molecular weight of the polymer.
• an anti-HTRAl antibody has a HEMA-PC polymer which has a molecular weight measured by Mw of between about 100 kDa and 1650 kDa is provided. In some embodiments, the molecular weight of the polymer as measured by Mw is between about 500 kDa and 1000 kDa. In some embodiments, the molecular weight of the polymer as measured by Mw is between about 600 kDa to about 900 kDa. In some embodiments, the polymer molecular weight as measured by Mw is 750 kDa plus or minus 15%.
• the antibody conjugate has an anti-HTRAl immunoglobulin G (IgG) bonded to a polymer, which polymer comprises MPC monomers, where the sequence of the anti-HTRAl heavy chain variable region (VH) includes at least one of the VH sequences as set forth in SEQ ID NOs:725, 742, 744, 745, 746, where the VH is C- terminally fused to a heavy chain constant region (HC) that includes the amino acid sequence of SEQ ID NO:536, where the polymer is bonded to C326 of SEQ ID NO:536 (as provided in Table 0.14), and the sequence of the anti-HTRAl light chain variable region includes at least one of the VL sequences as set forth in SEQ ID NO:748, where the VL is C-terminally fused to a light chain constant region (LC) comprising an amino acid sequence of SEQ ID NO:537.
• IgG anti-HTRAl immunoglobulin G
• the antibody conjugate has an anti-HTRAl immunoglobulin G (IgG) bonded to a polymer, which polymer comprises MPC monomers, where the sequence of the anti-HTRAl heavy chain variable region (VH) includes an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or about 100%, or a percentage in a range defined any two of the preceding values (e.g., 80-100%, 85- 99%, 90-97%, 95-100%, etc.), identical to at least one of the VH sequences as set forth in SEQ ID NOs: 725, 742, 744, 745, 746, where the VH is C-terminally fused to a heavy chain constant region (HC) that includes the amino acid sequence of SEQ ID NO:536, where the polymer is bonded to C326 of SEQ ID NO:536 (as provided in Table 0.14), and the sequence of the anti- HTRAl light chain variable region includes
• the antibody conjugate has an anti-HTRAl immunoglobulin G (IgG) bonded to a polymer, which polymer comprises MPC monomers, where the sequence of the anti-HTRAl heavy chain includes at least one of SEQ ID NOs: 819- 823, where the polymer is bonded to C445 of the at least one of SEQ ID NOs: 819-823, and the sequence of the anti-HTRAl light chain includes SEQ ID NO:824.
• the polymer has 9 arms and has a molecular weight of between about 600,000 to about 1,000,000 Da.
• compositions that contain an antibody or antibody conjugate of the present disclosure.
• the antibody conjugate is present in a liquid formulation.
• the antibody conjugate is combined with a pharmaceutically acceptable carrier.
• Therapeutic formulations of the anti-HTRAl antibodies and anti-HTRAl antibody conjugates used in accordance with the present invention are prepared for storage by mixing an antibody (or conjugate thereof) having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers, in the form of lyophilized formulations or aqueous solutions.
• Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may comprise buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine,
• Liposomes containing the anti-HTRAl antibody and/or anti-HTRAl antibody conjugate are prepared by methods known in the art. Liposomes with enhanced circulation time are known in the art. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through fdters of defined pore size to yield liposomes with the desired diameter.
• PEG-PE PEG-derivatized phosphatidylethanolamine
• the active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
• colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
• Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. fdms, or microcapsules.
• sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and 7 ethyl -L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3 -hydroxybutyric acid.
• polyesters for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)
• polylactides copolymers of L-glutamic acid and 7 ethyl -L-glutamate
• non-degradable ethylene-vinyl acetate non-
• the formulations to be used for in vivo administration must be sterile. This is readily accomplished by, for example, fdtration through sterile filtration membranes.
• Therapeutic anti-HTRAl antibody and/or antibody conjugate compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
• the antibody and/or antibody conjugate compositions are placed into a syringe to provide a prefilled syringe.
• the composition is a pyrogen-free composition which is substantially free of endotoxins and/or related pyrogenic substances.
• the endotoxin and pyrogen levels in the composition are less than 10 EU/mg, or less than 5 EU/mg, or less than 1 EU/mg, or less than 0.1 EU/mg, or less than 0.01 EU/mg, or less than 0.001 EU/mg.
• the compositions or methods provided herein allow for 0. lEU/eye/inj ection.
• the compositions or methods provided herein allow for 0.05EU/eye/inj ection.
• the compositions or methods provided herein allow for 0.02EU/eye/inj ection.
• the compositions or methods provided herein allow for O.OlEU/eye/injection.
• compositions according to the present invention may be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.
• the principal active ingredient is mixed with a pharmaceutical carrier, e.g. conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g. water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof.
• a pharmaceutical carrier e.g. conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g. water
• preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
• This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from about 0.1 to about 500 mg of the active ingredient of the present invention.
• the tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
• the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
• the two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release.
• enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
• Suitable surface-active agents include, in particular, non-ionic agents, such as Polysorbate or polyoxyethylenesorbitans (e g. TweenTM 20, 40, 60, 80 or 85) and other sorbitans (e.g. SpanTM 20, 40, 60, 80 or 85).
• Compositions with a surface-active agent will conveniently comprise between 0.01%, and 5% surface-active agent, and can be between 0.01 and 0.02% or 0.1 and 2.5% (polysorbate 20 or 80). It will be appreciated that other ingredients may be added, for example mannitol or other pharmaceutically acceptable vehicles, if necessary.
• Suitable emulsions may be prepared using commercially available fat emulsions, such as INTRALIPIDTM, LIPOSYNTM, INFONUTROLTM, LIPOFUNDINTM and LIPIPHYSANTM.
• the active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in an oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g. egg phospholipids, soybean phospholipids or soybean lecithin) and water.
• an oil e.g. soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil
• a phospholipid e.g. egg phospholipids, soybean phospholipids or soybean lecithin
• emulsions will typically contain up to 20% oil, for example, between 5 and 20%.
• the fat emulsion can comprise fat droplets between 0.1 and 1.0 pm, particularly 0.1 and 0.5 pm, and have a pH in the range of 5.5 to 8.0.
• the emulsion compositions can be those prepared by mixing an anti- HTRA1 antibody with IntralipidTM or the components thereof (soybean oil, egg phospholipids, glycerol and water).
• compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
• the liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above.
• the compositions are administered by the oral or nasal respiratory route for local or systemic effect.
• Compositions in preferably sterile pharmaceutically acceptable solvents may be nebulised by use of gases. Nebulised solutions may be breathed directly from the nebulising device or the nebulising device may be attached to a face mask, tent or intermittent positive pressure breathing machine.
• Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.
• polynucleotides encoding any of the antibodies, including antibody fragments and modified antibodies described herein. Also provided is a method of making any of the polynucleotides described herein. Polynucleotides can be made and expressed by procedures known in the art. Accordingly, provided herein are polynucleotides or compositions, including pharmaceutical compositions, comprising polynucleotides, encoding any of the anti-HTRAl antibodies provided herein.
• Polynucleotides complementary to any such sequences are also contemplated.
• Polynucleotides may be single-stranded (coding or antisense) or doublestranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules.
• RNA molecules include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one- to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.
• Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes an antibody or a fragment thereof) or may comprise a variant of such a sequence.
• Polynucleotide variants contain one or more substitutions, additions, deletions and/or insertions such that the immunoreactivity of the encoded polypeptide is not diminished, relative to a native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide may generally be assessed as described herein.
• variants exhibit at least about 70% identity, more preferably, at least about 80% identity, yet more preferably, at least about 90% identity, and most preferably, at least about 95% identity to a polynucleotide sequence that encodes a native antibody or a fragment thereof.
• Two polynucleotide or polypeptide sequences are said to be “identical” if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity.
• a “comparison window” as used herein refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, or 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
• Optimal alignment of sequences for comparison may be conducted using the MegAlign® program in the Lasergene® suite of bioinformatics software (DNASTAR®, Inc., Madison, WI), using default parameters.
• the "percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences.
• the percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e. the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
• polynucleotides of this disclosure can be obtained using chemical synthesis, recombinant methods, or PCR. Any suitable option for chemical polynucleotide synthesis, such as a commercial DNA synthesizer, can be used to produce the sequences provided herein.
• a polynucleotide comprising a desired sequence can be inserted into a suitable vector, and the vector in turn can be introduced into a suitable host cell for replication and amplification, as further discussed herein.
• Polynucleotides may be inserted into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, F-mating or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell genome. The polynucleotide so amplified can be isolated from the host cell by methods well known within the art.
• PCR allows reproduction of DNA sequences.
• RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using any suitable option.
• Suitable cloning vectors may be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors will generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector.
• Suitable examples include, without limitation, plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28.
• plasmids and bacterial viruses e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28.
• Expression vectors are further provided.
• Expression vectors generally are replicable polynucleotide constructs that contain a polynucleotide according to the present disclosure. It is implied that an expression vector must be replicable in the host cells either as episomes or as an integral part of the chromosomal DNA.
• Suitable expression vectors include but are not limited to plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vector(s).
• Vector components may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional controlling elements (such as promoters, enhancers and terminator). For expression (i.e., translation), one or more translational controlling elements are also usually required, such as ribosome binding sites, translation initiation sites, and stop codons.
• the vectors containing the polynucleotides of interest can be introduced into the host cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE- dextran, or other substances; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus).
• electroporation employing calcium chloride, rubidium chloride, calcium phosphate, DEAE- dextran, or other substances
• microprojectile bombardment e.g., where the vector is an infectious agent such as vaccinia virus.
• infection e.g., where the vector is an infectious agent such as vaccinia virus.
• the choice of introducing vectors or polynucleotides will often depend on features of the host cell.
• the present disclosure also provides host cells comprising any of the polynucleotides described herein. Any host cells capable of over-expressing heterologous DNAs can be used for the purpose of isolating the genes encoding the antibody, polypeptide or protein of interest.
• mammalian host cells include but not limited to COS, HeLa, HEK293, and CHO cells. Suitable non-mammalian host cells include prokaryotes (such as E. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe or K. lactis).
• the host cells express the cDNAs at a level of about 5 fold higher, more preferably, 10 fold higher, even more preferably, 20 fold higher than that of the corresponding endogenous antibody or protein of interest, if present, in the host cells.
• screening the host cells for a specific binding to HTRA1 is effected by an immunoassay or FACS. A cell overexpressing the antibody or protein of interest can be identified.
• an expression vector can be used to direct expression of any of the anti-HTRAl antibodies provided herein.
• a screening method can include immunizing a host animal, e.g., a mouse or rat, with human HTRA1, or a portion thereof.
• the host animal is immunized with at least the human HTRA1 protease domain.
• the host animal is immunized with the human HTRA1 protease domain having at least an amino acid sequence of SEQ ID NO:2.
• the screening method includes screening clones of antibody-producing cells (e.g., B cells) obtained from the immunized host animal based on one or more functional properties of the antibodies produced by the clones.
• the one or more functional properties include one or more of: binding to human HTRA1 (e g., a human HTRA1 N-terminal truncation (HTRA1 PD/PDZ)); inhibition of HTRA1 proteolytic activity; binding affinity for HTRA1 loop A; binding affinity for HTRA3; relative binding affinity to trimeric HTRA1 and monomeric HTRA1; and stoichiometry of binding to HTRA1.
• human HTRA1 e g., a human HTRA1 N-terminal truncation (HTRA1 PD/PDZ)
• inhibition of HTRA1 proteolytic activity e g., inhibition of HTRA1 proteolytic activity
• binding affinity for HTRA1 loop A e g., binding affinity for HTRA3
• relative binding affinity to trimeric HTRA1 and monomeric HTRA1 e stoichiometry of binding to HTRA1.
• clones are screened based on binding to human HTRA1, such as a human HTRA1 N-terminal truncation (HTRA1 PD/PDZ).
• human HTRA1 N-terminal truncation HTRA1 PD/PDZ
• the human HTRA1 N-terminal truncation has at least an amino acid sequence of SEQ ID NO: 3.
• one or more clones that produce antibodies that bind to human HTRA1 are selected. Binding to human HTRA1 can be measured using any suitable option, e.g., using a kinetic assay.
• clones are screened based on inhibition of HTRA1 proteolytic activity.
• inhibition of HTRA1 proteolytic activity is determined using an optimized substrate (H2-Opt) to measure proteolytic activity.
• the optimized substrate has at least an amino acid sequence Mca-Ile-Arg-Arg- Val-Ser-Tyr-Ser-Phe-Lys(Dnp)-Lys-OH (SEQ ID NO:750).
• inhibition of HTRA1 proteolytic activity is determined using casein, e.g., ⁇ -casein, as a substrate.
• one or more clones that produce antibodies that inhibit HTRA1 proteolytic activity are selected.
• clones are screened based on binding affinity for HTRA1 loop A.
• the HTRA1 loop A has at least an amino acid sequence of SEQ ID NO:4.
• one or more clones that produce antibodies that bind to HTRA1 loop A are selected.
• one or more clones that produce antibodies that do not bind HTRA1 loop A are selected. Binding to HTRA1 loop A, or lack thereof, can be determined using any suitable option, e.g., by ELISA.
• clones are screened based on binding affinity for HTRA3.
• the HTRA3 has at least an amino acid sequence of SEQ ID NO: 5.
• one or more clones that produce antibodies that do not bind to HTRA3 are selected. Lack of binding to HTRA3 can be determined using any suitable option, e g., by ELISA.
• clones are screened based on relative binding affinity to trimeric HTRA1 and monomeric HTRA1.
• the trimeric HTRA1 includes HTRA1 having at least an amino acid sequence of SEQ ID NOB.
• the monomeric HTRA1 includes HTRA1 having at least an amino acid sequence of SEQ ID NO:7.
• one or more clones that produce antibodies that preferentially bind trimeric HTRA1 over monomeric HTRA1 are selected.
• one or more clones that produce antibodies that bind trimeric HTRA1 and do not bind monomeric HTRA1 are selected. Binding to trimeric or monomeric HTRA is HTRA1 can be measured using any suitable option, e.g., using a kinetic assay.
• clones are screened based on stoichiometry of binding to HTRA1.
• the HTRA1 includes HTRA1 having at least an
• one or more clones that produce antibodies that bind to HTRA1 with a stoichiometry of about 0.3:1, about 0.7:1, about 1 :1, about 2: 1, about 3 : 1, or greater are selected.
• the stoichiometry of binding can be determined using any suitable option, e.g., size exclusion chromatography.
• the screening method of the present disclosure includes carrying out one or more rounds of affinity maturation on one or more of the anti- HTRA1 antibodies. Any suitable option for affinity maturation can be used.
• affinity maturation includes generating a library, e.g., a phage library, of heavy chain and light chain CDR variants of a parental heavy chain and light chain variable region pair, and selecting for variants that bind a suitable target over one or more rounds.
• selecting for target binding is carried out with increasing stringency over subsequent rounds, e.g., by reducing target concentration, increasing washing steps, increasing washing time, etc.
• the binding target is an HTRA1 protein.
• the binding target is a HTRA1 N-terminal truncation (HTRA1 PD/PDZ).
• the desired target has at least an amino acid sequence of SEQ ID NO:3.
• affinity maturation includes depleting the library for non-specific binders.
• depleting the library for non-specific binders includes contacting the variants with HTRA3 to remove non-specific binders.
• the HTRA3 includes at least an amino acid sequence of SEQ ID NO: 5.
• the cell line comprises a nucleic acid encoding a heavy chain comprising at least one of the heavy chain variable region (VH) amino acid sequences shown in Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7, 7.12, 7.17, and 8.1; and a nucleic acid encoding a light chain comprising at least one of the light chain variable region (VL) amino acid sequences shown in Figs.
• VH heavy chain variable region
• the heavy and light chains of the antibody are encoded on separate vectors. In some embodiments, the heavy and light chains of the antibody are encoded on the same vector.
• the anti-HTRAl antibodies can be produced by recombinant expression including (i) the production of recombinant DNA by genetic engineering, (ii) introducing recombinant DNA into prokaryotic or eukaryotic cells by, for example and without limitation, transfection, electroporation or microinjection, (iii) cultivating the transformed cells, (iv) expressing antibody, e.g. constitutively or on induction, and (v) isolating the antibody, e.g. from the culture medium or by harvesting the transformed cells, in order to (vi) obtain purified antibody.
• the anti-HTRAl antibodies can be produced by expression in a suitable prokaryotic or eukaryotic host system characterized by producing a pharmacologically acceptable antibody molecule.
• eukaryotic cells are mammalian cells, such as CHO, COS, HEK 293, BHK, SK-Hip, and HepG2.
• suitable expression systems are prokaryotic (e g., E. coli with pET/BL21 expression system), yeast (Saccharomyces cerevisiae and/or Pichia pastoris systems), and insect cells.
• vectors can be used for the preparation of the antibodies disclosed herein and are selected from eukaryotic and prokaryotic expression vectors.
• vectors for prokaryotic expression include plasmids such as, and without limitation, preset, pet, and pad, wherein the promoters used in prokaryotic expression vectors include one or more of, and without limitation, lac, trc, trp, recA, or araBAD.
• vectors for eukaryotic expression include: (i) for expression in yeast, vectors such as, and without limitation, pAO, pPIC, pYES, or pMET, using promoters such as, and without limitation, A0X1, GAP, GALI, or AUG1; (ii) for expression in insect cells, vectors such as and without limitation, pMT, pAc5, pIB, pMIB, or pBAC, using promoters such as and without limitation PH, plO, MT, Ac5, OpIE2, gp64, or polh, and (iii) for expression in mammalian cells, vectors such as, and without limitation, pSVL, pCMV, pRc/RSV, pcDNA3, or pBPV, and vectors derived from, in one aspect, viral systems such as and without limitation vaccinia virus, adeno-associated viruses, herpes viruses, or retroviruses, using promoters such as and without limitation
• the method includes conjugating an anti-HTRAl antibody as provided herein to a polymer, e.g., a phosphorylcholine containing polymer, wherein the anti-HTRAl antibody comprises a non-native cysteine residue outside a variable region of the antibody.
• the polymer comprises or consists of a phosphorylcholine containing polymer.
• the phosphorylcholine containing polymer comprises a sulfhydryl specific reacting group selected from the group consisting of a maleimide, a vinylsulfone, an orthopyridyl-disulfide, and an iodoacetamide.
• the sulfhydryl specific reacting group on the phosphorylcholine containing polymer reacts with the non-native cysteine residue on the anti-HTRAl antibody to make the antibody conjugate.
• the anti-HTRAl antibody is an immunoglobulin G (IgG) and the cysteine is in the Fc region of the antibody.
• the anti- HTRAl antibody comprises a light chain and a heavy chain, wherein the anti-HTRAl antibody heavy chain variable region comprises CDRHI : FYHVH (SEQ ID NO:830), CDRH2: SIYTSGYTEYASALES (SEQ ID NO:831), and CDRH3 : EGLQRVGVLDA (SEQ ID NO 834) or EGLQRVGVFDA (SEQ ID NO:832) or EGLQRVGVMDA (SEQ ID NO:833), and the anti-HTRAl antibody light chain variable region comprises CDRLI : RSSQSLLDEAGETYLA (SEQ ID NO:840), CDRL2: EVSLLES (SEQ ID NO:841), and CDRL3 : QQATYFPYT (SEQ ID NO:839).
• the anti-HTRAl antibody heavy chain isotype is IgGl .
• the anti-HTRAl antibody has one or more mutations relative to an IgGl constant region to modulate effector function, as provided herein.
• the anti-HTRAl antibody heavy chain comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 80% identical to SEQ ID NOs: 725, 742, 744, 745, 746
• the anti-HTRAl antibody light chain comprises a light chain variable region (VL) comprising an amino acid sequence at least 80% identical to SEQ ID NO:748.
• the non-native cysteine residue is selected from the group consisting of Q347C (EU numbering) and L443C (EU numbering).
• the sulfhydryl specific reacting group is maleimide.
• the polymer is selected from the group consisting of polyethylene glycol (PEG), branched PEG, PolyPEG® (Warwick Effect Polymers; Coventry, UK), polysialic acid (PSA), starch, hydroxylethyl starch (HES), hydroxyalkyl starch (HAS), carbohydrate, polysaccharides, pullulane, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, dextran, carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, polyethylene-co-maleic acid anyhydride, polystyrene-co-maleic acid anhydride
• PVA polysia
• the polymer is a zwitterionic polymer.
• the zwitterion is phosphorylcholine, i.e. a phosphorylcholine containing polymer.
• the polymer is composed of MPC units.
• the polymer, e.g., MPC polymer has three or more arms. In some embodiments, the polymer, e.g., MPC polymer, has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more arms. In some embodiments, the polymer, e g., MPC polymer, has 3, 6, or 9 arms. In some embodiments, the polymer, e g., MPC polymer, has 9 arms. In some embodiments, the polymer is synthesized with an initiator comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more polymerization initiation sites. In some embodiments, the polymer has three or more arms or is synthesized with an initiator comprising 3 or more polymerization initiation sites. In some embodiments, the polymer is synthesized with an initiator comprising 9 polymerization initiation sites.
• the polymer e.g., MPC polymer
• the polymer, e.g., MPC polymer has a molecular weight between about 500,000 and 1,000,000 Da, or between about 600,000 and 1,000,000 Da, or between about 600,000 to 900,000 Da, or between about 600,000 to 850,000 Da.
• the method of making the antibody conjugate has an additional step of contacting the antibody with a thiol reductant under conditions that produce a reduced cysteine sulfhydryl group.
• a thiol reductant under conditions that produce a reduced cysteine sulfhydryl group.
• the protein after purification is treated with a reducing agent, e.g., dithiothreitol.
• a reducing agent e.g., dithiothreitol.
• all native (e.g., inter and intra) Cys-Cys disulfides are reformed.
• the therapeutic protein is exposed to oxidizing conditions and/or oxidizing agents for a prescribed period of time, e.g., overnight. In some embodiments, ambient air exposure overnight can be used to achieve reformation of the native disulfide bonds.
• an oxidizing agent is employed to restore the native disulfides.
• the oxidizing agent is selected from the group consisting of aqueous CuSCh and dehydroascorbic acid (DHAA). In some embodiments, the oxidizing agent is DHAA. In some embodiments, the range of DHAA used is in the range of 5-30 equivalents. In some embodiments, the range is 10-20 equivalents. In some embodiments, the range is 15 equivalents.
• the thiol reductant is selected from the group consisting of: Tris[2-carboxyehtyl]phosphine hydrochloride (TCEP), dithiothreitol (DTT), di thioerythritol (DTE), sodium borohydride (NaBHi), sodium cyanoborohydride (NaCNBH3), P-mercaptoethanol (BME), 3,3',3''-Phosphanetriyltris(benzenesulfonic acid) trisodium (TPPTS), cysteine hydrochloride and cysteine.
• the thiol reductant is TCEP.
• the thiol reductant concentration is between 1 and 100 fold molar excess relative to the therapeutic protein concentration. In some embodiments, the thiol reductant concentration is between 20 to 50 fold molar excess relative to the therapeutic protein concentration. In some embodiments, the thiol reductant is removed following incubation with the therapeutic protein prior to oxidation of the therapeutic protein.
• the method includes a further step of purifying the antibody conjugate after conjugation.
• the antibody conjugate is purified using a technique selected from the group consisting of ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, and affinity chromatography or combinations thereof.
• the antibody conjugate retains at least 20% biological activity relative to unconjugated antibody. In some embodiments, the antibody conjugate retains at least 50% biological activity relative to unconjugated antibody. In some embodiments, the antibody conjugate retains at least 90% biological activity relative to native antibody. In some embodiments, the antibody conjugate retains at least 95% biological activity relative to native antibody. In some embodiments, the antibody conjugate retains about 100% biological activity relative to native antibody. [0314] In some embodiments, the antibody conjugate has an increased half-life relative to unconjugated antibody. In some embodiments, the antibody conjugate has at least a 1.5 fold increase in half-life relative to unconjugated antibody. In some embodiments, the antibody conjugate has at least a 5 fold increase in half-life relative to unconjugated antibody.
• the polymer is made from an initiator suitable for ATRP having one or more polymerization initiation sites.
• the polymer initiation site has a 2-bromoisobutyrate site.
• the initiator has 3 or more polymerization initiation sites.
• the initiator has 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more polymerization initiation sites.
• the initiator has 3, 6 or 9 polymerization initiation sites.
• the initiator has 9 polymerization initiation sites.
• the initiator is OG1786.
• the antibodies, conjugates thereof, and pharmaceutical compositions containing the same find use in many applications where antibody binding to HTRA1 and/or inhibition of HTRA1 enzymatic activity is desired.
• a method for treatment or prophylaxis of an ocular disease in subject in need thereof using an antibody or antibody conjugate of the present disclosure finds use in the treatment or prophylaxis of a disorder, e.g., an ocular disease, related to HTRA1, e.g., HTRA1 activity.
• the treatment or prophylaxis method can include administering an therapeutically effective dose of any antibody conjugate or antibody of the present disclosure to a subject in need thereof.
• the disease can be age-related macular degeneration (AMD).
• the disease can be wet AMD.
• the disease can be dry AMD.
• the ocular disease is selected from one or more of the group consisting of diabetic retinopathy, age-related macular degeneration (AMD) (e.g., early, intermediate, or advanced AMD), retinopathy of prematurity (ROP), dry AMD, geographic atrophy, wet AMD, polypoidal choroidal vasculopathy, and HTRA1 -associated disorders.
• AMD age-related macular degeneration
• ROP retinopathy of prematurity
• dry AMD geographic atrophy
• wet AMD e.g., wet AMD
• polypoidal choroidal vasculopathy e.g., retinopathy of prematurity
• HTRA1 -associated disorders e.g., diabetic retinopathy.
• the ocular disease is diabetic retinopathy.
• the ocular disease is an HTRA-1 -associated disorder.
• the ocular disease is dry AMD or geographic atrophy.
• the dry AMD comprises early, intermediate or advanced dry AMD.
• the anti-HTRAl antibody (or conjugate thereof) can be administered to a subject via any suitable route. It should be apparent to a person skilled in the art that the examples described herein are not intended to be limiting but to be illustrative of the techniques available. Accordingly, in some embodiments, the anti-HTRAl antibody (or conjugate thereof) is administered to a subject in accord with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, transdermal, subcutaneous, intra-articular, sublingually, intrasynovial, via insufflation, intrathecal, oral, inhalation or topical routes.
• intravenous administration e.g., as a bolus or by continuous infusion over a period of time
• intramuscular, intraperitoneal, intracerebrospinal transdermal, subcutaneous, intra-articular, sublingually, intrasy
• Administration can be systemic, e g., intravenous administration, or localized.
• Commercially available nebulizers for liquid formulations including jet nebulizers and ultrasonic nebulizers are useful for administration.
• Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution.
• anti-HTRAl antibody (or conjugate thereof) can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.
• the anti-HTRAl antibodies, anti-HTRAl antibody conjugates, and pharmaceutical compositions disclosed herein are used for prophylaxis or treatment of an ocular disease or condition.
• the conjugates are typically formulated for and administered by ocular, intraocular, and/or intravitreal injection, and/or juxtascleral injection, and/or subtenon injection, and/or suprachoroidal injection and/or topical administration in the form of eye drops and/or ointment.
• Such anti-HTRAl antibodies, anti- HTRAl antibody conjugates, and compositions can be delivered by any suitable option, e g. intravitreally as a device and/or a depot that allows for slow release of the compound into the vitreous.
• a device may be in the form of a minimum and/or a matrix and/or a passive diffusion system and/or encapsulated cells that release the compound for a prolonged period of time.
• the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic or substantially isotonic.
• Formulations for ocular, intraocular or intravitreal administration can be prepared by methods and using ingredients known in the art. Proper penetration into the eye is desirable for efficient treatment. Unlike diseases of the front of the eye, where drugs can be delivered topically, retinal diseases merit a more site-specific approach.
• the method of choice for drug delivery to treat retinal disease, such as AMD is direct intravitreal injection.
• intravitreal injections are repeated at intervals which depend on the patient's condition, and the properties and half-life of the drug delivered.
• the dosage of the active agent is from 0.01 mg/kg body weight, to typically around 1 mg/kg, for systemic administrations.
• dosage is typically 0.1 mg/eye/dose to 10 mg/eye/dose or more.
• the dosage is 100 ul/dose/eye.
• a needle can be used to administer the dosage.
• the needle can be, for example, a 30 gauge !4 inch needle or a ’A inch needle that is 27G or 29G.
• the physician can determine the actual dosage most suitable for an individual which depends on factors including the age, weight, sex and response of the individual, the disease or disorder being treated and the age and condition of the individual being treated.
• the above dosages are exemplary of the average case. There can, of course, be instances where higher or lower dosages are merited.
• the pharmaceutical composition may be administered once every one to thirty days.
• the antibody or antibody conjugate is administered no more frequently than once a month.
• the antibody or conjugate thereof is administered two times per month or weekly.
• the antibody or conjugate thereof is administered once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, or once every twelve months.
• the anti-HTRAl antibodies of the present disclosure may be employed by expression of such polypeptides in vivo in a patient, i.e., gene therapy.
• nucleic acid (optionally contained in a vector) into the patient's cells: in vivo and ex vivo.
• in vivo delivery the nucleic acid is injected directly into the patient, usually at the sites where the therapeutic protein is required, i.e., where biological activity of the therapeutic protein is needed.
• the patient's cells are removed, the nucleic acid is introduced into these isolated cells, and the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes that are implanted into the patient.
• nucleic acids there are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or transferred in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, transduction, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. Transduction involves the association of a replication-defective, recombinant viral (preferably retroviral) particle with a cellular receptor, followed by introduction of the nucleic acids contained by the particle into the cell. A commonly used vector for ex vivo delivery of the gene is a retrovirus.
• in vivo nucleic acid transfer techniques include transfection with viral or non-viral vectors (such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV)) and lipid-based systems (useful lipids for lipid- mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Chol).
• viral or non-viral vectors such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV)
• lipid-based systems useful lipids for lipid- mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Chol.
• the most preferred vectors for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses.
• a viral vector such as a retroviral vector includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post- translational modification of messenger.
• a viral vector such as a retroviral vector includes a nucleic acid molecule that, when transcribed in the presence of a gene encoding the therapeutic protein, is operably linked thereto and acts as a translation initiation sequence.
• Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used (if these are not already present in the viral vector).
• such vector typically includes a signal sequence for secretion of the PRO polypeptide from a host cell in which it is placed.
• the signal sequence for this purpose is a mammalian signal sequence, most preferably the native signal sequence for the therapeutic protein.
• the vector construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
• such vectors will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
• Other vectors can be used that are non- viral, such as cationic lipids, polylysine, and dendrimers.
• the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell-surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
• an agent that targets the target cells such as an antibody specific for a cell-surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
• proteins that bind to a cell-surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins that undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life. Any suitable technique of receptor-mediated endocytosis can be used.
• a method for treatment or prophylaxis of an ocular disease in a mammal in which a nucleic acid molecule that encodes an anti- HTRA1 antibody is administered.
• kits comprising any or all of the antibodies, antibody conjugates or pharmaceutical compositions and formulations described herein.
• Kits of the present disclosure include one or more containers comprising an anti-HTRAl antibody or conjugate described herein and instructions for use in accordance with any of the methods of the present disclosure. In some embodiments, these instructions comprise a description of administration of the anti-HTRAl antibody or conjugate for the therapeutic treatments disclosed herein.
• kits are provided for producing a single-dose administration unit.
• the kit can contain both a first container having a dried protein and a second container having an aqueous formulation.
• kits containing single and multi-chambered pre-filled syringes are included.
• the antibody is a human antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a monoclonal antibody.
• the instructions relating to the use of an anti- HTRA1 antibody or conjugate include information as to dosage, dosing schedule, and route of administration for the intended treatment.
• the containers may be unit doses, bulk packages (e.g., multi -dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
• kits are in suitable packaging.
• suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like.
• packages for use in combination with a specific device such as prefilled syringe, an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump.
• the kit has a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
• the container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
• At least one active agent in the composition is an anti-HTRAl antibody or conjugate.
• the container may further comprise a second pharmaceutically active agent.
• kits provide additional components such as buffers and interpretive information.
• the kits can include an additional syringe and needle used for back fill of the dosing syringe.
• the kit comprises a container and a label or package insert(s) on or associated with the container.
• An antibody conjugate comprising (1) an anti-HTRAl antibody and (2) a phosphorylcholine containing polymer, wherein the polymer is covalently bonded to the antibody at a non-native cysteine outside a variable region of the antibody.
• the anti-HTRAl antibody heavy chain comprises a heavy chain variable region (VH) comprising: a heavy chain complementarity determining region 1 (CDRHI) having an amino acid sequence of a CDRHI in any one of the VH sequences in Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7, 7.12, 7.17, and 8.1, or with a variation at no more than 5 residues thereof; a CDRH2 having an amino acid sequence of a CDRH2 in any one of the VH sequences in Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7, 7.12,
• VH heavy chain variable region
• the anti-HTRAl antibody light chain comprises a light chain variable region (VL) comprising: a light chain complementarity determining region 1 (CDRLI ) having an amino acid sequence of a CDRLI in any one of the VL sequences in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and 8.2, or with a variation at no more than 5 residues thereof; a CDRL2 having an amino acid sequence of a CDRL2 in any one of the VL sequences in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13,
• CDRL3 having an amino acid sequence of a CDRL3 in any one of the VL sequences in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and 8.2, or with a variation at no more than 5 residues thereof.
• the anti-HTRAl antibody VH comprises 3 CDRs each having an amino acid sequence of the corresponding CDRs in any one of the VH sequences in Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7, 7.12, 7.17, and 8.1, or with a variation at no more than 5 residues thereof
• the anti-HTRAl antibody VL comprises 3 CDRs each having an amino acid sequence of the corresponding CDRs in any one of the VL sequences in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and 8.2, or with a variation at no more than 5 residues thereof .
• the anti-HTRAl antibody heavy chain variable region comprises CDRHI : FYHVH (SEQ ID NO: SEQ ID NO:830), CDRH2: SIYTSGYTEYASALES (SEQ ID NO:831), and CDRH3 : EGLQRVGVLDA (SEQ ID NO:834) or EGLQRVGVFDA (SEQ ID NO:832) or EGLQRVGVMDA (SEQ ID NO:833)
• the anti-HTRAl antibody light chain variable region comprises CDRLI : RSSQSLLDEAGETYLA (SEQ ID NO:840), CDRL2: EVSLLES (SEQ ID NO:841), and CDRL3 : QQATYFPYT (SEQ ID NO:839).
• the anti-HTRAl antibody heavy chain variable region comprises CDRHI : GFSLTFYH (SEQ ID NO: SEQ ID NO:825), CDRH2: IYTSGYT (SEQ ID NO:826), and CDRH3 : AREGLQRVGVFDA (SEQ ID NO 827) or AREGLQRVGVMDA (SEQ ID NO: 828) or AREGLQRVGVLDA (SEQ ID NO:829)
• the anti-HTRAl antibody light chain variable region comprises CDRLI : QSLLDEAGETY (SEQ ID NO:837), CDRL2: EV (SEQ ID NO: 838), and CDRL3 : QQATYFPYT (SEQ ID NO: 839).
• the anti-HTRAl antibody heavy chain variable region comprises CDRHI : GFSLTFY (SEQ ID NO: SEQ ID NO:835), CDRH2: YTSGY (SEQ ID NO:836), and CDRH3 : EGLQRVGVLDA (SEQ ID NO:834) or EGLQRVGVFDA (SEQ ID NO:832) or EGLQRVGVMDA (SEQ ID NO:833)
• the anti-HTRAl antibody light chain variable region comprises CDRLI : RSSQSLLDEAGETYLA (SEQ ID NO:840), CDRi.2: EVSLLES (SEQ ID NO:841), and CDRL3 : QQATYFPYT (SEQ ID NO:839).
• the antibody conjugate according to arrangement 14, wherein the mutations are selected from the group consisting of (EU numbering): E233P, L234V, L234A, L235A, G237A, A327G, A33 OS, and P33 IS.
• the antibody conjugate according to arrangement 15, comprising the following mutations: L234A, L235A, and G237A (EU numbering).
• cysteine is in the anti-HTRAl antibody heavy chain and is Q347C (EU numbering) or L443C (EU numbering).
• the anti-HTRAl antibody heavy chain comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 80% identical to any one of SEQ ID NOs:725, 742, 744, 745, 746
• the anti-HTRAl antibody light chain comprises a light chain variable region (VL) comprising an amino acid sequence at least 80% identical to SEQ ID NO:748.
• the phosphorylcholine containing polymer comprises 2-(methacryloyloxyethyl)-2'- (trimethylammonium)ethyl phosphate (MPC) monomers as set forth below:
• An antibody conjugate comprising an anti-HTRAl immunoglobulin G (IgG) bonded to a polymer, which polymer comprises MPC monomers, wherein the anti-HTRAl antibody heavy chain comprises: a heavy chain variable region (VH) comprising an amino acid sequence at least 80% identical to any one of SEQ ID NOs:725, 742, 744, 745, 746, C-terminally fused to a heavy chain constant region (HC) comprising an amino acid sequence of SEQ ID NO:536, wherein the polymer is bonded to C326 of SEQ ID NO:536, and wherein the anti-HTRAl antibody light chain comprises: a light chain variable region (VL) comprising an amino acid sequence at least 80% identical to SEQ ID NO: 748, C-terminally fused to a light chain constant region (LC) comprising an amino acid sequence of SEQ ID NO:537.
• VH heavy chain variable region
• HC heavy chain constant region
• the anti-HTRAl antibody light chain comprises: a light
• each heavy chain of the anti-HTRAl antibody is denoted by the letter H, and each light chain of the anti-HTRAl antibody is denoted by the letter L; the polymer is bonded to the anti-HTRAl antibody through the sulfhydryl of C443 (EU numbering), which bond is depicted on one of the heavy chains;
• PC is , where the curvy line indicates the point of attachment to the rest of the polymer, where X is a) -OR where R is -H, methyl, ethyl, propyl, isopropyl; b) -H; c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) - NCS; and wherein either i) nl, n2, n3, n4, n5, n6, n7, n8 and n9 are the same or different and are integers from 0 to 3000; or ii) nl, n2, n3, n4, n5, n6, n7, n8 and n9 are the same or different such that the sum of nl, n2, n3, n4, n5, n6, n7, n8 and n9 is 2500 plus or minus 15%.
• An antibody conjugate comprising an anti-HTRAl immunoglobulin G (IgG) covalently bonded to a polymer, which polymer comprises MPC monomers
• the anti- HTRAl antibody heavy chain comprises: a heavy chain variable region (VH) comprising an amino acid sequence comprising SEQ ID NOs:725, 742, 744, 745, 746, C-terminally fused to a heavy chain constant region (HC) comprising an amino acid sequence of SEQ ID NO:536, wherein the polymer is bonded to C326 of SEQ ID NO:536, and wherein the anti-HTRAl antibody light chain comprises: a light chain variable region (VL) comprising an amino acid sequence comprising SEQ ID NO:748, C-terminally fused to a light chain constant region (LC) comprising an amino acid sequence of SEQ ID NO:537.
• VH heavy chain variable region
• HC heavy chain constant region
• LC light chain constant region
• An anti-HTRAl antibody which comprises a heavy chain and a light chain, wherein the heavy chain comprises: a CDRnl having an amino acid sequence of a CDRul in any one of the VH sequences in Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7, 7.12,
• the anti-HTRAl antibody light chain comprises a light chain variable region (VL) comprising: a CDRLI having an amino acid sequence of a CDRLI in any one of the VL sequences in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13,
• VL light chain variable region
• a CDRL2 having an amino acid sequence of a CDRL2 in any one of the VL sequences in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and 8.2
• a CDRL3 having an amino acid sequence of a CDRL3 in any one of the VL sequences in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and 8.2.
• the anti-HTRAl antibody VH comprises 3 CDRs each having an amino acid sequence of the corresponding CDRs in any one of the VH sequences in Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7, 7.12, 7.17, and 8.1
• the anti-HTRAl antibody VL comprises 3 CDRs each having an amino acid sequence of the corresponding CDRs in any one of the VL sequences in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and 8.2.
• the anti-HTRAl antibody VH comprises 3 CDRs each having an amino acid sequence of the corresponding CDRs in SEQ ID NO:725, 742, 744, 745, or 746
• the anti-HTRAl antibody VL comprises 3 CDRs each having an amino acid sequence of the corresponding 3 CDRs in SEQ ID NO: 748.
• An anti-HTRAl antibody which comprises a heavy chain and a light chain, wherein the heavy chain comprises CDRHI : FYHVH (SEQ ID NO:830), CDRH2: SIYTSGYTEYASALES (SEQ ID NO:831), and CDR H 3: EGLQRVGVLDA (SEQ ID NO 834) or EGLQRVGVFDA (SEQ ID NO:832) or EGLQRVGVMDA (SEQ ID NO:833), and the light chain comprises CDRLI : RSSQSLLDEAGETYLA (SEQ ID NO:840), CDRL2: EVSLLES (SEQ ID NO: 841), and CDR L 3 : QQATYFPYT (SEQ ID NO: 839), and the heavy chain isotype is IgGl, wherein the IgGl constant region comprises one or more of the following mutations to modulate effector function (EU numbering): L234A, L235A, and G237A.
• EU numbering effector function
• An anti-HTRAl antibody which comprises a heavy chain and a light chain, wherein the heavy chain comprises CDRHI : GFSLTFYH (SEQ ID NO: SEQ ID NO:825), CDRH2: IYTSGYT (SEQ ID NO:826), and CDRH3 : AREGLQRVGVFDA (SEQ ID NO:827) or AREGLQRVGVMDA (SEQ ID NO: 828) or AREGLQRVGVLDA (SEQ ID NO: 829), and the anti-HTRAl antibody light chain variable region comprises CDRLI : QSLLDEAGETY (SEQ ID NO:837), CDRL2: EV (SEQ ID NO:838), and CDR L 3 : QQATYFPYT (SEQ ID NO:839), and the heavy chain isotype is IgGl, wherein the IgGl constant region comprises one or more of the following mutations to modulate effector function (EU numbering): L234A, L235A, and
• An anti-HTRAl antibody which comprises a heavy chain and a light chain, wherein the heavy chain comprises CDRul : GFSLTFY (SEQ ID NO: SEQ ID NO:835), CDRH2: YTSGY (SEQ ID NO:836), and CDRH3 : EGLQRVGVLDA (SEQ ID NO:834) or EGLQRVGVFDA (SEQ ID NO:832) or EGLQRVGVMDA (SEQ ID NO:833), and the anti- HTRAl antibody light chain variable region comprises CDRLI : RSSQSLLDEAGETYLA (SEQ ID NO:840), CDRL2: EVSLLES (SEQ ID NO:841), and CDRL3 : QQATYFPYT (SEQ ID NO: 839), wherein the IgGl constant region comprises one or more of the following mutations to modulate effector function (EU numbering): L234A, L235A, and G237A.
• the heavy chain comprises CDRul : GF
• the anti- HTRAl antibody heavy chain comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 80% identical to at least one of SEQ ID NOs:725, 742, 744, 745, 746, and the anti-HTRAl antibody light chain comprises a light chain variable region (VL) comprising an amino acid sequence at least 80% identical to SEQ ID NO:748.
• VH heavy chain variable region
• VL light chain variable region
• An antibody comprising: a heavy chain amino acid variable region that comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOs:725, 742, 744, 745, 746; and a light chain amino acid variable region that comprises an amino acid sequence at least 80% identical to SEQ ID NO: 748.
• the antibody of arrangement 44 wherein the antibody is a human IgGl, and wherein a heavy chain constant region comprises one or more mutations that reduce an immune-mediated effector function.
• An antibody that binds to HTRA1 comprising: a CDRHI that is the CDRHI in any one of SEQ ID NOs:725, 742, 744, 745, 746; a CDRH2 that is the CDRH2 in any one of SEQ ID NOs:725, 742, 744, 745, 746; a CDRH3 that is the CDRH3 in any one of SEQ ID NOs:725, 742, 744, 745, 746; a CDRLI that is the CDRLI in SEQ ID NO:748; a CDRL2 that is the CDRL2 in SEQ ID NO:748; a CDRL3 that is the CDRL3 in SEQ ID NO:748; at least one of the following mutations (EU numbering): L234A, L235A, and G237A; and at least one of the following mutations(EU numbering): Q347C or L443C.
• the heavy chain comprises a heavy constant region having an amino acid sequence at least 80% identical to SEQ ID NO:536.
• a pharmaceutical composition comprising the antibody conjugate and/or the antibody according to any one of the preceding arrangements, in a liquid solution.
• a pharmaceutical composition comprising the antibody conjugate and/or the antibody according to any one of the preceding arrangements, and a pharmaceutically acceptable carrier.
• a method for treatment or prophylaxis of an ocular disease comprising administering the antibody conjugate and/or the antibody according to any one of arrangements 1-51, or the pharmaceutical composition according to arrangement 52 or 53.
• ocular disease is selected from the group consisting of diabetic retinopathy, age-related macular degeneration (AMD) (e.g., early, intermediate, or advanced AMD), retinopathy of prematurity (ROP), dry AMD, geographic atrophy, wet AMD, polypoidal choroidal vasculopathy, and HTRA1 -associated disorders.
• AMD age-related macular degeneration
• ROP retinopathy of prematurity
• a method of making an anti-HTRAl antibody conjugate comprising conjugating the anti-HTRAl antibody of any one of arrangements 35-51 to a phosphorylcholine containing polymer, wherein the phosphorylcholine containing polymer comprises a sulfhydryl specific reacting group selected from the group consisting of a maleimide, a vinylsulfone, an orthopyridyl-disulfide, and an iodoacetamide, wherein the sulfhydryl specific reacting group on the phosphorylcholine containing polymer reacts with the cysteine residue on the anti-HTRAl antibody to make the antibody conjugate. 62.
• a method of making an antibody conjugate comprising an anti-HTRAl antibody conjugated to a phosphorylcholine containing polymer comprising the step of: conjugating an anti-HTRAl antibody to a phosphorylcholine containing polymer, wherein the anti-HTRAl antibody comprises a non-native cysteine residue outside a variable region of the antibody, wherein the phosphorylcholine containing polymer comprises a sulfhydryl specific reacting group selected from the group consisting of a maleimide, a vinylsulfone, an orthopyridyl-disulfide, and an iodoacetamide, and wherein the sulfhydryl specific reacting group on the phosphorylcholine containing polymer reacts with the cysteine residue on the anti-HTRAl antibody to make the antibody conjugate.
• the anti-HTRAl antibody comprises a light chain and a heavy chain
• the anti-HTRAl antibody heavy chain comprises CDRnl : FYHVH (SEQ ID NO:830), CDRn2: SIYTSGYTEYASALES (SEQ ID NO:831), and CDRH3 : EGLQRVGVLDA (SEQ ID NO:834) or EGLQRVGVFDA (SEQ ID NO:832) or EGLQRVGVMDA (SEQ ID NO:833)
• the anti-HTRAl antibody light chain comprises CDRLI : RSSQSLLDEAGETYLA (SEQ ID NO:840), CDRL2: EVSLLES (SEQ ID NO:841), and CDRL3 : QQATYFPYT (SEQ ID NO:839).
• nonnative cysteine residue is selected from the group consisting of Q347C (EU numbering) and L443C (EU numbering).
• the anti- HTRA1 antibody heavy chain comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 80% identical to SEQ ID NO: 725, 742, 744, 745, 746 and the anti-HTRAl antibody light chain comprises a light chain variable region (VL) comprising an amino acid sequence at least 80% identical to SEQ ID NO: 748.
• VH heavy chain variable region
• VL light chain variable region
• the phosphorylcholine containing polymer comprises 2-(methacryloyloxyethyl)-2'- (trimethylammonium)ethyl phosphate (MPC) monomers as set forth below: 75.
• MPC 2-(methacryloyloxyethyl)-2'- (trimethylammonium)ethyl phosphate
• thiol reductant is selected from the group consisting of Tris[2-carboxyethyl]phosphine hydrochloride (TCEP), dithiothreitol (DTT), dithioerythritol (DTE), sodium borohydride (NaBH4), sodium cyanoborohydride (NaCNBHs), P-mcrcaptocthanol (BME), 3,3 ',3''- Phosphanetriyltris(benzenesulfonic acid) trisodium (TPPTS), cysteine hydrochloride, and cysteine.
• TCEP Tris[2-carboxyethyl]phosphine hydrochloride
• DTT dithiothreitol
• DTE dithioerythritol
• NaBH4 sodium borohydride
• NaCNBHs sodium cyanoborohydride
• P-mcrcaptocthanol BME
• TPTS 3,3 ',3''
• the isolated cell line of arrangement 96 wherein the cell line is selected from the group consisting of CHO, klSV, XCeed, CHOK1SV, and GS-KO. 98.
• a recombinant expression vector comprising the nucleic acid of arrangement 98.
• a host cell comprising the expression vector of arrangement 99.
• a method of producing an anti-HTRAl antibody comprising: culturing a cell line that recombinantly produces an anti-HTRAl antibody of any one of arrangements 35-51, under conditions wherein the antibody is produced; and recovering the antibody.
• a method of producing an anti-HTRAl antibody comprising: culturing a cell line comprising: a nucleic acid encoding a heavy chain comprising any one of the heavy chain variable region (VH) amino acid sequences shown in Figs. 21, 22A, 23 A, and 24A, and Tables 1.2, 6.3, 6.5, 7.1, 7.4, 7.7, 7.12, 7.17, and 8.1; and a nucleic acid encoding a light chain comprising any one of the light chain variable region (VL) amino acid sequences shown in Figs. 21, 22B, 23B, and 24B, and Tables 1.2, 6.3, 6.6, 7.1, 7.5, 7.8, 7.13, 7.18, and 8.2, under conditions wherein an antibody comprising the heavy chain and the light chain is produced; and recovering the antibody.
• VH heavy chain variable region
• the antibody of arrangement 105 wherein the antibody inhibits the protease activity of HTRAl with an ICso of about 2.0 x 10' 10 M or less.
• the antibody of arrangement 108 or 109, wherein the loop A peptide of HTRA1 comprises or consists of FRKLPFSKREVPV (SEQ ID NO:851).
• ocular disease is selected from the group consisting of diabetic retinopathy, age-related macular degeneration (AMD) (e.g., early, intermediate, or advanced AMD), retinopathy of prematurity (ROP), dry AMD, geographic atrophy, wet AMD, polypoidal choroidal vasculopathy, and HTRA1 -associated disorders.
• AMD age-related macular degeneration
• ROP retinopathy of prematurity
• This non-limiting example shows generation and screening of antibodies against HTRA1.
• HTRA1 PD his- tagged human HTRA1 protease domain
• an antibody that binds to HTRA1, e.g., human HTRA1 is obtained by immunizing a host animal, e.g., a mouse or rat, with human HTRA1 protease domain.
• an antibody that binds to HTRA1, e.g., human HTRA1 is obtained by selecting for a clone that binds to a human HTRA1 protease domain and/or binds or does not bind a HTRA1 loop A and/or does not bind HTRA3.
• an antibody that binds to HTRA1, e.g., human HTRA1 is obtained by selecting for a clone that binds to a human HTRA1 protease domain and/or a HTRA1 loop A and/or does not bind HTRA3.
• an antibody that binds to HTRA1, e.g., human HTRA1 is obtained by selecting for a clone that binds to a human HTRA1 protease domain and/or does not bind to a HTRA1 loop A and/or does not bind HTRA3.
• an anti-HTRAl antibody of the present disclosure has 3 heavy chain CDR (CDRH) of the 3 CDRH of at least one of the antibody clones set forth in Table 1.2, and has 3 light chain CDR (CDRL) of the 3 CDRL of the corresponding antibody clone.
• CDRH heavy chain CDR
• CDRL light chain CDR
• an anti- HTRAl antibody of the present disclosure has a VH having the amino acid sequence of the VH domain of at least one of the antibody clones set forth in Table 1.2, and has a VL having the amino acid sequence of the VL of the corresponding antibody clone.
• Loop A binding characterization Binding of antibodies to a sequence similar to a known HTRA1 epitope (loop A, US 2017/0145113 Al) was tested by ELISA (FIG. 1). Briefly, streptavidin plates (Cat.# 15120, Pierce) were coated with 50 pL of loop A peptide (OG2185) (30 pg/mL) overnight. On the next day, plates were washed five times with 300 pL PBS/0.05% Tween-20, then blocked with 200 pL of 2% BSA/PBS for 1 hour, then washed five additional times.
• FIG. 1 shows 14 out of 40 antibodies tested bound to loop A peptide.
• an anti-HTRAl antibody of the present disclosure binds to HTRA1, e.g., human HTRA1, with a KD of about 10' 8 M or less, about 10' 9 M or less, about IO 10 M or less, or about 10 11 M or less.
• an anti-HTRAl antibody of the present disclosure binds to loop A peptide of HTRA1.
• an anti- HTRAl antibody of the present disclosure does not bind to loop A peptide of HTRA1.
• H2-Opt assay Selected anti-HTRAl antibodies were screened for their capability to inhibit HTRA1 proteolytic activity using a FRET-based protease assay.
• FIG. 2 shows 33 antibodies inhibited at least 50% of HTRA1 activity under the studied conditions, in agreement with the observation that most of these molecules exhibit tight binding to the antigen (Table 2.2).
• a panel of 12 antibodies were tested on their ability to inhibit HTRA1 activity in a concentration-dependent manner.
• antibodies were serially diluted in assay buffer (from 28 nM to 0.5 pM, 1 :3 dilution), then experiment was carried on as described above.
• Slope values for the linear phase of the kinetic curve (RFU/s) were calculated and plotted against the concentration of antibody using GraphPad Prism software and fit with a non-linear regression curve.
• FIG.3 shows antibodies inhibit H2-Opt cleavage in a concentration-dependent fashion with nM to sub-nM ICso values (Table 3.1).
• FIG. 4 shows OG2161 degrades P-casein and substrate is intact in the absence of the enzyme.
• an anti-HTRAl antibody of the present disclosure inhibits HTRA1 protease activity with an ICso of about 10‘ 9 M or less, or 10‘ 10 M or less. In some embodiments, an anti-HTRAl antibody of the present disclosure inhibits digestion of P- casein by HTRA1.
• FIGs. 5A-5C show antibodies elute as single peaks and addition of HTRA1 change their elution profile. The mass of the complexes formed was dependent on the antibody tested.
• aHTRAl_26 panel A
• aHTRAl_30 panel B
• aHTRAl_36 panel C
• a complex -310 kDa
• 1 HTRA1 trimer 1 IgG
• the aHTRAl_36 antibody presents an advantageous complex stoichiometry since only one antibody molecules binds to HTRA1 trimer and it is enough to inhibit the enzyme activity.
• an anti-HTRAl antibody of the present disclosure binds to HTRA1 trimer. In some embodiments, an anti-HTRAl antibody of the present disclosure binds to HTRA1 trimer with 1 : 1 stoichiometry.
• Selected antibodies (non-loop A binders, high affinity, potent antagonistic activity) were conjugated with OG1802.
• the conjugation process involved two steps: an initial reduction reaction to deprotect cysteine thiol side chains (decapping), followed by a conjugation to the biopolymer OG1802 via maleimide-cysteine chemistry.
• the decapping was done by first reducing (60 min, room temperature) antibodies with 3 Ox molar excess of Tris(2- carboxyethyl)phosphine hydrochloride (TCEP) followed by buffer exchange into 20 mM Tris- HC1 pH 7.5, 100 mMNaCl to remove the cysteine cap and TCEP.
• TCEP Tris(2- carboxyethyl)phosphine hydrochloride
• the reduced antibodies were oxidized (60 min, room temperature) with 15x molar excess of dehydro-ascorbic acid (dHAA) followed by buffer exchange into 20 mM Tris pH 7.5, 100 mM NaCl to remove the oxidizing reagent.
• dHAA dehydro-ascorbic acid
• the conjugation process was done by mixing antibodies at final concentration of 2.4 mg/mL (or -20 pM) with 5x molar excess of the biopolymer OG1802 at pH 8.5, 50 mM NaCl, 2 mM EDTA. The conjugation process took place at 2-8 °C for a period of 2 to 3 days.
• Conjugated antibodies were purified from unconjugated material by cation exchange chromatography (CEX) with Poros XS resin using a step gradient of increasing salt concentrations (0-500 mM NaCl) in buffer containing 20 mM sodium acetate pH 5.5. The fractions containing the bioconjugate antibodies were pooled and buffer exchanged into PBS. The final yield ranged from 35% to 50%.
• SEC-MALS SEC-MALS analysis
• Table 5.1 SEC-MALS was carried on by injecting 10 pg (protein mass) at flow rate 1 mL/min into a size exclusion chromatography column (Shodex-SB806M-HQ) preequilibrated with PBS, in line with a light scattering (miniDAWN TREOS) and differential refractive index (Optilab T-rEX) detectors (Wyatt Technology). Data and protein conjugate analysis was done in ASTRA 7 (Wyatt Technology).
• H2-Opt assays (as described in example 3) were carried on to evaluate the effect of biopolymer on the antibody potency.
• FIG. 6B and Table 5.2 show OG1802 had no impact on antibody potency except for molecules aHTRAl_42 and aHTRAl_65.
• Binding kinetics were determined by measuring the binding of HTRA1 to protein A purified aHTRAl 36 and grafted hu aHTRAl 36 (Table 6.3) antibodies captured on a CM5 chip coated with anti -human IgG (as described in Example 1, kinetic screen). 5 concentrations (300, 100, 33.3, 11.1, 3.7 nM) of OG2161 (HTRA1) were flowed over the captured antibodies for 30 seconds at a flow rate of 30 pL/min and dissociated for 180 seconds in a multi and single cycle kinetics Biacore assay formats. Data analyses were performed using BIAevaluation software (GE). All sensorgrams were double reference-subtracted and fitted using a 1 : 1 Langmuir binding model. Table 6.4 shows grafted antibody presented diminished affinity in comparison to the native rat aHTRAl_36 molecule.
• a screening to optimize affinity of the grafted antibody was carried on by introducing changes to the CDRs and the framework. The latter was based on sequence similarity to heavy chain germlines IGHV3-30, IGHV3-NL and light chain germlines IGKV1- 16, IGKV1-39. A total of 10 heavy (Table 6.5) and 8 light (Table 6.6) chain variants were generated and 48 combinations were evaluated.
• HTRA1 binding was evaluated with a kinetics assay, in which 1 : 10 or 1 :20 dilutions of supernatant from transiently transfected Expi293 cultures were tested for binding to HTRA1 using a CM5 chip coated with anti-human IgG using 5 concentrations of OG2161 (HTRA1, 150, 50, 16.67, 5.56, 1.85 nM) as described in Example 1, kinetic screening section.
• Table 6.7 shows hu_aHTRAl_36_45 variant showed the best affinity recovery, therefore it was selected as an initial template for further affinity optimization by phage display.
• Example 7 Affinity maturation of humanized anti-HTRAl by phage display
• aHTRAl 36 45 a phage display Fab library with soft mutagenesis in all heavy (HCDR) and light (LCDR) CDRs was constructed and panned. Enriched outputs were screened by high throughput periplasmic extracts (PPE) ELISA, followed by kinetics measurements. Point mutations with improved affinity in individual clones were later combined and reformatted into full-length IgG. Antibodies were characterized for their binding affinity, expression level, stability, and potency.
• Fab library design and construction The Fab fragment of hu_aHTRAl_36_l was cloned into pCDisplay4_HV phagemid vector, which was engineered by replacing HA tag on pCDisplay4 (Cat. # VPT4024, Creative-bio) with V5 tag.
• the vector backbone was generated from hu_aHTRAl_36_l pCDisplay4 (pLL45) plasmid, by digesting out VL-CL-VH fragment with Sall and w'SI (FIG. 7A).
• the initial Fab library (GEN 1) was designed to target each amino acid position along all six CDRs defined in Table 7.1. It contained single point mutations (all residues, except Cys, Asn, and Met), plus limited dual mutations at one or two CDRs for the heavy (VH) and light (VL) chain variants. Those were precisely synthesized as oligo pools (Twist biosciences), which avoided stop codons and liability hot spots. The library diversity was approximately one million based on various combinations of heavy and light chains.
• VL and VH were amplified from the oligo pools and assembled with vector backbone and CL-pelB fragment (FIGS. 7B, 7C) by a HiFi assembly reaction according to supplier protocol (New England Biolabs).
• the resulting library was desalted and electroporated into E. coli TGI cells (Cat. # 60502-2, Lucigen), yielding ⁇ 5X10 7 transformants.
• Sequencing pool revealed that 71% of Fab genes in library Genl were unique and in the correct frame.
• Phage display panning selection The phage library was selected by insolution panning (Lou & Marks, 2010). The library was panned by using biotinylated OG2161 (HTRA1) and Dynabeads M-280 streptavidin (Cat# 11205D, Thermo). The phage library was blocked with 2% bovine serum albumin (BSA) in PBS, followed by depletion on Dynabeads or immobilized bio-OG2165 (HTRA3) to decrease non-specific binding and cross-reactivity to HTRA3 protein. To select high affinity binders, antigen concentrations were systematically reduced after each round, while the number of washing steps and the incubation time were increased.
• BSA bovine serum albumin
• Panning rounds were performed as outlined in Table 7.2.
• 100 nM biotinylated OG2161 was immobilized on the beads and then incubated with a depleted phage library.
• the beads were washed by PBST solution (PBST/0.2%BSA) for three times and PBS solution (PBST/0.2%BSA) for three times, with 5 minutes duration for each wash step.
• the bound phages were eluted with 100 mM TEA for 10 minutes and neutralized with 1 M Tris-HCl pH 7.5.
• the freshly grown mid log phase TGI cells were infected with eluted phage pool and plated on 2YTGC plates, then incubated at 32 °C overnight.
• the panning output was packaged into phage particles for further rounds of selection.
• bio-OG2161 was incubated with the phage pools first and then phage-bound bio-OG2161 was captured on the depleted beads.
• Five rounds of panning were performed in total with increasing stringencies.
• three arms were tested in parallel, two of which incorporated a large excess of unlabeled OG2161 to compete with the bio- OG2161 binding.
• one arm was screened at 37 °C, which would favor the binders with faster on rate.
• phage pools from each round of panning outputs were analyzed by ELISA.
• Pre-selection phage library phage preparations from parental hu_aHTRAl_36_45 (pLL44) served as the control.
• a Nunc 96-well MaxiSorp plate was coated with 50 pL of 2 pg/mL of HTRA1 (OG2161) or HTRA3 (OG2165) at 4 °C overnight. The plate was blocked with 2% BSA in PBS for 1 hour.
• the phage preparations were diluted to 10 12 pfu/mL in blocking buffer.
• Binding affinity of unique anti-HTRAl clones was determined using a Biacore T200 (Cytiva) at 25 °C. A series S sensor CM5 chip was activated with EDC and NHS according to the supplier’s instruction (Cytiva).
• Anti-V5 tag antibody (Cat. # ab27671, Abeam) was diluted into 10 mM sodium acetate (pH 5) to a concentration of 50 pg/mL and coupled to the activated chip surface at flow rate of 10 pL/min for 7 minutes, followed by quenching with 1 M ethanolamine. The immobilization of anti-V5 antibody achieved 4000-7900 RU on the surface.
• Binding kinetics The binding kinetics was measured on both CM5 anti- HuIgG chip and CM5 anti-Fab-VHHs chip by Biacore T200 at 25 °C.
• CM5 anti-HuIgG chip the preparation was same as described in Example 2.
• Purified antibodies (1 pg/mL) were captured at 10 pL/min flow rate for 25 seconds.
• 3-fold serial dilutions of HTRA1 (OG2161) in HBS-EP+ buffer were injected at increasing concentrations (0.56 nM to 45 nM) at 30 pL/min for 30 seconds in a single kinetic cycle and dissociated for 180 seconds.
• the sensor chip surface was regenerated by injecting 3 M MgCh at a flow rate of 30 pL/min for 60 seconds.
• the kinetic data was analyzed as previously described.
• Thermo at 30 pg/mL was injected at 5 pL/min flow rate for 200 seconds to achieve 2200-2600 RU.
• the sensor chip surface was regenerated by injecting 1.33M NaCl/IgG elution buffer (Cat. # 21004, Pierce/Thermo,) for 60 seconds at a flow rate of 50 pL/min.
• the kinetic data was analyzed as previously described.
• top variants Potency of top variants in FRET based H2-Opt cleavage assay.
• Top six antibody variants (HC4/LC1, HC1/LC1, HC7/LC1, HC4/LC3, HC4/LC4, HC1/LC3, HC7/LC4) were selected from the kinetics screening and the inhibition of HTRA1 enzyme activity were evaluated in an H2-Opt assay as described in Example 3.
• FIGS. 10A-10C show the antibodies have comparable ICso under testing conditions.
• Fab library Gen2 design and construction According to the kinetics and potency screening of library Genl, two heavy (HC4 and HC7) and two light (LC1 and LC4) chains were selected. To further increase the affinity, a second phage-display Fab library (Gen2) was designed based on these four chains. Besides single amino acid scanning, double mutations were also introduced, and the theoretical library size was ⁇ 5x 10 6 . The library construction and phage library packaging were performed as described in Example 7.1. The library size in E. coli TGI cells was 6.2xl0 7 and the phage library was 5.3xl0 13 pfu in total.
• phage pool ELISA was performed with all panning outputs as described in Example 7.1, except the phage preparations were diluted to 10 11 pfu/mL. The results are presented in FIGS 11A and 11B. HTRA1 binding signal systematically increased from R1 to R3. Round 4 selection did not result in further enrichment. The optimization of the blocking and binding buffer in Round 3 opt A and opt B did not increase the phage pool binding signal.
• PPEs of unique clones were selected for Fab affinity measurement using Biacore T200 at 25 °C.
• Human Fab capture kit (Cat. # 28-9583-25, Cytiva) was used for Fab capture.
• Anti -Hu-Fab IgG was immobilized on CM5 chip according to the instruction from supplier, achieving 7800-9200 RU on the surface.
• FIGS. 12A- 12C show the affinity improvement by phage display library Genl and Gen2 screening.
• Tagg and hydrodynamic radius of each IgG are reported in Table 7.21. Compared to the original rat aHTRAl 36 antibody, Taggs of the top clones were improved from 62 °C to 74 °C. Overall, light chain LC11 and heavy chains HC6, HC13 and HC14 yielded higher stability (Table 7.21).
• SYPRO Orange (Cat. # S5692, Sigma) binds to hydrophobic regions of proteins as they denature which results in increased fluorescence emission. Thermal shift assays were carried out with SYPRO Orange dye and the fluorescence emission was monitored with an RT-PCR machine (Quant studio 5, Applied Biosystems). 5 x SYPRO Orange was used to label 0.1 mg/mL antibodies in PBS in 20 pL reactions in 96- well plates. A sample without protein was used to examine SYPRO Orange dye background in PBS. Samples were equilibrated at 25 °C for 2 minutes, then temperature ramped to 99 °C at 0.05 °C/s, followed by a holding step at 99 °C for 2 minutes.
• Tm DI Protein Thermal Shift software 1.4
• Trimeric and monomeric HTRA1 binding test Native (or wild-type) HTRA1 is most abundant in trimeric format. This serine protease has a trimer-dependent activation cascade (Uemura, et al., 2019). The binding preference of five affinity matured variants from library Genl and Gen2 to the wild-type (OG2161) and a monomeric HTRA1 mutant (OG2241) (Uemura et al., 2019) were characterized by Biacore T200 at 25 °C. CM5 anti-HuIgG chip with immobilization level of 7600-8800 RU was used to capture the purified IgGs (1 pg/mL).
• Trimeric or monomeric HTRA1 was 3-fold serial diluted in HBS-EP+ buffer from 100 nM to 1.2 nM. HTRA1 dilutions were sequentially injected over the IgG captured surface at 30 pL/min for 30 seconds and dissociated for 1800 seconds. The sensor chip surface was regenerated between each cycle. Data was analyzed as previously described.
• FIGS. 13A and 13B and Table 7.23 show the tested antibodies have comparable affinity for the wild type HTRA1 (OG2161). aHTRAl_36 (rat) and HC6 variants have preferential binding to HTRA1 in the trimeric form, in contrast to variant HC13 heavy chain sequence.
• the expression yield in substitution variants was measured by Biacore T200 at 25 °C as previously described using CM5 anti-HuIgG Chip.
• the captured antibody ligand level was measured from culture supernatants after 1 :20 dilution.
• the variants with ligand level ⁇ 50 RU were not selected for purification, except HC23/LC22 and HC29/LC21.
• the kinetics screening of purified variants was performed on CM5 anti-HuIgG chip with immobilization level of 8400-9100 RU.
• the purified IgGs (1 pg/mL) were captured on the surface at 10 pL/min for 25 seconds.
• HTRA1 3-fold serial diluted HTRA1 (OG2161) in HBS-EP+ buffer was injected at 30 pL/min for 30 seconds and dissociated for 1800 seconds. The kinetics data were analyzed as previously described.
• Trimeric and monomeric HTRA1 binding test The variants with HC17 on HC6 background, HC20 and HC21 on HC13/HC14 background, all pared with LC21, were tested for binding to the wild type trimeric HTRA1 (OG2161) and the monomeric HTRA1 mutant (OG2241) by Biacore T200 at 25 °C as described in Example 7.3.
• Table 8.5 shows HC17/LC21, HC20/LC21 and HC21/LC21 have comparable affinity for the wild type HTRA1 (OG2161).
• HC17/LC21 have very low binding level (Rmax) to HTRA1 monomeric mutant, in contrast to HC20/LC21 and HC21/LC21.
• Trimeric HTRA1 is the active form of the enzyme (Truebestein et al., 2011). Without being bound by theory, preferential binding to the active HTRA1 could improve the potency of the antibody.
• CHO cell expression and binding affinity To evaluate the IgG expression and production, four clones (HC37/LC21, HC39/LC21, HC40/LC21 and HC41/LC21) were transfected into 125 mL of ExpiCHO cells. The cultures were harvested on the fifth day and purified through a protein A column followed by a polishing step with a CEX column using standard protocols.
• FIG. 14A is a sequence alignment of regions containing the catalytic domain, showing the similarity of HTRA1 and its homologs.
• the assays were performed on Biacore T200 at 25 °C according to Example 8.
• CM5 anti-HuIgG chip with immobilization level at 8000-8600 RU was used to capture tested antibodies (1 pg/mL).
• HTRA1 OG2161
• HTRA2 Cat.
• HTRA3 (Cat. # abl34450, Abeam) and HTRA4 (Cat. # abl34444, Abeam) were 3-fold serial diluted in HBS-EP+ buffer from 45 nM to 0.56 nM.
• the increasing concentrations of HTRA1 and its homologues were flowed at 30 pL/min for 30 seconds in single kinetic method and dissociated for 1800 seconds. All sensorgrams were double subtracted and fitted with a 1 : 1 Langmuir binding model.
• FIGS. 14B and 14C show the tested antibodies bound uniquely to HTRA1.
• BVP assay BVP (Cat# E3001, MEDNA) stock was diluted to 1 :200 with 50 mM sodium carbonate (pH 9.6) according to the supplier’s instruction.
• 96-well high binding plate Cat. # 9018, Corning
• the unbound BVPs were removed, and the remaining steps were performed at room temperature.
• the plate was blocked with 150 pL of blocking buffer (0.5% BSA in PBS) for 1 hour, followed by washing three times with PBS.
• 50 pL of 1 pM antibodies in blocking buffer were added to the wells and incubated for 1 hour, followed by washing six times with PBS.
• HRP Goat Anti-human IgG Fc
• cardiolipin (10 pg/mL, Cat. # C0563, Sigma), KLH (5 pg/mL, Cat. # H8283, Sigma), LPS (10 pg/mL, Cat. # L2637, Sigma), ssDNA (1 pg/mL, by heating dsDNA at 95 °C for 30 minutes), dsDNA (1 pg/mL, Cat. # D3664, Sigma), and insulin (5 pg/mL, Cat. # 19278, Sigma) were coated onto a 96-well high binding plate (Cat. # 9018, Corning) at 50 pL per well overnight at 4 °C.
• FIGS. 15A-15D The results are presented in FIGS. 15A-15D.
• A_S574 was a positive control for polyreactivity (Mouquet et al., 2010; Irimia et al., 2016; Haynes et al., 2005).
• OG1950 was used as a negative control in these assays (OG1950 is disclosed in U.S. App. Pub. No. 2017/0190766, which is incorporated herein by reference in its entirety).
• Clones HC41/LC21 and HC17/LC21 had low binding to tested antigen panel and BVP particles.
• FIGS. 16A-16C show polymer conjugation of HC17/LC21, HC40/LC21 and HC41/LC21 does not affect the antibody potency.
• the mass of the complexes was approximately 300 kDa, as expected for a complex of 1 HTRA1 trimer and 1 antibody.
• FIG. 17B shows the 1 : 1 stoichiometry is maintained after polymer conjugation, though molar mass of complex is lower than anticipated (1,100 kDa). Without being bound by theory, this could be due to a mixture of free antibody conjugate with antibody conjugate HTRA1 complex that is not resolved by the size exclusion column and limitations to accurately measure the complex molar mass using the protein conjugation analysis module in ASTRA.
• aHTRAl antibodies HC40/LC21 and HC41/LC21
• HTRA1 S328A
• antibody and HTRA1 S328A
• Individual antibodies or HTRA1 (S328A) sample was prepared at a concentration of 30 nM. Samples were 3-fold diluted before loading onto One MP mass photometer. The landing events of single particles on the coverslip were recorded with AcquireMP software (Refeyn Ltd) and analyzed using DiscoverMP software (Refeyn Ltd).
• FIGS. 18A-18C show tested antibodies and HTRA1 samples appear to be 150 kDa close to their expected molecular weights (HTRA1 trimer 150kD and aHTRAl 146 kD).
• FIGS. 18A-18C show tested antibodies and HTRA1 samples appear to be 150 kDa close to their expected molecular weights (HTRA1 trimer 150kD and aHTRAl 146 kD).
• 18D and 18E show antibody and HTRA1 mixtures present two major peaks, one peak at around 150 kDa from unbound antibodies and/or HTRA1, and the other peak at approximately 300 kDa, which agrees with the stoichiometry of one IgG bound to one HTRA1 trimer.
• azlactone beads coated with HTRA1 were used to capture a portion of free HC41/LC21 or its bioconjugate from an equilibrated sample with titrated HTRA1 (OG2704). Captured HC41/LC21 or its bioconjugate was detected with a fluorescently labeled anti-Human IgG (Jackson Immuno Research). The fluorescent signal was converted to a voltage signal that is directly proportional to the amount of free antibody in the equilibrated samples.
• azlactone-OG2704 beads were also used as the capture reagent for HC41/LC21 kinetic experiments. 100 pM HC41/LC21 was incubated with 125 pM HTRA1 (OG2704). The amount of free HC41/LC21 in the sample was measured pre-equilibrium, yielding data points that monitor the decrease in free HC41/LC21 as the sample moves toward equilibrium over time.
• the KinExA Pro software performs a least squares analysis on the measured data to fit optimal solutions for the KD and the activity of the constant binding partner to a curve representative of a 1 :1 reversible bi-molecular interaction.
• the results from this assay show that HC41/LC21 and its bioconjugate bind to HTRA1 with double digit pM affinity at 37 °C (Table 9.4).
• Loop A peptide (FIG. 26A), which is the same as parental aHTRAl_36.
• Human HTRA1-PD (OG2138), mouse HTRA1-PD (OG3349), ratHTRAl-PD (OG3350) and dogHTRAl-PD (OG3351) were 3-fold serial diluted in HBS-EP+ buffer from 45 nM to 0.56 nM.
• the increasing concentrations of HTRA1 PD proteins were flowed at 30 pL/min for 30 seconds in single kinetic method and dissociated for 1800 seconds. All sensorgrams were double subtracted and fitted with a 1 : 1 Langmuir binding model.
• Table 11.1 shows the tested antibodies tightly bound to human and dog HTRA1.
• the binding to mouse and rat HTRA1 were significantly decreased with lower binding signal and fast dissociation rate (FIGS. 28B and 28C).
• There are three conserved amino acids substitutions in both mouse and rat HTRA1 PD which results in a significant change in the binding affinity. This suggests those amino acids on human HTRA1 are important for the selected antibody binding.
• Hotzel I., Theil , F.-P., Bernstein , L. J., Prabhu , S., Deng , R., Quintana , L., Lutman , J., Sibia , R., Chan , P., Bumbaca , D., et al. (2014).
• Irimia , A., Sarkar , A., Stanfield , R., and Wilson I. (2016). Crystallographic Identification of Lipid as an Integral Component of the Epitope of HIV Broadly Neutralizing Antibody 4E10. Immunity 44, 21-31.
• HTRA1 an age-related macular degeneration protease, processes extracellular matrix proteins EFEMP1 and TSP1. Aging. Cell. 17, el2710-. Lou , J., and Marks , J. D. (2010). Affinity Maturation by Chain Shuffling and Site Directed Mutagenesis. 377-396. Lu , Z., Lin , V., May , A., Che , B., Xiao , X., Shaw , D. H., Su , F., Wang , Z , Du , H, and Shaw , P.
• HTRA1 synergizes with oxidized phospholipids in promoting inflammation and macrophage infiltration essential for ocular VEGF expression.

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