BR122024004106A2 - USE OF AN ANTIBODY THAT BINDS TO AN ACTIVE PLASMA KALYCREIN (PKAL) AND DOES NOT BIND TO HUMAN PREKALYKREIN IN THE TREATMENT OF RETINAL DISEASES - Google Patents

Abstract

The use of antibodies that specifically bind to an active plasma kallikrein (PKal), but not human prekallikrein, in the manufacture of a drug to treat a retinal disease such as diabetic macular edema (DME), age-related macular degeneration is described. (AMD), retinal vein occlusion (RVO), uveitis, endophthalmitis, or polypoidal choroidal vasculopathy (PCV). Said antibody inhibits the activity of active PKal by at least 80%, has an apparent Ki (Ki,app) lower than 1 nM and has a binding affinity (KD) for active PKal of less than 10-6 M, being formulated into a composition for intravitreal injection.

Description

CROSS REFERENCE TO RELATED ORDERS

[0001] This application claims the benefit of American priority No. US 61/971,170, filed on March 27, 2014, which is incorporated by reference in this document in its entirety.

BASICS OF THE INVENTION

[0002] Retinal diseases affect the area of the retina that serves as central vision. While many retinal diseases share common symptoms, each has unique characteristics.

[0003] Diabetic macular edema (DME) is a swelling of the macula caused by leaking retinal blood vessels that occurs in patients with diabetes. DME is the leading cause of vision loss in people with diabetic retinopathy. People with diabetes have a 10 percent risk of developing DME in their lifetime. DME affects up to 30% of people who have had diabetes for more than 20 years. If left untreated, DME can result in moderate to severe vision loss.

SUMMARY OF THE INVENTION

[0004] The present disclosure is based, in part, on studies showing that animal models of diabetic macular edema and retinal diseases can be treated with DX-2944, a Fab antibody that binds to active PKal.

[0005] Some aspects of the disclosure relate to a method for treating a retinal disease, such as diabetic macular edema (DME), age-related macular degeneration, retinal vein occlusion, uveitis, endophthalmitis, polypoidal choroidal vasculopathy (PCV) or any other retinal disease presenting with macular edema in a subject, the method comprising administering (e.g., via intravitreal injection, intraocular injection, or subcutaneous injection) an effective amount of a composition comprising an antibody that specifically targets binds to active plasma kallikrein (PKal) in a subject in need of it.

[0006] In some embodiments, the antibody does not bind to human prekallikrein. In some embodiments, the antibody specifically binds to the catalytic domain of human PKal. In some embodiments, the antibody interacts with one or more amino acid residues in active human PKal and inhibits its activity by at least 50%. The one or more amino acid residues may be one or more of V410, L412, T413, A414, Q415, R416, L418, C419, H434, C435, F436, D437, G438, L439, W445, Y475, K476, V477, S478 , E479, G480, D483, F524, E527, K528, Y552, D554, Y555, A564, D572, A573, C574, K575, G576, S578, T596, S597, W598, G599, E600, G601, C602, A603, 604 , Q607, P608, G609, V610, and Y611. In some embodiments, the antibody binds to an epitope comprising the segment of V410-C419, H434-L439, Y475-G480, F524-K528, Y552-Y555, D572-S578, T596-R604, or Q607-Y611.

[0007] In some embodiments, the antibody inhibits the activity of active PKal by at least 80%. In some embodiments, the antibody has an apparent Ki (Ki,app) of less than about 1 nM. In some embodiments, the antibody has a Ki, app of less than 0.1 nM. In some embodiments, the antibody has a Ki,app of less than about 0.05 nM. In some embodiments, the antibody has a binding affinity (KD) for active PKal of less than 10-6 M. In some embodiments, the antibody preferentially binds active PKal over an active PKal mutant that contains one or more mutations at positions R551, Q553, Y555, T558 and R560.

[0008] In some embodiments, the antibody comprises a heavy chain variable region that comprises a complementarity determining region 1 (HC CDR1), a complementarity determining region 2 (HC CDR2), and a complementarity determining region 3 (HC CDR3). , and where the HC CDR3 comprises the themes X99R100X101G102X103P104R105X106X107X108X109X110X111, where: X99 is R or Q; X101 is T, I, R, S or P; X103 is V, I or L; X106 is R or W; X107 is D or N; X108 is A, S, D, E or V; X109 is F or L; X110 is D, E, or N, and X111 is I, N, M, or S (SEQ ID NO:15). In some embodiments, X99 is Q and X101 is I, R, S, or P. In some embodiments, X106 is W and X111 is N, M, or S. In some embodiments, X101 is I, or L. In some embodiments, X101 is I and X103 is I or L. In some embodiments, X103 is I or L, and X110 is D, E, or N. In some embodiments, the heavy chain variable region includes H31 in HC CDR1. In some embodiments, the heavy chain variable region includes F27, F29, or both in the framework region 1 (FR1).

[0009] In some embodiments, the antibody additionally comprises a light chain variable region comprising a complementarity determining region 1 (LC CDR1), a complementarity determining region 2 (LC CDR2) and a complementarity determining region 3 (LC CDR3 ). In some embodiments, the LC CDR2 includes K50, L54, E55, S56, or a combination thereof. In some embodiments, the light chain variable region additionally includes G57 in framework region 3 (FR3). In some embodiments, the light chain variable includes N45 or K45 in the framework 2 (FR2) region.

[00010] In some embodiments, the antibody binds to the same epitope as DX-2944, or competes for binding to active PKal with DX-2944. Such an antibody may comprise a heavy chain (HC) CDR1, HC CDR2, and HC CDR3 of DX-2930 and a light chain (LC) CDR1, LC CDR2, and LC CDR3 of DX-2930. In some embodiments, the antibody comprises the HC variable domain of DX-2930 (SEQ ID NO:3) and the LC variable domain of DX-2930 (SEQ ID NO:4). In one example, the antibody is DX-2944.

[00011] In any of the methods described in this document, the antibody can be a complete antibody or an antigen-binding fragment thereof. In some embodiments, the antibody is a Fab. In some embodiments, the antibody is a human antibody or a humanized antibody.

[00012] Still within the scope of the present disclosure are (i) pharmaceutical compositions for use in the treatment of retinal diseases (e.g., DME, AMD, RVO, uveitis, endophthalmitis or PCV), the compositions comprising one or more antibodies binding to PKal active. (e.g., those described herein) and a pharmaceutically acceptable carrier, and (ii) uses of such compositions or antibodies for producing a medicament for use in treating retinal diseases.

[00013] Details of one or more embodiments of the invention are set out in the attached figures and in the description below. Other features or advantages of the present invention will be apparent from the description, the drawings, and also from the claims.

BRIEF DESCRIPTION OF FIGURES

[00014] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood with reference to one or more of these drawings in combination with the detailed description of specific embodiments presented in this document.

[00015] Figure 1A shows a reduction in the fluorescein angiography signal in animals treated with an intraocular injection of an anti-VEGF antibody (n=5, p<0.05 by t-test).

[00016] Figure 1B shows similar reduction in signal in animals treated with DX-2944 (n = 3 for vehicle group, n = 4 for test article group, p < 0.05 by t-test).

[00017] Figure 2A shows an exemplary photograph after laser-induced CNV treatment of a brown Norway rat eye treated by NaCl vehicle.

[00018] Figure 2B shows an exemplary photograph after laser-induced CNV of a brown Norway rat eye treated by DX-2944.

[00019] Figure 3 shows the amino acid sequence of the heavy chain variable region (VH) and light chain variable region (VL) of a parental antibody, M0162-A04, from which DX2930 was derived, and its alignment with VH corresponding germinated and VL genes as indicated. Variations in M0162-A04, compared to the germinated sequences, are indicated (bold). The sequences in Figure 3, from top to bottom, correspond to SEQ ID NOs: 16 to 18 (V light gene) and SEQ ID NOs: 19 to 21.

[00020] Figure 4 shows an amino acid sequence of the human plasma kallikrein catalytic domain (residues 391 to 638 of complete human PKal) (SEQ ID NO:22). Bold and underlined residues refer to those that are involved in the interaction with the Fab fragment of DX2930, as identified by the crystal structure discussed in Example 2 below.

[00021] Figures 5A to 5D are a series of graphs showing the apparent Ki (Ki, app) of a number of mutant antibodies derived from M0162-A04 against human PKal, including: X135-A01 and X135-A03 (figure 5A), M162-A04 and X133-B02 (figure 5B), X133-D06 and X133-F10 (figure 5C), and X133-G05 and M199-A08 (figure 5D).

[00022] Figure 6 is a series of graphs showing the apparent Ki (Ki, app) of mutant X115-F02 (see table 2 below) against wild-type PKal and a number of mutant PKal

[00023] Figures 7A to 7B show the amino acid sequences of a number of mutant PKal (catalytic domain), which were produced in Pichia cells. The sequences, from top to bottom, correspond to SEQ ID NOs: 23 to 27.

[00024] Figure 8 shows the effect of DX-2944 compared to the anti-VEGF positive control on CNV laser in Norwegian brown rats on day 15. The observed reduction in the fluorescein angiography signal in animals treated with an intraocular injection of an antibody anti-VEGF was comparable to the reduction in signal observed with animals treated with DX 2944 (n =7, p<0.05 by t-test).

[00025] Figure 9 shows the effect of DX-2944 compared to the anti-VEGF positive control on laser CNV in Norwegian brown rats on day 22. The observed reduction in the fluorescein angiography signal in animals treated with an intraocular injection of an antibody anti-VEGF was comparable to the reduction in signal observed with animals treated with DX 2944 (n =7, p<0.05 by t-test).

DETAILED DESCRIPTION OF THE INVENTION

[00026] Millions of people suffer from varying degrees of vision loss due to retinal diseases, in which the delicate layer of tissue lining the inner fundus of the eye is damaged, reducing its ability to send light signals to the brain. Retinal diseases can be caused by several factors, including genetic and age-related factors and other diseases, such as diabetes. Diabetic retinopathy is a condition that occurs in people who have diabetes, both type I and type II diabetes. Diabetic retinopathy is believed to be the result of hyperglycemia-induced damage to the retinal microvasculature. This damage causes the retinal vessels to become more permeable. In some cases, damaged blood vessels leak fluids, proteins and/or lipids into the macula, which causes swelling and thickening of the macula. The swelling and thickening of the macula is referred to as diabetic macular edema (DME). Symptoms of DME include blurred vision, distorted vision, and spots in the field of vision (sometimes referred to as "floaters").

[00027] The standard treatment for DME is laser photocoagulation. This treatment has undesirable side effects, including partial loss of peripheral vision and/or night vision.

[00028] The disclosure is based, in part, on a study showing that antibodies that bind to active plasma kallikrein (PKal) are therapeutically effective in an animal model of retinal diseases, such as DME, AMD, RVO, uveitis, endophthalmitis or PCV. Thus, in some aspects, the disclosure relates to compositions and methods for treating a retinal disease, such as DME, AMD, RVO, uveitis, endophthalmitis or PCV using antibodies capable of binding to active PKal (e.g., PKal active human).

Binding of Antibodies to Active PKal

[00029] The present disclosure provides isolated antibodies that specifically bind active PKal, for example, the catalytic domain of PKal. In some embodiments, the antibody described herein does not bind to prekallikrein (e.g., human prekallikrein).

[00030] Plasma kallikrein is a serine protease component of the contact system (Sainz I.M. et al., Thromb Haemost 98, 77 83, 2007). The contact system is activated by either factor XIIa upon exposure to external or negatively charged surfaces or on endothelial cell surfaces by prolylcarboxypeptidases (Sainz I.M. et al., Thromb Haemost 98, 77-83, 2007). Activation of plasma kallikrein amplifies intrinsic coagulation through its feedback activation of factor XII and accentuates inflammation through production of the pro-inflammatory nonapeptide bradykinin. As the primary kininogenase in the circulation, plasma kallikrein is largely responsible for the generation of bradykinin in the vasculature.

[00031] Exemplary plasma kallikrein sequences may include human, mouse or rat plasma kallikrein amino acid sequences, a sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to one of these sequences, or a fragment thereof, for example, a sequence provided below.

[00032] An exemplary sequence of a mature human plasma kallikrein is shown below (see, for example, Tang et al. (2005) Expression, Crystallization, and Three-dimensional Structure of the Catalytic Domain of Human Plasma Kallikrein. J of Biol Chem 280(49): 41077-41089, which is incorporated herein by reference). This exemplary sequence comprises a mutation (S484; in bold) in order to facilitate the production of a homogeneous product.

[00033] GCLTQLYENAFFRGGDVASMYTPNAQYCQMRCTFHPRC LLFSFLPASSINDMEKRFGCFLKDSVTGTLPKVHRTGAVSGHSLKQCGHQI SACHRDIYKGVDMRGVNFNVSKVSSVEECQKRCTSNIRCQFFSYATQTFH KAEYRNNCLLKYSPGGTPTAIKVLSNVESGFSLKPCALSEIGCHMNIFQHL VARVLTPDAFVCRTICTYHPNCLFFTFYTNVWKIESQRNVCLLKT SESGTPSSSTPQENTISGYSLLTCKRTLPEPCHSKIYPGVDFGGEELNVTF VKGVNVCQETCTKMIRCQFFTYSLLPEDCKEEKCKCFLRLSMDGSPTRIA YGTQGSSGYSLRLCNTGDNSVCTTKTSTR/IVGGTNSSWGEWPWQVSLQ VKLTAQRHLCGGS LIGHQWVLTAAHCFDGLPLQDVWRIYSGILNLSDITKD TPFSQIKEIIIHQNYKVSEGNHDIALIKLQAPLNYTEFQKPISLPSKGDTSTIYT NCWVTGWGFSKEKGEIQNILQKVNIPLVTNEECQKRYQDYKITQRMVCAG YKEGGKDACKGDSGGPLVCKHNGMWRLVGITSWGEGCARREQPGVYTK VA EYMDWILEKTQSSDGKAQMQSPA (SEQ ID NO:11)

[00034] Factor XIIa activates prekallikrein by cleaving the polypeptide sequence at a single site (between Arg371-Ile372, cleavage site marked by "/" in the sequence above) to generate active plasma kallikrein, which then consists of two linked polypeptides to disulfide; a heavy chain of approximately 52 kDa and a catalytic domain of approximately 34 kDa [Colman and Schmaier, (1997) "Contact System: A Vascular Biology Modulator With Anticoagulant, Profibrinolytic, Antiadhesive, and Proinflammatory Attributes” Blood, 90, 3819-3843] .

[00035] Exemplary amino acid sequences of human, mouse and rat prekallikrein (including signal peptides) are illustrated below. The sequences of prekallikrein are the same as plasma kallikrein, except that active plasma kallikrein (pKal) has the single polypeptide chain cleaved at a single position (indicated by the "/") to generate two chains. The sequences provided below are complete sequences that include signal sequences. Upon secretion from the expressing cell, the signal sequences are expected to be removed.

Human plasma kallikrein (ADHERENCE: NP_000883.2)

[00036] >gi|78191798|ref|NP_000883.2| plasma kallikrein B1 precursor [Homo sapiens] MILFKQATYFISLFATVSCGCLTQLYENAFFRGGDVASMYTPNAQYC QMRCTFHPRCLLFSFLPASIND MEKRFGCFLKDSVTGTLPKVHRTGAVSGHSLKQCGHQISACHRDIY KGVDMRGVNFNVSKVSSVEECQKR CTSNIRCQFFSYATQTFHKAEYRNNCLLKYSPGGTPTAIK VLSNVES GFSLKPCALSEIGCHMNIFQHLA FSDVDVARVLTPDAFVCRTICTYHPNCLFFTFYTNVWKIESQRNVCL LKTSESGTPSSSTPQENTISGYS LLTCKRTLPEPCHSKIYPGVDFGGEELNVTFVKGVNVCQETCTKMIR CQFFTYSLLPEDCKEEKCKCFLR LSMDGSPTRIAYGTQGSSGYSLRLCNTGDNSVCTTKTS TR/IVGGTN SSWGEWPWQVSLQVKLTAQRHLCG GSLIGHQWVLTAAHCFDGLPLQDVWRIYSGILNLSDITKDTPFSQIKE IIIHQNYKVSEGNHDIALIKLQ APLNYTEFQKPICLPSKGDTSTIYTNCWVTGWGFSKEKGEIQNILQK VNIPLVTNEECQKRYQDYKITQR MVCAGYKEGGKDACKG DSGGPLVCKHNGMWRLVGITSWGEGCA RREQPGVYTKVAEYMDWILEKTQSSDG KAQMQSPA(SEQ ID NO:12) Mouse Plasma Kallikrein (ADHESSION: NP_032481.1)

[00037] >gi|6680584|ref|NP_032481.1| kallikrein B, plasma 1 [Mus musculus] MILFNRVGYFVSLFATVSCGCMTQLYKNTFFRGGDLAAIYTPDAQY CQKMCTFHPRCLLFSLAVTPPKE TNKRFGCFMKESITGTLPRIHRTGAISGHSLKQCGHQISACHRDIYK GLDMRGSNFNISKTDNIEECQKL CTNNFHCQFFTYATSAFYRPEYRKKCLLKHSASGTPTSIKSA DNLVS GFSLKSCALSEIGCPMDIFQHSA FADLNVSQVITPDAFVCRTICTFHPNCLFFTFYTNEWETESQRNVCF LKTSKSGRPSPPIPQENAISGYS LLTCRKTRPEPCHSKIYSGVDFEGEELNVTFVQGADVCQETCTKTIR CQFFIYSLLPQDCKEEGCKCSLR LSTDGSPTRITYGMQGSSGYSLRLCKLVDSPDCTKINAR/ MICAGYKEGGTDACKGDSGGPL VCKHSGRWQLVGITSWGEGCGR KDQPGVYTKVSEYMDWILEKTQSSDV RALETSSA(SEQ ID NO:14) Rat plasma kallikrein (ADHESION: NP_036857.2)

[00038] >gi|162138905|ref|NP_036857.2| kallikrein B, plasma 1 [Rattus norvegicus] MILFKQVGYFVSLFATVSCGCLSQLYANTFFRGGDLAAIYTPDAQHC QKMCTFHPRCLLFSFLAVSPTKE TDKRFGCFMKESITGTLPRIHRTGAISGHSLKQCGHQLSACHQDIYE GLDMRGSNFNISKTDSIEECQKL CTNNIHCQFFTYATKAFHRPEYRKSCLLKRSSSGTP TSIKPVDNLVS GFSLKSCALSEIGCPMDIFQHFA FADLNVSHVVTPDAFVCRTVCTFHPNCLFFTFYTNEWETESQRNVC FLKTSKSGRPSPPIIQENAVSGYS LFTCRKARPEPCHFKIYSGVAFEGEELNATFVQGADACQETCTKTIR CQFFTYSLLPQDCKAEGCKCSLR LSTDGSPTRITYEAQGSSGYSLRLCKVVESSDCTTKINAR /IVGGTNS SLGEWPWQVSLQVKLVSQNHMCG GSIIGRQWILTAAHCFDGIPYPDVWRIYGGILNLSEITNKTPFSSIKELII HQKYKMSEGSYDIALIKLQ TPLNYTEFQKPICLPSKADTNTIYTNCWVTGWGYTKERGETQNILQK ATIPLVPNEECQKKYRDYVITKQ MICAGYKEGGIDACKGDSGG PLVCKHSGRWQLVGITSWGEGCARK EQPGVYTKVAEYIDWILEKIQSSKE

[00039] RALETSPA(SEQ ID NO:13)

[00040] Antibodies can be used in the methods described in this document, for example, in a method of treating a retinal disease. The term "isolated antibody" used herein refers to an antibody substantially free of naturally associated molecules, that is, naturally associated molecules constituting at most 20% by dry weight of a preparation containing the antibody. Purity can be measured by any suitable method, for example column chromatography, polyacrylamide gel electrophoresis and HPLC. In some examples, the antibody disclosed herein specifically binds active PKal or an epitope thereof.

[00041] An antibody that "specifically binds" (used interchangeably herein) to a target or an epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is known to exhibit "specific binding" if it reacts or associates more frequently and rapidly, with greater duration and/or with greater affinity with a particular target antigen compared to alternative targets. An antibody "specifically binds" to a target antigen if it binds with greater affinity, avidity, readily, and/or longer duration than other substances. For example, an antibody that specifically (or preferentially) binds to an active human PKal or an epitope thereof, is an antibody that binds to this target antigen with greater affinity, avidity, more readily, and/or longer duration than binds to other antigens or other epitopes on the same antigen. It is also understood when reading this definition that, for example, an antibody that specifically binds to a first target antigen may or may not bind specifically or preferentially to a second target antigen. As such, "specific binding" or "preferred binding" does not necessarily require (although it may include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.

[00042] An antibody (used interchangeably in the plural form) is an immunoglobulin molecule capable of specifically binding to a target, such as carbohydrates, polynucleotides, lipids, polypeptides, etc., through at least one site of recognition of antigen located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses not only intact (e.g., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, Fv). , single chain (scFv), mutants thereof, fusion proteins comprising an antibody moiety, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule comprising an antigen recognition site of the required specificity, including antibody glycosylation variants, antibody amino acid sequence variants, and covalently modified antibodies. An antibody includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or a subclass thereof), and the antibody need not be of any particular class. Depending on the amino acid sequence of the antibody and its heavy chains, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of them can be divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains that correspond to different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[00043] The antibodies described in this document can also inhibit PKal activity. In some cases, the antibodies described herein can inhibit PKal activity by at least 50%, e.g., 60%, 70%, 80%, 90%, 95%, or greater. The inhibition constant (Ki) provides a measure of the potency of the inhibitor; is the concentration of inhibitor required to reduce enzyme activity by half and is not dependent on enzyme or substrate concentrations. The inhibitory activity of an anti-PKal antibody can be determined by routine methods, such as the method described in example 3 below.

[00044] In some examples, the inhibitory activity of an anti-PKal antibody is determined by the apparent Ki value (Ki,app). The Ki value (Ki,app) of an antibody obtained at different substrate concentrations, when measuring the inhibitory effect of different concentrations of the antibody on the extent of the reaction (e.g., enzyme activity); fitting the change in the pseudo-first order rate constant as a function of inhibitor concentration to the Morrison equation (equation 1) produces an estimate of the apparent Ki value. For a competitive inhibitor, Ki is obtained from the point of intersection with the y-axis extracted from a linear regression analysis of a plot of Ki,app versus substrate concentration.

[00045] In some examples, the anti-PKal antibodies described herein have a Ki,app value lower than 1nM, for example, 0.5 nM, 0.2 nM, 0.1 nM, 0.09nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, 0.01 nM, or lower. The Ki,app value of an antibody can be estimated by following the methods known in the art and described in this document (example 2).

[00046] The antibodies described in this document may be murine, rat, human or of any other origin (including chimeric or humanized antibodies). In some examples, the antibody comprises a modified constant region, such as a constant region that is immunologically inert, for example, does not activate complement-mediated lysis, or does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC activity can be assessed using methods disclosed in United States Patent No. 5,500,362. In other embodiments, the constant region is modified, as described in Eur. J. Immunol. (1999) 29:2613-2624; PCT Application No. PCT/GB99/01441; and/or UK Patent Application No. 9809951.8.

[00047] Any of the antibodies described in this document can be either monoclonal or polyclonal. A "monoclonal antibody" refers to a homogeneous antibody population and a "polyclonal antibody" refers to a heterogeneous antibody population. These two terms do not limit the source of an antibody or the way in which it is made.

[00048] In one example, the antibody used in the methods described herein is a humanized antibody. Humanized antibodies refer to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain a minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues of a complementary determining region (CDR) of the recipient are replaced by residues of a CDR of a non-human species (donor antibody), such as mouse, rat or rabbit, having the desired capacity, affinity and specificity. In some cases, human immunoglobulin Fv framework region (FR) residues are replaced by corresponding non-human residues. Additionally, the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further improve and optimize the performance of the antibody. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all, or substantially all, of the CDR regions correspond to those of a non-human immunoglobulin, and all, or substantially all, of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody will optimally also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Antibodies may have modified Fc regions, as described in WO 99/58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) that are altered from the original antibody, which are also called one or more CDRs "derived from" one or more CDRs of original antibody. Humanized antibodies may also involve affinity maturation.

[00049] In another example, the antibody described in this document is a chimeric antibody, which includes a constant heavy region and a constant light region of a human antibody. Chimeric antibodies refer to antibodies having a variable region or part of a variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable region of both the light and heavy chains mimic the variable region of antibodies derived from a mammalian species (e.g., a non-human mammal, such as a mouse, rabbit, and rat), while the constants are homologous to antibody sequences derived from other mammals, such as human. In some embodiments, amino acid modifications can be made in the variable region and/or the constant region.

[00050] In some embodiments, the anti-PKal antibodies described herein have a suitable binding affinity for a PKal or catalytic domain thereof. As used herein "binding affinity" refers to the apparent association constant of KA. Ka is the reciprocal of the dissociation constant (KD). The antibody described herein may have a binding affinity (KD) of at least 10 5, 10-6, 10-7, 10-8, 10-9, 10-10 M or less. A higher binding affinity corresponds to a decrease in KD. Greater affinity binding of an antibody to a first target relative to a second target may be indicated by a greater Ka (or a lower numerical KD value) for binding the first target than the KA (or numerical KD value) for binding to the second target. In such cases, the antibody has specificity for the first target (e.g., a protein in a first conformation or mimic thereof) relative to the second target (e.g., the same protein in a second conformation or mimic thereof; or a second protein ). Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100 , 500, 1000, 10,000 or 105 times.

[00051] Binding affinity can be determined by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance or spectroscopy (for example, using a fluorescence assay). Exemplary conditions for assessing binding affinity are in HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v/v) Surfactant P20) These techniques can be used to measure binding protein concentration bounded as a function of target protein concentration. The concentration of bounded binding protein ([Bounded]) is related to the concentration of free target protein ([Free]) and the concentration of binding sites for the binding protein on the target, where (N) is the number of sites binding per target molecule by the following equation:

[00052] It is not always necessary to make an exact determination of KA, although, since it is sometimes sufficient to obtain a quantitative measurement of affinity, for example, determination using a method such as ELISA or FACS analysis is proportional to KA, and, thus, it can be used for comparisons such as determining whether a higher affinity is, for example, 2-fold higher, in order to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, for example, by activity in an assay. functional, for example, an in vitro or in vivo assay.

[00053] In some embodiments, the anti-PKal antibody comprises heavy and light CDRs, or heavy and light chain variable regions of DX-2930. The complete heavy chain and light chain sequences of DX-2930 are shown below. The sequences of the heavy chain variable domain and the light chain variable domain are also shown below. The CDR sequences of DX-2930 are shown in Table 1.

[00054] DX-2930 Heavy Chain Amino Acid Sequence (451 amino acids)

[00055] EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWV RQAPGKGLEWVSGIYSSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCAYRRIGVPRRDEFDIWGQGTMVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG (SEQ ID NO:1)

[00056] DX-2930 Light Chain Amino Acid Sequence (amino acids 213, 23419.08 Da)

[00057] DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQ KPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQ QYNTYWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH K VYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:2)

[00058] Amino acid sequence of DX-2930 heavy chain variable domain

[00059] EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWV RQAPGKGLEWVSGIYSSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCAYRRIGVPRRDEFDIWGQGTMVTVSS (SEQ ID NO:3)

[00060] Amino acid sequence of DX-2930 light chain variable domain

[00061] DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQ KPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQ QYNTYWTFGQGTKVEIK (SEQ ID NO:4)

[00062] Table 1. CDRs for DX-2930.

[00063] In some embodiments, the anti-PKal antibody is a Fab comprising the same CDRs or heavy and light chain variable regions as DX-2930. For example, DX-2944 described in example 1 below is the Fab portion of DX-2930.

[00064] DX-2930 is a fully human IgG derived from paternal clone M0162-A04. The amino acid sequences of VH and VL of M0162-A04 are shown in figure 3. Their alignment with the corresponding germline VH gene (VH3_3-23) and VL gene (VK1_L12) is also shown in figure 3. Compared to the HC CDR3 of M0162-A04, the HC CDR3 of DX-2930 includes the variations of T101I, I103V and A108E (see table 3 below, the HC CDR3 of DX-2930 being identical to M0199-A08). The Chothia Numbering Scheme is used in the present disclosure. www.bioinf.org.uk/abs/.

[00065] Table 2 below provides structural information of DX 2930, its paternal antibody M0162-A04 and respective variants thereof.

[00066] Table 2. DX-2930 Variant Sequence Properties

[00067] Antibodies Targeting Specific Residues in Human Plasma Kallikrein

[00068] In some embodiments, the antibody that specifically binds active PKal interacts with one or more of the residues (e.g., at least 3.5, 8, 10, 15, 20, 25, 30, 35, 40, or 45 ) in the catalytic domain of human PKal, including V410, L412, T413, A414, Q415, R416,L418, C419, H434, C435, F436, D437, G438, L439, W445, Y475,K476, V477, S478, E479, G480 , D483, F524, E527, K528, Y552,D554, Y555, A564, D572, A573, C574, K575, G576, S578, T596,S597, W598, G599, E600, G601, C602, A603, R604, Q607, 608 ,G609, V610, and/or Y611 (numbers based on the complete prekallikrein amino acid sequence). The positions of these residues are indicated in figure 4 (bold and underlined). These residues are identified as interacting with one or more residues in DX-2930, according to the crystal structures described in example 2 below.

[00069] Interacting means that the distance between two residues in a complex formed by two ligand pairs is less than a predetermined value, for example, <; 6 Â, <; 4 Â, or <; 2 Â. For example, an interacting residue in a ligand pair may have at least 1 atom within a given threshold (e.g., <;6 Â, <;4 Â, or <;2 Â) of at least 1 atom from a residue from another linking pair in the complex structure. Interaction does not require real connection. Interacting residues are suggested to be involved in antibody recognition.

[00070] In some embodiments, the antibodies described herein bind human active PKal to an epitope comprising one or more of the residues listed above. An "epitope" refers to the site on a target compound which is bound by an antibody, such as a Fab or full-length antibody. An epitope can be linear, which is typically 6 to 15 aa in length. Alternatively, the epitope may be conformational.

[00071] In some examples, the antibody that specifically binds active PKal, described herein, binds an epitope comprising the following segments: V410-C419, H434-L439, Y475-G480, F524-K528, Y552-Y555, D572-S578, T596-R604, or Q607-Y611. In some examples, the antibody (e.g., a non-DX-2930 antibody) binds the same epitope as DX-2930 or competes for binding with active PKal such as DX-2930.

[00072] In one example, the anti-PKal antibodies described herein preferentially bind wild-type PKal compared to a mutant that includes mutations in one or more of R551, Q553, Y555, T558 and R560, e.g., mutant 2 described in example 4. Such antibodies can bind wild-type PKal at a much higher affinity compared to the mutant (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold -folds, 500-folds, 1,000-folds top). Alternatively or in addition, the antibodies exhibit much greater inhibitory activity than wild-type PKal relative to the mutant (e.g., at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold , 200-ply, 500-ply, 1,000-ply higher).

[00073] In other examples, the anti-PKal antibodies described herein bind to active wild-type PKal and functional variants thereof. The antibody may preferentially bind an active PKal over its binding to an inactive mutant. The antibody may preferentially bind active PKal over prekallikrein.

[00074] Anti-Plasma Kallikrein Antibodies Having Specific Motifs and/or Residues

[00075] In some embodiments, the anti-PKal antibody described herein comprises a VH and a VL, each of which comprises three CDRs flanked by framework regions (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; see figure 3). The heavy chain CDR3 may comprise the motif: or N, X108 is A, S, D, E, or V, X109 is F or L, X110 is D, E, or N, and In some examples, X99 is Q and X101 is I, R, S, or P. Alternatively, or additionally, X106 is W and X111 is N, M, or S. In other examples,

[00076] X101 is I, X108 is E, and X103 is I or L; or X101 is I and X103 is I or L. In still other examples,

[00077] X103 is I or L and X110 is D, E or N.

[00078] In addition, such anti-pKal antibody may include one or more other residues that are identified based on the crystal structures discussed herein as being involved in interaction with the catalytic domain of human PKal. These residues can be located in VH or in the VL chain. Examples include E1, V2, F27, T28, F29 and S30 in FR1 of VH, H31 in HC CDR1; S31 and W32 in the LC CDR1, Y49 in the FR1 of the VL chain, K50, T53, L54 and E55 and S56 in LC CDR2 and G57 and V58 the FR3 of the VL chain.

[00079] Anti-PKal antibodies, as described above, can use any germinal heavy chain or light chain V gene as a structure. Heavy chain V genes include, but are not limited to, IGHV1-2, IGHV1-3, IGHV1-8, IGHV1-18, IGHV1-24, IGHV1-45, IGHV1-46, IGHV1-58, IGHV1-69, IGHV2 -5, IGHV2-26, IGHV2-70, IGHV3-7,IGHV3- 9, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-20, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-33 , IGHV3-43, IGHV3-48, IGHV3-49, IGHV3-53, IGHV3-64, IGHV3-66, IGHV3-72, IGHV3-73, IGHV3-74, IGHV4-4, IGHV4-28, IGHV4-31, IGHV4 -34, IGHV4-39, IGHV4-59, IGHV4-61, IGHV4-B, IGHV5-51, IGHV6-1, and IGHV7-4-1.

[00080] In some examples, the antibody uses a K light chain. Light chain VK genes include, but are not limited to, V genes for IGKV1-05, IGKV1-06, IGKV1-08, IGKV1-09, IGKV1-12 , IGKV1-13, IGKV1- 16, IGKV1-17, IGKV1-27, IGKV1-33, IGKV1-37, IGKV1-39, IGKV1D-16, IGKV1D-17, IGKV1D-43, IGKV1D-8, IGKV2-24, IGKV2 -28, IGKV2-29, IGKV2-30, IGKV2-40,IGKV2D-26, IGKV2D-29, IGKV2D-30, IGKV3-11, IGKV3-15,IGKV3-20, IGKV3D-07, IGKV3D-11, IGKV3D-20 , IGKV4-1,IGKV5-2, IGKV6-21, and IGKV6D-41. In other examples, the antibody uses a light chain, for example, any one of IGLV1-IGLV10.

[00081] The antibody can also use any germline heavy J segment (e.g., IGJH1-IGJH6 heavy chain) and light chain J segments (e.g., IGJK1, IGJK2, IGJK3, IGJK4, or IGJK5), which can be subjected to variations, such as deletions at the C-terminus, N-terminus, or both.

[00082] Germline antibody gene/segment sequences are well known in the art. See, for example, www.vbase2.org/vbstat.php.

[00083] In some examples, the anti-PKal antibody described herein uses VH3_3-23 and/or VK1_L12 as the framework for the heavy chain and/or light chain. May include substantially similar HC CDR1, HC CDR2, HC CDR3 and/or LC CDR1, LC CDR2, and/or LC CDR3 as those in M0162-A04 (Figure 3), for example, containing up to 5, 4, 3, 2 or 1 amino acid residue variations compared to the corresponding CDR region in M0162-A04.

[00084] In other examples, the anti-PKal antibody comprises a VH chain that includes a VH CDR1, VH CDR2 and VH CDR3 at least 75% (e.g., 80%, 85%, 90%, 95% or 98% ) identical to the corresponding VHCDRs of M0162-A04 and a VL chain that includes a VL CDR1, VL CDR2, and VL CDR3 at least 75% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to VL CDRs of corresponding M0162-A04.

[00085] Alternatively, the anti-PKal antibody comprises a VH chain at least 75% (e.g., 80%, 85%, 90%, 95% or 98%) identical to the VH chain (mature or precursor) of M0162-A04 and/or a VL chain at least 75% (e.g., 80%, 85%, 90%, 95% or 98%) identical to the VL (precursor mature) chain of M0162-A04.

[00086] The "percent identity" of two amino acid sequences is determined using the Karlin and Altschul Proc algorithm. Natl. Academic. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Academic. Sci. USA 90:5873-77, 1993. This algorithm is incorporated into the NBLAST and XBLAST (version 2.0) programs by Altschul, et al. J. Mol. Biol. 215:403-10, 1990. Protein BLAST searches can be performed with the program XBLAST, score = 50, word length = 3 to obtain homologous amino acid sequences for the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be used, as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g. XBLAST and NBLAST) can be used.

[00087] In some cases, conservative mutations may be introduced into the CDRs in M0162-A04, for example, in positions where the residues are not likely to be involved in interaction with PKal, as determined based on the crystal structure. As used herein, "a conservative amino acid substitution" refers to an amino acid substitution that does not alter relative charge or size characteristics of the protein in which the amino acid substitution is made. The variants can be prepared according to methods for altering the sequence of polypeptides known to the person skilled in the art, as are found in references compiling such methods, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Publishers, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions made among amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[00088] Use of anti-PKal Antibodies for Treatment of Diabetic Macular Edema (DME)

[00089] Aspects of the disclosure relate to the treatment of the subject, suspected of having, or at risk of having, a retinal disease, for example, DME, AMD, RVO, uveitis, endophthalmitis or PCV. In some embodiments, methods for treating these subjects are provided, in which a composition that includes an effective amount of an antibody that specifically binds to active PKal, as described herein, is administered to the subject via an appropriate route.

[00090] To practice a method disclosed herein, an effective amount of a composition (e.g., a pharmaceutical composition) described herein may be administered to a subject (e.g., a human) in need of treatment via a suitable route. , such as intravenous administration (e.g., as a bolus or by continuous infusion over a period of time), by subcutaneous injection, intravitreal injection, or intraocular injection. The composition may comprise one or more antibody binding to active human PKal. Alternatively, the composition may comprise nucleic acid(s) encoding the anti-PKal antibody, which may be operably linked to an appropriate promoter. Such nucleic acid can be an expression vector.

[00091] The subject to be treated by the compositions and methods described herein may be a mammal, more preferably a human, for example, a human having diabetes. Mammals include, but are not limited to, farm animals, sporting animals, pets, primates, horses, dogs, cats, mice and rats. A human subject in need of treatment may be a human patient having or at risk or suspected of having a retinal disease, including DME, AMD, RVO, uveitis, endophthalmitis or PCV. Age-related macular degeneration (AMD) is a deterioration or breakdown of the macula of the eye. With macular degeneration, a subject may have symptoms such as blurred vision, dark areas or distortion in central vision, and, optionally, permanent loss of central vision. Retinal vein occlusion (RVO) is a blockage of the small veins that carry blood from the retina. It is often caused by hardening of the arteries (atherosclerosis) and the formation of blood clots. Diabetic macular edema (DME) is the proliferative form of diabetic retinopathy, characterized by swelling of the retinal layers, neovascularization, vascular leakage, and thickening of the retina in diabetes mellitus due to leakage of fluid from blood vessels within the macula. Polypoidal choroidal vasculopathy (PCV) is a disease of the choroidal vasculature. It is present in both men and women of many ethnicities, characterized by serosanguineous detachments of the pigmented epithelium and exudative changes that can commonly lead to subretinal fibrosis. Uveitis is swelling and irritation of the uvea, the middle layer of the eye. The uvea provides most of the blood supply to the retina. It can be caused by autoimmune diseases, including rheumatoid arthritis or ankylosing spondylitis. It can also be caused by infection or exposure to toxins. In many cases the cause is unknown. Endophthalmitis is an inflammatory condition of the intraocular cavity (i.e., the aqueous and/or vitreous humor) usually caused by infection.

[00092] A subject having such retinal disease can be identified by a routine medical examination, for example, a visual acuity test, tonometry, optical coherence tomography, color stereo retinography, a fluorescein angiogram, or combinations thereof. A subject suspected of having a retinal disease may show one or more symptoms of the disease, for example, blurred vision, distorted vision, or spots in the field of vision. A subject at risk for a retinal disease may be a subject having one or more risk factors. For example, a subject at risk for DME may have one or more of the following risk factors: hypertension, fluid retention, hypoalbuminemia, or hyperlipidemia. Risk factors associated with RVO include atherosclerosis, diabetes, high blood pressure (hypertension), and other eye conditions such as glaucoma, macular edema, or vitreous hemorrhage.

[00093] In some embodiments, a subject may be treated with an antibody, as described herein, in combination with other treatments for DME. Non-limiting examples of treatment for DME include laser photocoagulation, steroids, VEGF targeting agents (e.g., Lucentis® (ranibizumab) or Eylea® (aflibercept)), and/or anti-PDGF agents.

[00094] "An effective amount" as used herein refers to the amount of each active agent necessary to confer a therapeutic effect on the subject, either alone or in combination with one or more other active agents. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, individual patient parameters, including age, physical condition, size, gender and weight, the duration of treatment, the nature of the simultaneous therapy (if any), the specific route of administration and similar factors within the knowledge and expertise of the healthcare professional. These factors are well known to those skilled in the art, and can be addressed with no more than standard experimentation. It is generally preferable that a maximum dose of individual components or combinations thereof be used, i.e. the highest dosage, according to medical judgement. It will be understood by those skilled in the art, however, that a patient may insist on a lower or tolerable dose for medical reasons, psychological reasons or for other reasons.

[00095] Empirical considerations, such as half-life, will generally contribute to dose determination. For example, antibodies that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, can be used to extend the half-life of the antibody and to prevent the antibody from being attacked by the host's immune system. The frequency of administration can be determined and adjusted throughout therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or improvement and/or delay of DME. Alternatively, sustained release formulations of an anti-PKal may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[00096] In one example, dosages of an anti-PKal antibody, as described herein, can be determined empirically in individuals who have received one or more administrations of the antibody. Incremental doses of the antibody are given to individuals. To evaluate the effectiveness of the antibody, an indicator of a retinal disease must be followed.

[00097] In general, for the administration of any of the antibodies described herein, a first candidate dosage may be about 2 mg/kg. For the purpose of the present disclosure, a typical daily dose may range from about 0.1 μg/kg to 3 μg/kg to 30 μg/kg to 300 μg/kg to 3 mg/kg to 30 mg/kg to 100 mg/kg or more depending on the factors mentioned above. For repeated administrations over several days or more, depending on the condition, treatment is continued until a desired suppression of symptoms occurs, or until sufficient therapeutic levels are achieved to alleviate the DME, or a symptom thereof. An exemplary dosage regimen comprises administering an initial dose of approximately 2 mg/kg, followed by a weekly maintenance dose of about 1 mg/kg of the antibody, or followed by a maintenance dose of about 1 mg/kg every two weeks. weeks. However, other dosage regimens may be useful depending on the pattern of pharmacokinetic decay the practitioner wishes to achieve. For example, dosing 1 to 4 times per week is contemplated. In some embodiments, the dosage ranges from about 3 μg/mg to about 2 mg/kg (such as, about 3 μg/mg, about 10 μg/mg, about 30 μg/mg, about 100 μg /mg, about 300 μg/mg, with 1 mg/kg and about 2 mg/kg) can be used. In some embodiments, dosage frequency is once every week, every two weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once a month, every 2 months, or every 3 months, or more. The progress of this therapy is easily monitored by conventional assays and techniques. The dosage regimen (including the antibody used) may vary over time.

[00098] In some embodiments, for a normal weight adult patient, doses of about 0.3 to 5.00 mg/kg can be administered. The particular dosage regimen, e.g., dose, time, and repetition, will depend on the particular individual and the individual's medical history, as well as the properties of the individual agents (such as the half-life of the agent and other considerations well known in the art). ).

[00099] For the purposes of the present invention, the appropriate dosage of an anti-PKal antibody will depend on the specific antibody (or compositions thereof) employed, the type and severity of retinal disease (e.g., DME, AMD, RVO, endophthalmitis uveitis or PCV), whether the antibody is administered for preventive or therapeutic purposes, prior therapy, the patient's clinical history, response to the antibody, and the physician's discretion. Typically, the doctor will administer an anti-PKal antibody until a dose is reached to achieve the desired result. Administration of an anti-PKal antibody may be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic and other factors known to those skilled in the art. Administration of an anti-PKal antibody may be essentially continuous over a predetermined period of time or may be in a series of spaced doses, for example, either before, during or after developing retinal disease.

[000100] As used herein, the term "treat" refers to the application or administration of a composition including one or more active agents to a subject, who has DME, a symptom of a retinal disease (e.g., DME, AMD, RVO, uveitis, endophthalmitis or PCV), or a predisposition to retinal disease, for the purpose of curing, healing, relieving, altering, remedying, improving or affecting the retinal disease, the symptom of the disease or the predisposition to the illness.

[000101] Alleviating a retinal disease, such as DME, AMD, RVO, uveitis, endophthalmitis or PCV includes slowing the development or progression of the disease, or reducing its severity. Alleviating disease does not necessarily require curative results. As used herein to "delay" the development of a retinal disease means to postpone, prevent, delay, delay and/or postpone the progression of the disease. This delay can be of varying lengths of time depending on the history of the disease and/or individuals being treated. A method that "delays" or alleviates the development of disease, or delays the onset of disease, is a method that reduces the probability of developing one or more symptoms of the disease in a given period of time, and/or reduces the progression of symptoms in a given period of time compared to not using the methods. Such comparisons are typically based on clinical studies, using a sufficient number of subjects to give a statistically significant result.

[000102] "Development" or "progression" of a disease means initial manifestations and/or that followed the progression of the disease. Development of the disease can be detectable and evaluated using standard clinical techniques, also known in the art. However, development also refers to progression that may be undetectable. For the purposes of this disclosure, development or progression refers to the biological evolution of symptoms. "Development" includes occurrence, recurrence, and onset. As used herein, "onset" or "occurrence" of a retinal disease includes initial and/or recurrent onset.

[000103] In some embodiments, the anti-PKal antibody described herein is administered to a subject in need of treatment in an amount sufficient to inhibit activity or active PKal by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) in vivo. In other embodiments, the antibody is administered in an amount effective to reduce the PKal level by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) .

[000104] Conventional methods known to those skilled in the art of medicine can be used to administer the pharmaceutical composition to the subject, depending on the type of disease to be treated or the site of the disease. This composition may also be administered by other conventional routes, for example, administration orally, parenterally, spray inhalation, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parental" as used herein includes intravitreal, subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intra-sternal, intrathecal, intralesional and intracranial injection or infusion techniques. In addition, it may be administered to the subject via injectable depot administration routes, such as using 1-, 3-, or 6-month injectable or biodegradable depot materials and methods. In some embodiments, the composition as described herein is administered to an eye of a patient in which treatment is necessary. In one example, it is administered topically. In another example, it is injected intraocularly or intravitreally.

[000105] Injectable compositions may contain various carriers, such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol and polyols (glycerol, propylene glycol, liquid polyethylene glycol and the like). By intravenous injection, water-soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients. Intramuscular preparations, for example, a sterile formulation of a suitable soluble salt form of the antibody, can be dissolved and administered in a pharmaceutical excipient, such as water injection, 0.9% saline or 5% glucose solution.

[000106] In one embodiment, an anti-PKal antibody is administered via specific or targeted local delivery techniques. Examples of site-specific or target-site delivery techniques include various implantable depot sources of anti-PKal antibody or local delivery catheters, such as infusion catheters, an indwelling catheter, or a needle catheter, synthetic grafts, adventitial wraps. , shunts and stents or other implantable devices, site-specific carriers, direct injection or direct application. See, for example, PCT Publication No. WO 53211/00 and Pat. of the United States No. 5,981,568.

[000107] Targeted delivery of therapeutic compositions containing a presense polynucleotide, expression vector or subgenomic polynucleotides can also be used. Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al, Trands Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol Chem. (1988) 263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Academic. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338.

[000108] Therapeutic compositions containing a polynucleotide (e.g., those encoding the anti-PKal antibodies described herein) are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol . In some embodiments, the concentration is in the range of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg. of DNA or more can also be used during the gene therapy protocol.

[000109] Anti-PKal antibodies described herein can be delivered using gene delivery vehicles. The gene delivery vehicle may be of viral or non-viral origin (see, generally, Jolly, CancerGene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, HumanGene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and/or enhancers. Expression of the coding sequence can be constitutive or regulated.

[000110] Virus-based vectors for delivering a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary virus-based vehicles include, but are not limited to, recombinant retroviruses (see, for example, PCT publication nos. WO 90/07936; WO 94/03622; WO 93/25698; WO93/25234; WO 93/ 11230; WO 93/10218; US Pat. Nos. 5,219,740 and 4,777,127; Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelanequine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV) vectors (see, for example, PCT Publication Nos. WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). Administration of DNA linked to killed adenoviruses as described in Curiel, Hum. Gene Ther. (1992) 3:147 can also be used.

[000111] Non-viral delivery vehicles and methods may also be employed, including, but not limited to, condensed polycationic DNA bound or unbound to killed adenoviruses alone (see, for example, Curiel, Hum. Gene Ther. (1992 ) 3:147); Linker-bound DNA (see, for example, Wu. J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicle cells (see, e.g., United States Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO 95/30763; and WO 97 /42338) and neutralization of nucleic acids from charge or fusion with cell membranes. Free DNA can also be used. Methods of introducing exemplary free DNA are described in PCT Publication No. WO 90/11092 and United States Patent No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Pat. European No. 5,422,120; PCT publication no. WO 95/13796; WO 94/23697; WO 91/14445; and European Patent No. 0524968. Additional approaches are described in Philip, Mol. Cell. Biol. (1994) 14:2411, and in Woffendin, Proc. Natl. Academic. Sci. (1994) 91:1581.

[000112] The particular dosage regimen, for example, dose, time and repetition, used in the method described herein will depend on the particular subject and the subject's medical history.

[000113] In some embodiments, more than one anti-PKAL antibody, or a combination of an anti-PKal antibody and another suitable therapeutic agent, can be administered to a subject in need of treatment. The antagonist can be the same type or different from each other. The anti-PKal antibody can also be used in conjunction with other agents that serve to enhance and/or complement the effectiveness of the agents.

[000114] Efficacy of treatment for a retinal disease can be assessed by methods known in the art, for example, by fluorescein angiography.

Antibody preparation

[000115] Antibodies capable of binding PKal, as described herein, can be made by any method known in the art. See, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[000116] In some embodiments, antibodies specific to a target antigen (e.g., a human PKal or its catalytic domain) can be made by conventional hybridoma technology. The complete target antigen or a fragment thereof, optionally coupled to a carrier protein, such as KLH, can be used to immunize a host animal for generating antibody binding to that antigen. The route and schedule of immunization of the host animal are generally in accordance with established and conventional techniques for antibody stimulation and production, as further described herein. General techniques for producing humanized, human and mouse antibodies are known in the art and are described herein. It is contemplated that any mammalian subject, including humans, or antibody producing cells thereof may be manipulated to serve as a basis for the production of mammalian, including human hybridoma cell lines. Typically, the host animal is inoculated intraperitoneally intramuscularly, orally, subcutaneously, and/or intradermally with an amount of antigen, including as described herein.

[000117] Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975) Nature 256:495-497 or as modified by Buck, D. W., et al, In Vitro, 18:377-381 (1982). Available myeloma lines, including but not limited to X63-Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, California, USA, can be used in hybridization. Generally, the technique consists of fusing myeloma cells and lymphoid cells using a fusogen such as polyethylene glycol, or by electrical means known to those skilled in the art. After fusion, cells are separated from the fusion medium and cultured in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate unhybridized parental cells. Any of the media described herein, supplemented with or without serum, can be used for culturing hybridomas that secrete monoclonal antibodies. As another alternative to the cell fusion technique, EBV-immortalized B cells can be used to produce the anti-PKal monoclonal antibodies described here. Hybridomas are expanded and subcloned, if desired, and supernatants are tested for anti-antigen activity by standard immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).

[000118] Hybridomas that can be used as a source of antibodies include all derivatives, progeny cells of parental hybridomas that produce monoclonal antibodies capable of interfering with PKal activity. Hybridomas that produce such antibodies can be grown in vitro or in vivo using known procedures. Monoclonal antibodies can be isolated from culture medium or body fluids by conventional immunoglobulin purification procedures, such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography and ultrafiltration, if desired. Unwanted activity, if present, can be removed, for example, by carrying out the preparation on adsorbents made from antigens bound to a solid phase and eluting or releasing the desired antibodies off the antigen. Immunization of a host animal with a target antigen or a fragment containing the target amino acid sequence conjugated to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or keyhole trypsin inhibitor. soy using a bifunctional or derivatizing agent, for example, sulfosuccinimide maleimidobenzoyl ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl or R1N=C=NR, where R and R1 are different alkyl groups, can produce a population of antibodies (e.g. monoclonal antibodies).

[000119] If desired, an antibody (monoclonal or polyclonal) of interest (e.g., produced by a hybridoma) can be sequenced and the polynucleotide sequence can then be cloned into an expression or propagation vector. The antibody coding sequence of interest can be maintained in vector in a host cell and the host cell can be expanded and frozen for future use. Alternatively, the polynucleotide sequence may be used for genetic manipulation to "humanize" the antibody or to improve the affinity (affinity maturation) or other characteristics of the antibody. For example, the constant region can be designed to more closely resemble human constant regions to avoid immune response if the antibody is used in human clinical trials and treatments. It may be desirable to genetically manipulate the antibody sequence to obtain greater affinity for the target antigen and greater efficacy in inhibiting PKal activity. It will be apparent to one skilled in the art that one or more polynucleotide changes can be made to the antibody and still maintain its binding specificity to the target antigen.

[000120] In other embodiments, fully human antibodies can be obtained using commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals that are engineered to produce one or more more desirable robust immune responses (e.g., fully human antibodies) can also be used to generate humanized or human antibodies. Examples of such technology are Xenomouse RTM from Amgen Inc. (Fremont, Calif.) and HuMAb-Mouse RTM and TC Mouse TM from Medarex Inc. (Princeton, N.J.). In another alternative, antibodies can be made recombinant by phage display or yeast technology. See, for example, U.S. Pat. No. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al. (1994) Annu. Rev. Immunol. 12:433 455, and . Alternatively, phage display technology (McCafferty et al. (1990) Nature 348:552-553) can be used to produce human antibodies and antibody fragments in vitro from variable immunoglobulin domain gene repertoires ( V) from non-immunized donors.

[000121] Antigen-binding fragments of an intact antibody (complete antibody) can be prepared using routine methods. For example, F(ab') 2 fragments can be produced by pepsin digestion of an antibody molecule, and Fab fragments can be generated by reducing the disulfide bonds of F(ab') 2 fragments.

[000122] Genetically manufactured antibodies, such as humanized antibodies, chimeric antibodies, single chain antibodies, Fabs and bispecific antibodies, can be produced through, for example, conventional recombinant technology. In one example, a DNA encoding monoclonal antibodies specific to a target antigen can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of the antibodies). monoclonal). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into one or more expression vectors, which are then transfected into host cells, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma that do not otherwise produce immunoglobulin protein to obtain the synthesis of monoclonal antibodies in recombinant host cells. See, for example, PCT Publication No. WO 87/04462. The DNA can then be modified, for example, by substituting the coding sequence for human light and heavy chain constant domains in place of the murine homologous sequences, Morrison et al, (1984) Proc. Nat. Acad. Sci. 81:6851, or by covalently attaching to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Thus, genetically altered antibodies, such as "chimeric" or "hybrid" antibodies; can be prepared that have binding specificity for a target antigen.

[000123] Techniques for producing Fabs are also known in the art (see, for example, PCT Publication Nos. WO1993006217 and WO2005038031, which are incorporated by reference in this document). A variety of host expression vector systems can be used to recombinantly express a Fab. Such host expression systems represent cells that can, when transformed or transfected with the appropriate nucleotide coding sequences, express a Fab described in this document. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing coding sequences encoding a described Fab antibody. in this document; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing sequences encoding a Fab antibody described herein; insect cell systems infected with recombinant virus expression vectors (e.g., baclovirus) that contain coding sequences for a Fab antibody described herein; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) that contains the sequences encoding a Fab antibody described herein; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphocytic cells harbor recombinant expression constructs encoding a Fab antibody described herein. In some embodiments, a Fab described herein is recombinantly expressed E. coli. Once a Fab has been recombinantly expressed, it can be purified by any method known in the art for purifying polypeptides or antibodies. , for example, by chromatography (e.g., ion exchange, affinity, or sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of polypeptides or antibodies.

[000124] Techniques developed for the production of "chimeric antibodies" are well known in the art. See, for example, Morrison et al. (1984) Proc. Natl. Academic. Sci. USA 81, 6851; Neuberger et al. (1984) Nature 312, 604; and Takeda et al. (1984) Nature 314:452.

[000125] Methods for creating humanized antibodies are also known in the art. See, for example, Queen et al, Proc. Natl. Academic. Sci. USA, 86:10029-10033 (1989). In one example, VH and VL variable regions of a parental non-human antibody are subject to three-dimensional molecular modeling analysis, following methods known in the art. Next, backbone amino acid residues important for the formation of the correct CDR structures are identified using the same molecular modeling analysis. In parallel, human VH and VL chains having amino acid sequences that are homologous to those of parental non-human antibodies are identified from any antibody gene database using the parental VH and VL sequences as search queries. VH and VL acceptor genes are then selected.

[000126] The CDR regions within the selected human acceptor genes can be replaced with the CDR regions from the non-human parental antibody or functional variants thereof. When necessary, residues within the framework regions of the parental chain that are predicted to be important in interacting with the CDR regions (See description above) can be used to replace the corresponding residues in the human acceptor genes.

[000127] A single-chain antibody can be prepared through recombinant technology by linking a nucleotide sequence coding for a heavy chain variable region and a nucleotide sequence coding for a light chain variable region. Preferably, a flexible linker is incorporated between the two variable regions. Alternatively, techniques described for the production of single-chain antibodies (U.S. Patent Nos. 4,946,778 and 4,704,692) can be adapted to produce phage or yeast scFv libraries and scFv clones specific for a PKal can be identified. from the library, following routine procedures. Positive clones can be subjected to additional screening to identify those that inhibit PKal activity.

[000128] Antibodies obtained following a method known in the art and described in this document can be characterized using methods known in the art. For example, it is a method for identifying the epitope to which the antigen binds, or "epitope mapping." There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including solving the crystal structure of an antibody complex. -antigen, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999. In a further example, epitope mapping can be used to determine the sequence to which an antibody binds. The epitope can be a linear epitope, that is, contained in a single stretch of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids that cannot necessarily be contained in a single stretch (linear sequence of primary structure). Peptides of varying lengths (e.g., at least 4-6 amino acids in length) can be isolated or synthesized (e.g., recombinant) and used for binding assays with an antibody. In another example, the epitope to which the antibody binds can be determined in a systematic scan by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody. According to gene fragment expression assays, the open reading frame encoding the target antigen is fragmented, either randomly or by genetic constructs, and the reactivity of the expressed fragments of the antigen within the antibody to be tested is determined. Gene fragments can, for example, be produced by PCR and then transcribed and translated into proteins in vitro, in the presence of radioactive amino acids. Antibody binding to radioactively labeled antigen fragments is then determined by gel electrophoresis and immunoprecipitation. Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in simple binding assays. In a further example, mutagenesis of an antigen-binding domain, domain swapping experiments and alanine scanning mutagenesis can be performed to identify residues required, sufficient and/or required for epitope binding. For example, domain swap experiments can be performed using a mutant of a target antigen in which several fragments of the PKal polypeptide have been replaced (swapped) with sequences from a closely related but antigenically distinct protein (e.g., another member of the PKal family). of neurotrophin proteins). By evaluating antibody binding to mutant PKal (e.g., those mutants described in example 2 below), the importance of the particular antigen fragment for antibody binding can be assessed.

[000129] Alternatively, competition assays can be performed using other antibodies known to bind the same antigen to determine whether an antibody binds to the same epitope as the other antibodies. Competition trials are well known to those skilled in the art.

[000130] Any of the suitable methods known in the art, for example, epitope mapping methods, as described herein, can be applied to determine whether the anti-PKal antibody binds to one or more of the specific residues/segments in PKal as described in this document. Furthermore, the interaction of the antibody with one or more of the residues defined in PKal can be determined by routine technology. For example, a crystal structure can be determined following the method disclosed in example 1 below and the distances between the residues in PKal and one or more residues in the antibody can be determined accordingly. Based on such a distance, whether a specific residue in PKal interacts with one or more residues in the antibody can be determined. Additionally, appropriate methods such as competition assays and target mutagenesis assays can be applied to determine preferential binding of a candidate anti-PKal antibody to PKal compared to another target, such as a mutant PKal.

Pharmaceutical compositions

[000131] One or more of the anti-PKal antibodies described above can be mixed with a pharmaceutically acceptable carrier (excipient), including the buffer, to form a pharmaceutical composition for use in relieving DME. "Acceptable" means that the carrier must be compatible with the active ingredient of the composition (and, preferably, capable of stabilizing the active ingredient) and not harmful to the subject being treated. Pharmaceutically acceptable excipients (carriers), including buffers, which are known in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover. In one example, a pharmaceutical composition described herein contains more than one anti-PKal antibody that recognizes different epitopes/residues of active PKal.

[000132] The pharmaceutical compositions to be used in the present methods may comprise pharmaceutically acceptable carriers, excipients or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). acceptable carriers, excipients, or stabilizers are non-toxic to recipients at doses and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants include ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol ); low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrans; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g. protein-Zn complexes); and non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are described herein.

[000133] In some examples, the pharmaceutical composition described in this document comprises liposomes containing the anti-PKal antibody, which can be prepared by methods known in the art, as described in Epstein, et al, proc. Natl. Academic. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl. Academic. Sci. USA 77:4030 (1980); and Pat. United States Nos. 4,485,045 and 4,544,545. Liposomes with improved circulation time are disclosed in U.S. Pat. European No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and derivatized phosphatidylethanolamine-PEG (PEG-PE). Liposomes are extruded through filters of defined pore size to produce liposomes of the desired diameter.

[000134] The anti-PKal antibody can also be entrapped in microcapsules prepared, for example, through coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose microcapsules or gelatin and poly-(methylmethacylate) microcapsules, respectively, in systems of colloidal drug delivery (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are known in the art, see, for example, Remington, The Science and Practice of Pharmacy, 20th Edition Editora Mack (2000).

[000135] In other examples, the pharmaceutical composition described in this document can be formulated in sustained release form. Suitable examples of sustained release preparations include semipermeable solid hydrophobic polymer matrices containing the antibody, such matrices are in the form of molded articles, for example, films or microcapsules. Examples of sustained release matrices, polyesters, hydrogels (e.g. poly(2-hydroxyethyl methacrylate) or poly(v nyl alcohol)), polylactactates (European Pat. No. 3,773,919), copolymers of L-glutamic acid and 7 ethyl -L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-) -3-hydroxybutyric acid.

[000136] Pharmaceutical compositions to be used for in vivo administration must be sterile. This is easily done by, for example, filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle.

[000137] The pharmaceutical compositions described in this document may be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions or suppositories, for oral, parenteral or rectal administration, by inhalation or insufflation.

[000138] For the preparation of solid compositions, such as tablets, the main active ingredient can be mixed with a drug carrier, for example, conventional tablet ingredients, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, for example, 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. When referring to these preformulation compositions as homogeneous, it means that the active ingredient is dispersed evenly throughout the composition so that the composition can be easily subdivided into equally effective units of 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 0.1 to about 500 mg of the active ingredient of the present invention. Tablets or pills of the new composition may be coated, or otherwise compounded, to provide a dosage form providing the advantage of prolonged action. For example, the tablet or pill may comprise an internal dosage and an external dosage component, the latter being in the form of an envelope. The two components may be separated by an enteric layer that serves to resist disintegration in the stomach and allows the internal component to pass intact into the duodenum or delay release. A variety of materials can be used for such 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.

[000139] Suitable surfactants include, particularly, non-ionic agents, such as 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 surfactant will conveniently comprise between 0.05 and 5% surfactant and may be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added, for example mannitol or other pharmaceutically acceptable carriers, if necessary.

[000140] Suitable emulsions can be prepared using commercially available fat emulsions, such as IntralipidTM, LiposynTM, InfonutrolTM, LipofundinTM and LipiphysanTM. The active ingredient may be either dissolved in a premixed emulsion composition or, alternatively, may be dissolved in an oil (e.g., soybean oil, safflower oil, sunflower oil, sesame oil, corn oil or almond) and an emulsion formed by mixing with a phospholipid (for example, egg phospholipids, soy phospholipids or soy lecithin) and water. It will be appreciated that other ingredients may be added, for example glycerol or glucose, in order to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, for example between 5 and 20%. The fat emulsion may comprise fat droplets between 0.1 and 1.0. im, particularly 0.1 and 0.5. im, and has a pH in the range of 5.5 to 8.0.

[000141] Emulsion compositions can be prepared by mixing an anti-PKal antibody with IntralipidTM or components thereof (soybean oil, egg phospholipids, glycerol and water).

[000142] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable solvents, aqueous or organic, or mixtures thereof and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set forth above. In some embodiments, the compositions are administered orally or via the nasal respiratory route for local or systemic effect.

[000143] Compositions in preferably sterile pharmaceutically acceptable solvents can be nebulized by the use of gases. Nebulized solutions may be breathed directly from the nebulizer device or the nebulizer device may be associated with a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension or powder compositions may preferably be administered orally or nasally from devices that deliver the formulation in a suitable manner. Kits for Use in the Treatment of Retinal Diseases

[000144] The present disclosure also provides kits for use in treating a retinal disease, such as DME, AMD, RVO, uveitis, endophthalmitis or PCV. Such kits may include one or more containers comprising an anti-PKal antibody, for example, any of those described herein, for example, DX-2944.

[000145] In some embodiments, the kit may comprise instructions for use in accordance with any of the methods described herein. The included instructions may comprise a description of administering the anti-PKal antibody to treat, delay the onset of, or alleviate a retinal disease. The kit may further comprise a description of selecting a suitable subject for treatment based on identifying that the subject has or is at risk of having a retinal disease. In still other embodiments, the instructions include a description of administering an antibody to an individual at risk for the target disease.

[000146] Instructions relating to the use of an anti-PKal antibody generally include information such as dosage, dose planning and route of administration for the intended treatment. Containers may be in unit doses, large volumes (e.g., multi-dose packets) or sub-unit doses. Instructions provided in kits of the invention are typically instructions written on a label or packaging insert (e.g., a sheet of paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk ) are also acceptable.

[000147] The kits in this disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jugs, flexible packaging (e.g., sealed Mylar or plastic bags) and the like. Packages for use in combination with a specific device, such as a syringe or an infusion device, such as a minipump, are also contemplated. A kit may have a sterile access port (for example, the container may be a bag of intravenous solution or a vial with a cap pierceable by a hypodermic injection needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a cap pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-PKal antibody such as those described herein.

General Techniques

[000148] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombination techniques), microbiology, cell biology, biochemistry and immunology, which are within the state of the art. Such techniques are explained in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Publisher; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Editora Humana; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Publisher; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Editora Plenum; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Publishing, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene TransferVectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Publishing, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publications, 1995).

[000149] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments should therefore be construed as merely illustrative and not limiting the remainder of the disclosure in any respect. All publications cited in this document are incorporated by reference for the purposes or subject matter mentioned in this document.

EXAMPLES

[000150] Example 1: Effect of DX-2944 on a Laser induced Choroidal Neovascularization Disease Model (CNV Laser)

[000151] DX-2944 is a recombinant Fab version of DX-2930 that was expressed and purified from an E. coli expression system. The Laser CNV model is an established connector model of complications associated with human retinal diseases, such as age-related macular degeneration (AMD), central retinal vein occlusions, and macular edema. The experimental design used for this study is described below.

Experimental Project

[000152] Day 1: Bilateral Laser Treatment to Produce 3 Lesions Per Eye

[000153] Day 3: Bilateral intravitreal injection of test agent, vehicle, or positive control (anti-VEGF Ab)

[000154] Day 22: In-vivo fluorescein angiography

[000155] The results in figures 1A to 1B and figures 2A to 2B indicate that DX-2944 reduces observed CNV to approximately the same extent as the positive control (an anti-VEGF antibody). The mean fluorescein angiography signal for the anti-VEGF treated group was 7023 fluorescence units, which is similar to that observed for the DX-2944 treated group at 7071 fluorescence units.

[000156] Example 2: Identification of Critical Residues in the Catalytic Domain of Human Plasma Kallikrein Based on Crystal Structures of the DX-2930-PKal Complex.

[000157] The catalytic domain of human plasma kallikrein fused to a his-tag was expressed in insect cells and initially purified by a nickel affinity column. The his-tag was removed from plasma kallikrein by trypsin digestion and free plasma kallikrein was purified by a benzamidine affinity column followed by an SEC column. The purified product was examined on a PAGE gel. The result indicates that the catalytic domain of human plasma kallikrein was properly expressed and purified.

[000158] DX-2930 was prepared using standard recombinant technology and purified. A recombinant Fab fragment of DX 2930 was produced by the standard method and purified.

[000159] The Fab fragment of DX-2930 and the catalytic domain of human plasma kallikrein were mixed in various concentrations under suitable conditions, allowing the formation of antibody-PKal complexes. The complexes thus formed were examined using HPLC to determine the proportion of antibodies-PKal in the complexes. Consequently, suitable concentrations of both antibodies and PKal were identified for formation of a 1:1 complex.

[000160] The antibody-PKal complex was maintained under various conditions, allowing crystallization. Diffraction analysis was performed on the crystallized complex. The crystal structures (2.1 Â and 2.4 Â) were determined based on diffraction statistics.

[000161] According to the crystal structures, residues of the catalytic domain of human PKal that are involved in the interaction with DX-2930 were identified. These residues are indicated (bold and underlined) in Figure 4, which provides the amino acid sequence of the catalytic domain of human PKal (residues 391 to 638 of human PKal).

[000162] Additionally, residues in DX-2930 that interact with PKal were also identified based on the crystal structure, including E1, V2, F27, T28, F29, S30, H31, R100, I101, G102, V103, P104, R105 , R106, D107, G107, K108, and D111 in the heavy chain variable region and S31, W32, Y49, K50, T53, L54, E55, S56, G57, and V58 in the light chain variable region.

[000163] These results indicate that the HC CDR3 of DX-2930 is the main region that interacts with PKal and a pair of residues in the HC CDR1 and FR1 may also contribute to the interaction with PKal. In the light chain, the LC CDR2 region contributed to the interaction.

[000164] Additionally, the results also indicate that variations in certain positions with the HC CDR3 region may be allowed. For example, position 103 requires small hydrophobic residues, such as V or I. As another example, R106 can be replaced with W, and E108 can be replaced with S or D without substantially affecting PKal binding activity. Similarly, D110 can be replaced by E.

[000165] Example 3: Affinity Maturation Results Corresponding to Crystal Structure Structural Information Derivatives

[000166] The variable region of the heavy chain, particularly the HC CDR3 region of antibody M0162-A04, was subject to affinity maturation. Several mutants having amino acid variations at one or more positions in the HC CDR3 region were generated, and their Ki,app values were determined following standard methods.

[000167] Briefly, PKal and a Fab at various concentrations are incubated together for 1 hour at 30 ° C. A substrate peptide (cleavable by PKal) is added to this PKal-Fab mixture. The rate of substrate peptide/proteolysis cleavage is then measured and plotted against Fab concentrations. This plot is then plugged into the Morrison equation, which calculates the value of Ki, app. The results thus obtained are shown in figures 5A to 5D and in table 3 below:

[000168] Table 3. Summary of Hv-CDR3 Affinity Maturation Results

[000169] Affinity maturation results indicate that variations at certain positions within the HC CDR3 region result in high affinity/inhibitory anti-PKal antibodies compared to the parental clone M0162-A04. These results correspond to the structural information provided in example 2 above. Note that the HC CDR3 region of clone M0199-A08 is identical to that of DX-2930.

[000170] Example 4: Impact of Mutations in Plasma Kallikrein on Antibody Inhibitory Activity

[000171] The inhibitory activities of the X115-F02 mutant against various PKal mutants were examined.

[000172] ). The binding specificity and affinity of X115-F02 is the same as DX-2930.

[000173] The wild type and four plasma kallikrein mutants used in this study (figures 7A to 7B) are catalytic domains expressed recombinantly and purified by pichia pastoris. Mutant 1 contains the following mutations in the S3 subsite of the active site: S478A, N481A, S506A, Y507A) (numbers based on the complete prekallikrein amino acid sequence). Mutant 2 contains the following mutations in the S1' subsite of the active site: R551A, Q553A, Y555A, T558A, R560A. Mutant 4 contains the following mutations that are distal to the active site: N396A, S398A, W399A. Mutant 3 was inactive, and therefore was not tested in the activity assay. Mutant 3 contains the following mutations in the S1' subsite of the active site: D572A, K575A, D577A.

[000174] The inhibitory activity of X115-F02 against wild-type PKal and mutants was performed using the method described in example 3 above and Ki,app values were determined. As shown in Figure 6, mutations in Mutant 1 and 4 did not significantly affect the potency of X115-F02 inhibition of plasma kallikrein. Mutations in Mutant 2 reduced potency by approximately 65-fold. These results indicate that residues R551A, Q553A, Y555A, T558A, R560A and their respective adjacent residues may be important for the inhibitory activity of X115-F02 (DX-2930).

[000175] Example 5: Effect of DX-2944 on a Disease Model (CNV Laser) of Laser-induced Choroidal Neovascularization - Study 3

[000176] DX-2944, as described herein, was expressed and purified from an E. coli expression system. The CNV Laser model used in this study is an established connector model of complications associated with human retinal diseases, such as age-related macular degeneration (AMD), central retinal vein occlusions, and macular edema. The experimental design carried out is summarized below.

[000177] Experimental Project: Laser-induced Choroidal Neovascularization (CNV) in rats

[000178] Day 1: Bilateral Laser Treatment to Produce 3 Lesions Per Eye

[000179] Day 3: Bilateral intravitreal injection of test agent, vehicle and positive control

[000180] Day 10: Bilateral intravitreal injection of test agent, vehicle and positive control

[000181] Day 15: In-vivo fluorescein angiography

[000182] Day 22: In-vivo fluorescein angiography

[000183] The results shown in Figure 8 indicate that reduced DX-2944 CNV was observed to approximately the same extent as the positive control (an anti-VEGF antibody) on Day 15 of the study. The mean fluorescein angiography signal for the anti-VEGF treated group was 4627 fluorescence units, which was similar to that observed for the DX-2944 treated group at 4917 fluorescence units. The results shown in Figure 9 indicate that reduced DX-2944 CNV was observed to approximately the same extent as the positive control (an anti-VEGF antibody) on Day 22 of the study. The mean fluorescein angiography signal for the anti-VEGF treated group was 4551 fluorescence units, which was similar to that observed for the DX-2944 treated group at 5011 fluorescence units.

[000184] These results show that DX-2944 was effective in reducing CNV in the animal model, indicating that this antibody would be effective in treating human retinal diseases such as age-related macular degeneration (AMD), central retinal vein occlusions and macular edema.

[000185] Other sequences:

[000186] Heavy chain variable domain (VH) of M202-H03

[000187] EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWV RQAPGKGLEWVSGIYSSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCAFRRTGVPRWDDFDIWGQGTMVTVSSASTKGPSVFPLAPS SKS (SEQ ID NO: 61)

[000188] Light chain variable domain (VL) of M202-H03

[000189] QDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQ QKPGKAPNLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQYNTYWTFGQGTKVEIK (SEQ ID NO: 62)

OTHER MODALITIES

[000190] All features disclosed in this specification can be combined in any combination. Each resource disclosed in this specification may be replaced by an alternative resource serving the same, equivalent or similar purpose. Therefore, unless expressly stated otherwise, each feature disclosed is only one example of a generic series of equivalent or similar features.

[000191] From the above description, one skilled in the art can easily determine the essential characteristics of the present invention, and without deviating from its spirit and scope, can make various changes and modifications to the invention to adapt it for various uses and conditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

[000192] Various inventive embodiments have been described and illustrated in this document, those skilled in the art will readily appreciate a variety of other means and/or structures for performing the function and/or obtaining results and/or one or more of the advantages described in this document , and each of such variations and/or modifications is considered within the scope of the inventive embodiments described herein. More generally, those skilled in the art will understand that all parameters, dimensions, materials and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials and/or configurations will depend on the specific application or applications to which the teachings are made. inventive are used. Those skilled in the art will recognize, or be able to determine, using no more than routine experimentation, many equivalents to the specific embodiments described herein. Therefore, it should be understood that the above embodiments are presented by way of example, and that, within the scope of the appended claims and equivalents thereof, inventive embodiments may be practiced other than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit and/or method described herein. In addition, any combination of two or more such resources, systems, articles, materials, kits, and/or methods, if such resources, systems, articles, materials, kits, and/or methods are not mutually incompatible, is included within the scope invention of the present disclosure.

[000193] All definitions, as defined and used in this document, are to be understood as controlling dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

[000194] The indefinite articles "one" and "an", as used herein, in the specification and in the claims, unless otherwise specified, should be understood to mean "at least one".

[000195] The phrase "and", "as used herein, in the specification and in the claims, should be understood as "one or both" of the elements together, that is, elements that are conjunctions present in some cases and alternatives present in other cases. Multiple elements listed with "and/or" should be interpreted in the same way, e.g. "one or more" of the elements are joined. Optionally, elements other than the elements specifically identified by the "and/or" clause may be present, related or unrelated to those specifically identified elements. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with general language such as "comprising" may refer, in one embodiment, to just A (optionally including elements that are not B); in another embodiment, for just B (optionally including elements other than A); in another embodiment, for A and B (optionally including other elements); etc.

[000196] As used herein in the specification and in the claims, "or" is to be understood as having the same meaning as "and/or" as defined above. For example, when separating items in a list, "either" or "and/or" should be interpreted as being inclusive, that is, the inclusion of at least one, but also including more than one, of a number or a list of elements, and, optionally, of additional unlisted items. Only terms clearly indicating otherwise, such as "just one of" or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements . In general, the term "or" as used herein should only be construed to indicate exclusive alternatives (i.e., "one or the other, but not both") when preceded by exclusive terms such as "or", " one of,""unique", or "exactly one of". "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[000197] As used herein in the specification and claims, the phrase "at least one", in reference to a list of one or more elements, is to be understood as at least one element selected from any one or more of the elements in the element list, but not necessarily including at least one of each element specifically listed within the element list and not excluding any combinations of elements in the element list. This definition also allows elements other than specifically identified elements within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those specifically identified elements, to optionally be present. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and/or B") may refer is, in one embodiment, at least one, optionally including more than one A, with no B present (and optionally including elements other than B); in another embodiment, at least one, optionally including more than one B, without A present (and optionally including elements other than A); in another embodiment, at least optionally including more than one A and at least one, optionally including more than one B (and optionally including other elements); etc.

[000198] It should also be understood that, unless clearly indicated otherwise, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or method acts are cited.

[000199] In the claims, as well as in the above specification, all transitional phrases such as "comprising", "including", "carrying", "having", "containing," "involving", "maintaining", " composed of" and the like should be understood as being broad, that is, including, but not limited to. Only the transitional phrases, "consisting of" and "consisting essentially of" must be closed or semi-closed transitional phrases, respectively.

Claims (11)

1. Use of an antibody that specifically binds to an active plasma kallikrein (PKal) and does not bind to human prekallikrein, characterized by the fact that it is in the manufacture of a medicine to treat a retinal disease in a subject in need thereof, wherein the antibody comprises a heavy chain complementarity determining region 1 (HC CDR1) shown as HYIMM (SEQ ID NO: 5), an HC CDR2 shown as GIYSSGGITVYADSVKG (SEQ ID NO: 6), and an HC CDR3 shown as RRTGVPRWDDFDI (SEQ ID NO: 45); a light chain complementarity determining region 1 (LC CDR) shown as RASQSISSWLA (SEQ ID NO: 8), an LC CDR2 shown as KASTLES (SEQ ID NO: 9), and an LC CDR3 shown as QQYNTYWT (SEQ ID NO: 10). 2. Use according to claim 1, characterized in that the retinal disease is selected from the group consisting of diabetic macular edema (DME), age-related macular degeneration (AMD), retinal vein occlusion ( RVO), uveitis, endophthalmitis, or polypoidal choroidal vasculopathy (PCV). 3. Use according to claim 1, characterized by the fact that the retinal disease is DME. 4. Use according to claim 1 or 2, characterized in that the antibody specifically binds to the catalytic domain of human PKal. 5. Use according to any one of claims 1 to 4, characterized in that the antibody inhibits the activity of active PKal by at least 80%. 6. Use according to any one of claims 1 to 5, characterized in that the antibody has an apparent Ki (Ki,app) lower than 1 nM. 7. Use according to any one of claims 1 to 6, characterized in that the antibody has a binding affinity (KD) for active PKal of less than 10-6 M. 8. Use according to any one of claims 1 to 7, characterized in that the antibody comprises a heavy chain variable domain (HC) set forth as EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYIMMWVRQAPGKGLEWVS GIYSSGGITVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAFRR TGVPRWDDFDIWGQGTMVTVSSASTKGPSVFPLAPS SKS (SEQ ID NO: 61); and a light chain variable domain (LC) set forth as QDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPNLLIYK ASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYWTFGQG TKVEIK (SEQ ID NO: 62). 9. Use according to any one of claims 1 to 8, characterized in that the antibody is a complete antibody or an antigen-binding fragment thereof; and/or the antibody is a human antibody or humanized antibody. 10. Use according to any one of claims 1 to 9, characterized in that the antibody is a Fab. 11. Use according to any one of claims 1 to 10, characterized by the fact that the medicine is formulated for intravitreal injection.