CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62/473,996, filed March 20, 2017, entitled "Heavy Chain Only Antibodies to VEGF," the content of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The described invention generally relates to antibodies and their therapeutic methods of use.

BACKGROUND

[0003] Angiogenesis, the formation of new blood vessels from preexisting vasculature, is a major component in several retinal vascular diseases causing blindness, such as retinopathy of prematurity, proliferative diabetic retinopathy, diabetic macular edema, and age-related macular degeneration. Ocular neovascularization is the abnormal or excessive formation of blood vessels in the eye. Ocular neovascularization has been shown to be relevant in both diabetic retinopathy and age-related macular degeneration.

[0004] Age-related macular degeneration (AMD) is a leading cause of blindness in the elderly population and is recognized as dry and wet AMD forms. The dry, or nonexudative, form involves both atrophic and hypertrophic changes of the retinal pigment epithelium (RPE). The dry form is characterized by macular drusen which are pigmented areas containing dead cells and metabolic products that distort the retina and eventually cause loss of acute vision. Patients with nonexudative AMD (dry form) can progress to the wet, or exudative or neovascular, AMD, in which pathologic choroidal neovascular membranes (CNVM) develop under the retina, leak fluid and blood, and, ultimately, cause a centrally blinding disciform scar over a relatively short time frame if left untreated. Choroidal neovascularization (CNV), the growth of new blood vessels from the choroid capillary network across the Bruch's membrane/RPE interface into the neural retina, results in retinal detachment, subretinal and intraretinal edema, and scarring.

[0005] Diabetes can affect the eye in a number of ways. Diabetic retinopathy (DR) is a complication of diabetes that results from damage to the blood vessels of the light-sensitive tissue at the back of the eye (the retina). At first, diabetic retinopathy may cause no symptoms or only mild vision problems. Eventually, however, diabetic retinopathy can result in blindness. Diabetic macular edema (DME) is the swelling of the retina in diabetes mellitus due to leaking of fluid from blood vessels within the macula.

SUMMARY

[0006] Disclosed herein are monospecific heavy chain only antibodies (HCAb) having specificity for VEGF, bispecific antibodies having specificity for VEGF and either ANG-2 or PDGF, or trispecific antibodies having specificity for VEGF, PDGF, and ANG-2.

[0007] In some embodiments, heavy chain only antibodies (HCAb) are disclosed with specificity for VEGF. In some embodiments, the VEGF is VEGF-A. In certain embodiments, the HCAb has the variable heavy (VH) region sequence of one of SEQ ID NOs: 5, 9, or 13. In other embodiments, the HCAb VH region sequence has at least 85% or 95% identity to the full length sequence of one of SEQ ID NOs: 5, 9, or 13.

[0008] In some embodiments, one or more of the complementarity determining regions (CDR) of the HCAb are selected from SEQ ID NOs:6-8 and 10-12. In certain embodiments, the HCAb CDRs have at least 85% or 95% identity to the full length CDR sequence selected from SEQ ID NOs:6-8 and 10-12.

[0009] In some embodiments, the HCAb with specificity for VEGF-A comprises the CDR of SEQ ID NO:6. In some embodiments, the HCAb with a specificity for VEGF-A comprises the CDR of SEQ ID NO:7. In some embodiments, the HCAb with specificity for VEGF-A comprises the CDR of SEQ ID NO:8. In some embodiments, the HCAb with specificity for VEGF-A comprises the CDR of SEQ ID NO:10. In some embodiments, the HCAb with specificity for VEGF-A comprises the CDR of SEQ ID NO: 11. In some embodiments, the HCAb with specificity for VEGF-A, comprises the CDR of SEQ ID NO:12.

[0010] In some embodiments, at least one amino acid of the VH region is substituted, added, or deleted and the HCAb retains its specificity for VEGF-A.

[0011] Also disclosed are human or humanized antibodies which compete for binding to VEGF-A with HCAb LA-C5, LA-D6, and/or Hu-C5.

[0012] In some embodiments, bispecific antibodies having a first specificity for VEGF and a second specificity for PDGF are provided. In certain embodiments, the first specificity is represented by the HCAb of any one of claims 1-14. In certain embodiments, the second specificity is represented by a VH region selected from P36F3 (SEQ ID NO: 16), P36E10 (SEQ ID NO:17), P36E8 (SEQ ID NO:18), P36C12 (SEQ ID NO:19), P36A4 (SEQ ID NO:20), P36A3 (SEQ ID NO:21), P36D9 (SEQ ID NO:22), P36E4 (SEQ ID NO:23), P36E9 (SEQ ID NO:24), P36G9 (SEQ ID NO:25), and P36H4 (SEQ ID NO:26).

[0013] In some embodiments, a bispecific antibody having a first specificity for VEGF and a second specificity for ANG-2 is provided. In certain embodiments, the first specificity is represented by the HCAb of any one of claims 1-14. In some embodiments, the second specificity is represented by a VH region selected from A33A8 (SEQ ID NO:27), A1G2 (SEQ ID NO:28), A1F8 (SEQ ID NO:29), A2B6 (SEQ ID NO:30), and A1B1 (SEQ ID NO:31). In one embodiment, the bispecific antibody has the amino acid sequence of SEQ ID NO: 15.

[0014] Also disclosed is a trispecific antibody having a first specificity for VEGF, a second specificity for ANG-2, and a third antigen for PDGF. In some embodiments, the trispecific antibody has a heavy chain sequence of SEQ ID NO:32. In certain embodiments, the trispecific antibody optionally includes a light chain sequence of SEQ ID NO:33.

[0015] Also disclosed herein are methods of treating ophthalmologic disorders comprising administering to a subject in need thereof, a VEGF-binding HCAb having a VH region disclosed herein, or a bispecific or trispecific antibody disclosed herein.

[0016] Also disclosed herein is the use of a VEGF-binding HCAb having a VH region disclosed herein, or a bispecific or trispecific antibody disclosed herein, in the manufacture of a medicament for treating an ophthalmologic disorder in a subject in need thereof.

[0017] In some embodiments, the ophthalmologic disorder comprises age-related macular degeneration (AMD), choroidal neovascularization (CNV), cystoid macula edema (CME), myopia-associated choroidal neovascularization, vascular streaks, diabetic macular edema (DME), macular edema, retinal vein occlusion, abnormal corneal angiogenesis, pterygium conjunctivae, subretinal edema, or intraretinal edema. In some embodiments, the abnormal corneal angiogenesis is as a result of keratitis, corneal transplantation, keroplasty or hypoxia.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 depicts the HCAb antibody structure.

[0019] FIG. 2A-G depicts several forms of multivalent antibodies including bispecific single domain VH (FIG. 2A); bi-specific HCAb antibodies (FIG. 2B and 2C); tetrameric antibody/VH bispecific antibodies (FIG. 2D); bispecific Fab/VH antibodies (FIG. 2E); heavy/light chain bispsecific antibodies (FIG. 2F); and a trispecific antibody (FIG. 2G). The second and third antigen-binding domains are depicted as ovals. Optional locations for the second antigen- binding domain are indicated by asterisks.

[0020] FIG. 3 depicts the gene structure of humanized HCAb disclosed herein.

[0021] FIG. 4 depicts the effects of Hu-C5 on VEGF-dependent calcium mobilization in human umbilical vein endothelial cells.

[0022] FIG. 5 depicts the structure of a bispecific anti-VEGF-A/ANG-2 antibody.

[0023] FIG. 6A-C depicts to different assays of affinity binding of bispecific anti-VEGF- A/ANG-2 HCAb using a forteBio OCTET® QK ^e system equipped with anti-hlgG Fc capture biosensor tips and strepavidin-coated biosensors.

[0024] FIG. 7A-F depicts six different assays of Octet real-time binding of the trispecific antibody to VEGF-A, PDGF BB and ANG-2.

[0025] FIG. 8 depicts an exemplary trispecific antibody.

DETAILED DESCRIPTION

[0026] Disclosed herein are monospecific heavy chain only antibodies (HCAb) having specificity for vascular endothelial growth factor (VEGF), bispecific antibodies having specificity for VEGF and angiopoietin-2 (ANG-2), or for VEGF and platelet derived growth factor (PDGF), and trispecific antibodies having specificity for VEGF, PDGF, and ANG-2.

[0027] The VEGF family in mammals is comprised of five members: VEFG-A, placenta growth factor (PGF), VEGF-B, VEGF-C, and VEGF-D. All members of the VEGF family stimulate cellular responses by binding to tyrosine kinase receptors (the VEGFRs) on the cell surface, causing them to dimerize and become activated through transphosphorylation, although to different sites, times, and extents. The VEGF receptors have an extracellular portion consisting of seven immunoglobulin-like domains, a single transmembrane spanning region, and an intracellular portion containing a split tyrosine-kinase domain. VEGF-A binds to VEGFR- 1 (Flt-1) and VEGFR-2 (KDR/Flk-1). VEGFR-2 appears to mediate almost all of the known cellular responses to VEGF. The function of VEGFR-1 is less well-defined, although it is thought to modulate VEGFR-2 signaling. Another function of VEGFR-1 may be to act as a dummy/decoy receptor, sequestering VEGF from VEGFR-2 binding (this appears to be particularly important during vasculogenesis in the embryo). VEGF-C and VEGF-D, but not VEGF-A, are ligands for a third receptor (VEGFR-3/Flt4), which mediates lymphangiogenesis. VEGFR-3 is the site of binding of main ligands (VEGF-C and VEGF-D), which mediates perpetual action and function of ligands on target cells. VEGF-C stimulates lymphangiogenesis (via VEGFR-3) and angiogenesis via VEGFR-2. VEGF-A is a 232 amino acid sequence (UniProtKB - P15692; M N FLLSWVH WSLALLLYLH H AKWSQAAPM AEGGGQN H H EWKFM D VYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQ IMRIK PHQGQHIGEMSFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVYVGAR CCLMPWSLPGPHPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKP R

R; SEQ ID NO:1).

[0028] Thus, disclosed herein are monospecific and bispecific HCAb antibodies to VEGF and methods of treating ophthalmological disorders using the disclosed antibodies.

[0029] Antibodies for treatment of diseases are well known in the art. As used herein, the term "antibody" refers to a monomeric or multimeric protein comprising one or more polypeptide chains that comprise antigen-binding sites. An antibody binds specifically to an antigen and may be able to modulate the biological activity of the antigen. As used herein, the term "antibody" can include "full length antibody" and "antibody fragments." The terms "binding site" or "antigen- binding site" as used herein denote the region(s) of an antibody molecule to which a ligand actually binds.

[0030] Antibody specificity refers to selective recognition of the antibody for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. The term "monospecific" antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen. The monovalent, monospecific antibodies disclosed herein are specific for VEGF. As used herein, "specificity" and "antigen- binding specificity" are used interchangeably and refer to the same activity.

[0031] "Bispecific antibodies" refers to antibodies which have two different antigen-binding specificities. Bispecific antibodies disclosed herein have a first specificity for VEGF and a second specificity for PDGF or ANG-2.

[0032] The term "trispecific antibodies" refers to antibodies which have three different antigen-binding specificities. Trispecific antibodies disclosed herein have specificities for VEGF, PDGF, and ANG-2.

[0033] The term "valenf as used herein denotes the presence of a specified number of binding sites in an antibody molecule. As such, the terms "bivalent", "tetravalent" , and "hexavalenf denote the presence of two binding site, four binding sites, and six binding sites, respectively, in an antibody molecule. The bispecific antibodies disclosed herein are "bivalent." However, monospecific bivalent antibodies are within the scope of the present disclosure in which the two antigen-binding sites bind the same antigen. The antigen-binding sites of monospecific bivalent antibodies can bind either the same epitope or different epitopes on the antigen. The trispecific antibodies disclosed herein are "trivalent."

[0034] By "full length antibody" herein is meant the structure that constitutes the natural biological form of an antibody, including variable and constant regions. For example, in most mammals, including humans and mice, the full length antibody of the IgG class is a tetramer and consists of two identical pairs of two immunoglobulin chains, each pair having one light and one heavy chain, each light chain comprising immunoglobulin domains VL and CL, and each heavy chain comprising immunoglobulin domains VH, CH1, CH2, and CH3. In some mammals, for example in camels and llamas, IgG antibodies consist of only two heavy chains (HCAb), each heavy chain comprising a variable domain attached to the Fc region (CH2 and CH3 domains).

[0035] Natural antibody structural units typically comprise a tetramer. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one "light" (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). Each of the light and heavy chains are made up of two distinct regions, referred to as the variable and constant regions. For the IgG class of immunoglobulins, the heavy chain is composed of four immunoglobulin domains linked from N- to C-terminus in the order VH-CH1-CH2-CH3, referring to the heavy chain variable domain, heavy chain constant domain 1, heavy chain constant domain 2, and heavy chain constant domain 3 respectively (also referred to as VH-CY1-CV2-CV3, referring to the heavy chain variable domain, constant gamma 1 domain, constant gamma 2 domain, and constant gamma 3 domain respectively). The IgG light chain is composed of two immunoglobulin domains linked from N- to C-terminus in the order VL-CL, referring to the light chain variable domain and the light chain constant domain respectively. The constant regions show less sequence diversity, and are responsible for binding a number of natural proteins to elicit important biochemical events.

[0036] The variable region of an antibody contains the antigen binding determinants of the molecule, and thus determines the specificity of an antibody for its target antigen. The variable region is so named because it is the most distinct in sequence from other antibodies within the same class. In the variable region, three loops are gathered for each of the V domains of the heavy chain and light chain to form an antigen-binding site. Each of the loops is referred to as a complementarity-determining region (hereinafter referred to as a "CDR"), in which the variation in the amino acid sequence is most significant. There are six CDRs total, three each per heavy and light chain, designated VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. The variable region outside of the CDRs is referred to as the framework (FR) region. Although not as diverse as the CDRs, sequence variability does occur in the FR region between different antibodies. Overall, this characteristic architecture of antibodies provides a stable scaffold (the FR region) upon which substantial antigen binding diversity (the CDRs) can be explored by the immune system to obtain specificity for a broad array of antigens.

[0037] Determination of CDR regions in an antibody is well known to those skilled in the art. There are at least two techniques for determining CDRs: (1) an approach based on cross- species sequence variability (Kabat et al. Sequences of Proteins of Immunological Interest, 5th ed., 1991, National Institutes of Health, Bethesda Md.); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Chothia et al. (1989) Nature 342:877; Al-lazikani et al (1997) J. Molec. Biol. 273:927-948)). As used herein, a CDR may refer to CDRs defined by either approach or by a combination of both approaches.

[0038] The genes encoding the immunoglobulin locus comprise multiple V region sequences along with shorter nucleotide sequences named "D" and "J" and it is the combination of the V, D, and J nucleotide sequence that give rise to the VH diversity.

[0039] Antibodies are grouped into classes, also referred to as isotypes, as determined genetically by the constant region. Human constant light chains are classified as kappa (CK) and lambda (CA) light chains. Heavy chains are classified as mu (μ), delta (δ), gamma (v), alpha (a), or epsilon (ε), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG class is the most commonly used for therapeutic purposes. In humans this class comprises subclasses lgG1, lgG2, lgG3, and lgG4. In mice this class comprises subclasses lgG1, lgG2a, lgG2b, lgG3. IgM has subclasses, including, but not limited to, lgM1 and lgM2. IgA has several subclasses, including but not limited to lgA1 and lgA2. Thus, "isotype" as used herein is meant any of the classes or subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. The known human immunoglobulin isotypes are lgG1, lgG2, lgG3, lgG4, lgA1, lgA2, lgM1, lgM2, IgD, and IgE. The disclosed HCAb antibodies and bispecific antibodies can have constant regions comprising all, or part, of the above-described isotypes. [0040] Also within the scope of the present disclosure are antibody fragments including, but are not limited to, (i) a Fab fragment comprising VL, CL, VH, and CH1 domains, (ii) a Fd fragment comprising VH and CH1 domains, (iii) a Fv fragment comprising VL and VH domains of a single antibody; (iv) a dAb fragment comprising a single variable region, (v) isolated CDR regions, (vi) a F(ab') ₂ fragment, a bivalent fragment comprising two linked Fab fragments, and (vii) a single chain Fv molecule (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site. In certain embodiments, antibodies are produced by recombinant DNA techniques. In additional embodiments, antibodies are produced by enzymatic or chemical cleavage of naturally occurring antibodies.

[0041] By "humanized" antibody as used herein is meant an antibody comprising a human framework region (FR) and one or more complementarity determining regions (CDR's) from a non-human (usually mouse or rat but other species are possible) antibody. The non-human antibody providing the CDR's is called the "donor" and the human immunoglobulin providing the framework is called the "acceptor." In certain embodiments, humanization relies principally on the grafting of donor CDRs onto acceptor (human) VL and VH frameworks. This strategy is referred to as "CDR grafting." "Backmutation" of selected acceptor framework residues to the corresponding donor residues is often required to regain affinity that is lost in the initial grafted construct. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, and thus will typically comprise a human Fc region. Humanization or other methods of reducing the immunogenicity of nonhuman antibody variable regions may include resurfacing methods. In one embodiment, selection based methods may be employed to humanize and/or affinity mature antibody variable regions, that is, to increase the affinity of the variable region for its target antigen. Other humanization methods may involve the grafting of only parts of the CDRs, including but not limited to methods described in US 6,797,492, incorporated by reference herein for all it discloses regarding CDR grafting. Structure-based methods may be employed for humanization and affinity maturation, for example as described in US 7,117,096, incorporated by reference herein for all it discloses regarding humanization and affinity maturation.

[0042] In various embodiments herein, the antibodies are heavy chain only antibodies (HCAb). Camelids (camels, dromedary, and llamas) contain, in addition to normal heavy and light chain antibodies (2 light chains and 2 heavy chains in one antibody), single chain antibodies containing only heavy chains (see FIG. 1). These are coded for by a distinct set of VH segments referred to as VHH genes. The VH and VHH are interspersed in the genome (i.e., they appear mixed in between each other). The identification of an identical D segment in a VH and VHH cDNA suggests the common use of the D segment for VH and VHH. Natural VHH- containing antibodies are missing the entire CH1 domain of the constant region of the heavy chain. The exon coding for the CH1 domain is present in the genome but is spliced out due to the loss of a functional splice acceptor sequence at the 5' side of the CH1 exon. As a result the VDJ region is spliced onto the CH2 exon. When a VHH is recombined onto such constant regions (CH2, CH3), an antibody is produced that acts as a single chain antibody (i.e., an antibody of two heavy chains without a light chain interaction). Binding of an antigen is different from that seen with a conventional antibody, but high affinity is achieved the same way, i.e., through hypermutation of the variable region and selection of the cells expressing such high affinity antibodies.

[0043] In an exemplary embodiment, HCAb are produced by immunizing a transgenic mouse in which endogenous murine antibody expression has been eliminated and human transgenes have been introduced. HCAb mice are disclosed in US8,883,150, US8,921,524, US8.921.522, US8.507.748, US8.502.014, US 2014/0356908, US2014/0033335, US2014/0037616, US2014/0356908, US2013/0344057, US2013/0323235, US2011/0118444, and US2009/0307787, all of which are incorporated herein by reference for all they disclose regarding heavy chain only antibodies and their production in transgenic mice. The HCAb mice are immunized and the resulting primed spleen cells fused with a murine myeloma cells to form hybridomas. The resultant HCAb can then be made fully human by replacing the murine CH2 and CH3 regions with human sequences.

[0044] In other exemplary embodiments, HCAb are produced by immunizing a camelid, such as a llama, with the desired antigen and obtaining primed peripheral blood mononuclear cells (PBMC) from the animal. RNA isolated from the PBMC can be used to generate a phage display library to isolate antigen-specific VHH sequences.

[0045] Also disclosed herein are bifunctional antibodies in which two antigen binding domains are joined in a single bispecific molecule. Bifunctional antibodies can take many forms including (i) bi-specific Fv fragments (FIG. 2A); (ii) HCAb of a first specificity having associated therewith a second VH domain having a second specificity (FIG. 2B and 2C); (iii) tetrameric monoclonal antibodies with a first specificity having associated therewith with a second VH domain having a second specificity, wherein the second VH domain is associated with a first VH domain (FIG. 2D); and (iv) Fab fragments (VH-CH1/VLJCL) of a first specificity having associated therewith a second VH domain with a second specificity (FIG. 2E). Exemplary Fab fragments are depicted in FIG. 2E in which the second VH sequence having the second specificity is associated with the C-terminus or the N-terminus of the first VH domain, or the C- terminus or the N-terminus of the first CH1 or first CL domains. In additional embodiments also depicted in FIG. 2E, VH sequences having a second and/or a third specificity can be associated with the C-terminus or the N-terminus of the first VH domain, or the C-terminus or the N- terminus of the first CH1 or first CL domains. Also disclosed herein are antibodies in which three antigen binding domains are joined in a single trispecific molecule. The same methods used to produce bispecific antibodies can be used to produce trispecific antibodies.

[0046] Bispecific and trispecific antibodies may include linker sequences linking a sequence of a VEGF-binding antibody disclosed herein, to a VH region with a second (or a third) specificity which allows for proper folding of the sequences to generate the desired three- dimensional conformation and antigen binding profiles. Suitable linkers include, but are not limited to, EPKSCD (SEQ ID NO:2), ASTKGP (SEQ ID NO:3), and (GGGGS)„ (SEQ ID NO:4), wherein n is an integer between 0 and 8. In one embodiment, n is 1.

[0047] The bispecific antibodies disclosed herein are bivalent comprising a first specificity to VEGF, and a second specificity can include, but is not limited to, PDGF or ANG-2. Within the scope of the present disclosure are bispecific antibodies wherein the first specificity and the second specificity are independently ANG-2, VEGF, or PDGF, with the only limitation that the first and second specificity cannot be the same.

[0048] Human angiopoietins-1 and -2 (ANG-1 and ANG-2 (UniProtKB-015123; alternatively abbreviated with ANGPT2 or ANG2)) were discovered as ligands for Ties, a family of tyrosine kinases that is selectively expressed within the vascular endothelium. There are four definitive members of the angiopoietin family. Angiopoietin-3 and -4 (ANG-3 and ANG-4) may represent widely diverged counterparts of the same gene locus in mouse and man. ANG-1 and ANG-2 were originally identified in tissue culture experiments as agonist and antagonist, respectively. All of the known angiopoietins bind primarily to Tie-2. ANG-1 supports endothelial cell (EC) survival and to promote endothelium integrity, whereas ANG-2 had the opposite effect and promoted blood vessel destabilization and regression in the absence of the survival factors VEGF or basic fibroblast growth factor. However, many studies of ANG-2 function have suggested a more complex situation. ANG-2 might be a complex regulator of vascular remodeling that plays a role in both vessel sprouting and vessel regression. Supporting such roles for ANG-2, expression analysis reveals that ANG-2 is rapidly induced, together with VEGF, in adult settings of angiogenic sprouting, whereas ANG-2 is induced in the absence of VEGF in settings of vascular regression. Consistent with a context-dependent role, ANG-2 specifically binds to the same endothelial-specific receptor, Tie-2, which is activated by ANG-1, but has context-dependent effects on its activation.

[0049] ANG-1 and ANG-2 have similar effects in corneal angiogenesis assays, acting synergistically with VEGF to promote growth of new blood vessels. At high concentration, ANG- 2 acts as an apoptosis survival factor for endothelial cells during serum deprivation apoptosis through activation of Tie-2 via PI-3 Kinase and Akt pathway. The role of ANG-1 is thought to be conserved in the adult, where it is expressed widely and constitutively. In contrast, ANG-2 expression is primarily limited to sites of vascular remodeling where it is thought to block the constitutive stabilizing or maturing function of ANG-1, allowing vessels to revert to, and remain in, a plastic state which may be more responsive to sprouting signals. ANG-2 is expressed during development at sites where blood vessel remodeling is occurring. In adult individuals, ANG-2 expression is restricted to sites of vascular remodeling as well as in highly vascularized tumors. ANG-2 is required for postnatal angiogenesis. Developmental^ programmed regression of the hyaloid vasculature in the eye does not occur in ANG-2 knockout mice and their retinal blood vessels fail to sprout out from the central retinal artery. Deletion of ANG-2 results in profound defects in the patterning and function of the lymphatic vasculature. Genetic rescue with ANG-1 corrects the lymphatic, but not the angiogenesis defects.

[0050] PDGF plays a significant role in blood vessel formation (angiogenesis), the growth of blood vessels from already-existing blood vessel tissue. PDGF is a potent mitogen for cells of mesenchymal origin, including fibroblasts, smooth muscle cells and glial cells. PDGF is a dimeric glycoprotein comprised of two A (-AA) or two B (-BB) subunits, or a combination of the two (-AB). The A subunit is a 211 amino acid sequence (UniProtKB - Ρ04085;) and the B subunit is a 241 amino acid sequence (UniProtKB - P01127). Thus, in various embodiments, an antibody is disclosed having specificity for PDGF-AA, PDGF-BB, and/or PDGF-AB, or a fragment thereof. In both mouse and human, the PDGF signaling network consists of four ligands, PDGFA-D, and two receptors, PDGFRalpha and PDGFRbeta (receptor tyrosine kinases). All PDGFs function as secreted, disulfide-linked homodimers, but only PDGFA and B can form functional heterodimers. Thus, in certain embodiments, the PDGF is PDGF-AA, PDGF-BB, or PDGF-AB.

[0051] Thus, disclosed herein are HCAb specific for VEGF, bispecific antibodies specific for both VEGF and ANG-2 or specific for both VEGF and PDGF. In other embodiments, the VEGF/PDGF bispecific antibody is formed from the human anti-VEGF A HCAb antibody Hu-C5 disclosed herein and the VH and/or VL regions of any human or humanized PDGF-specific antibody. Exemplary humanized anti-PDGF antibodies are disclosed in WO2014/072876, WO2005087812, and WO2014109999, which are incorporated by reference herein for all they disclose regarding PDGF-specific antibodies. Exemplary PDGF antibodies include VH regions represented by P36F3, P36E10, P36E8, P36C12, P36A4, P36A3, P36D9, P36E4, P36E9, P36G9, and P36H4 which are disclosed in International patent application PCT/US2016/046595 filed on August 11, 2016, which is incorporated by reference for all it discloses regarding PDGF- specific antibodies.

[0052] In other embodiments, the VEGF/ANG-2 bispecific antibody is formed from the human anti-VEGF-A HCAb antibody Hu-C5 disclosed herein and the VH and/or VL regions of any human or humanized ANG-2-specific antibody. Exemplary humanized anti-ANG-2 antibodies are disclosed in, for example, US2010/0159587 and US20130259868, which are incorporated by reference herein for all they disclose regarding anti-ANG-2 antibodies. Exemplary ANG-2-binding antibodies include VH regions represented by A33A8, A1G2, A1F8, A2B6, and A1B1 which are disclosed in International patent application PCT/US2016/044838 filed on July 29, 2016, which is incorporated by reference for all it discloses regarding ANG-2- binding antibodies.

[0053] Also disclosed herein are trispecific antibodies which have specificity for all of VEGF, ANG-2, and PDGF. Trispecific antibodies as disclosed herein include three VH sequences disclosed herein, wherein each of the VH regions has a different specificity. Optionally one of the VH regions can be part of an antibody heavy chain. Optionally between the VH regions and/or heavy chain is one or more linkers as disclosed herein. In one embodiment, the trispecific antibody is represented by VH (specificity 1)-heavy chain (specificity 2)-linker-VH (specificity 3). The first, second, and third specificities can be represented by any of SEQ ID NOs.:39, 43, 47, 49-60, and 61-65. The trispecific antibody can be a HCAb or comprise both a heavy chain and a light chain.

[0054] An exemplary trispecific antibody has an amino acid sequence having at least 85%, 90%, 95%, or 98% sequence identity to the full length heavy chain of SEQ ID NO:48 and optionally light chain of SEQ ID NO:49.

[0055] Also within the scope of the present disclosure are amino acid sequence variants of the human anti-VEGF monospecific, bispecific, or trispecific antibodies prepared by introducing appropriate nucleotide changes into the antibody DNA, or by peptide synthesis. Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibodies of the examples herein. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter post- translational processes of the humanized or variant antibodies, such as changing the number or position of glycosylation sites.

[0056] A useful method for identification of certain residues or regions of the antibodies that are preferred locations for mutagenesis is called "alanine scanning mutagenesis." A residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis is conducted at the target codon or region and the expressed antibody variants are screened for the desired activity.

[0057] Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an anti-VEGF antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag. Other insertional variants of the antibody molecules include the fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide which increases the serum half-life of the antibody.

[0058] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions." If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions" in Table 1, or as further described below in reference to amino acid classes, may be introduced and the products screened. Table 1

[0059] Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:

(1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, lie;

(2) Neutral hydrophilic: Cys, Ser, Thr;

(3) Acidic: Asp, Glu;

(4) Basic: Asn, Gin, His, Lys, Arg;

(5) Residues that influence chain orientation: Gly, Pro; and

(6) Aromatic: Trp, Tyr, Phe. [0060] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0061] Any cysteine residue not involved in maintaining the proper conformation of the monospecific, bispecific, or trispecific anti-VEGF antibodies also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

[0062] Another type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene Mil product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identified hypervariable region residues contributing significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and human VEGF. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.

[0063] Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody.

[0064] Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0065] Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0066] Nucleic acid molecules encoding amino acid sequence variants of the monospecific, bispecific, or trispecific anti-VEGF antibodies are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of an anti-VEGF antibody.

[0067] Other modifications of the monospecific, bispecific, or trispecific anti-VEGF antibodies are contemplated. For example, it may be desirable to modify the antibodies with respect to effector function, so as to enhance the effectiveness of the antibody in treating disease, for example. For example cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement- mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers. Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities.

[0068] Also within the scope of the present disclosure are anti-VEGF HCAb having at least 85% identity, at least 90% identity, at least 93% identity, at least 96% identity, or at least 98% identity to sequence of an anti-VEGF HCAb disclosed herein.

[0069] Exemplary variants, or mutated anti-VEGF HCAb, comprise one or more of the sequences of SEQ ID NOs:5, 9, and 13, or one or more of the CDRs selected from SEQ ID NOs: 6-8 and 10-12, wherein one or more amino acids are substituted with an amino acid according to Table 1. In some embodiments, up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 12, up to 14, up to 16, up to 18, or up to 20 amino acids are added, substituted, or deleted with the proviso that the substituted sequence retains the binding specificity, or has an increased binding specificity, of the un-substituted sequence. In other embodiments, up to 1 , up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 12, up to 14, up to 16, up to 18, or up to 20 amino acids are added, substituted, or deleted with the proviso that the substituted sequence retains at least 85% identity, at least 90% identity, at least 93% identity, at least 96% identity, or at least 98% identity to entire length of the un-substituted sequence.

[0070] In certain embodiments, the variant, or mutated anti-VEGF HCAb includes one or more additions, substitutions, or deletions in the variable region. In some embodiments, the variant, or mutated anti-VEGF HCAb includes one or more additions, substitutions, or deletions in one or more complementarity determining regions. In some embodiments, the variant, or mutated anti-VEGF HCAb includes one or more additions, substitutions, or deletions in one or more framework region. In yet more embodiments, the variant, or mutated anti-VEGF HCAb includes one or more additions, substitutions, or deletions in one or more constant regions.

[0071] In certain embodiments, the variant, or mutated anti-VEGF HCAb includes one or more additions, substitutions, or deletions in CDR1. In some embodiments, the variant, or mutated anti-VEGF HCAb includes one or more additions, substitutions, or deletions in CDR2. In some embodiments, the variant, or mutated anti-VEGF HCAb includes one or more additions, substitutions, or deletions in CDR3.

[0072] In certain embodiments, the variant, or mutated anti-VEGF HCAb includes one or more additions, substitutions, or deletions in the framework 1 region. In some embodiments, the variant, or mutated a anti-VEGF HCAb includes one or more additions, substitutions, or deletions in the framework 2 region. In some embodiments, the variant, or mutated anti-VEGF HCAb includes one or more additions, substitutions, or deletions in the framework 3 region. In some embodiments, the variant, or mutated anti-VEGF HCAb includes one or more additions, substitutions, or deletions in the framework 4 region. In some embodiments, the variant, or mutated anti-VEGF HCAb includes one or more additions, substitutions, or deletions in the hinge region. [0073] Also disclosed herein are immunoconjugates comprising the antibody described herein conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

[0074] Chemotherapeutic agents useful in the generation of such immunoconjugates have been described. Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, botulinum toxins, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated monospecific or bispecific anti- PDGF antibodies. Examples include ^{2 2} Bi, ¹³¹ l, ³ ln, °°Y, and ^18e Re.

[0075] Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis- azido compounds (such as bis (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6- diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Carbon- 14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX- DTPA) is an exemplary chelating agent for conjugation of radionucleotide to an antibody.

[0076] In another embodiment, an antibody may be conjugated to a "receptor" (such streptavidin) for utilization in pre-targeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "Ngand" (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a radionuclide).

[0077] The monospecific, bispecific, or trispecific anti-VEGF antibodies disclosed herein may also be formulated in liposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in US4,485,045, US4,544,545, and US5.013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibodies can be conjugated to the liposomes via a disulfide interchange reaction.

[0078] Covalent modifications of the monospecific, bispecific, or trispecific anti-VEGF antibodies are also included within the scope of this disclosure. They may be made by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if applicable. Other types of covalent modifications of the antibodies are introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues. Exemplary covalent modifications of polypeptides are described in US5,534,615, specifically incorporated herein by reference for all it discloses regarding covalent modifications of polypeptides. An exemplary type of covalent modification of the antibody comprises linking the antibody to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in US4,640,835, US4,496,689, US4,301,144, US4.670.417, US4.791.192, or US4.179.337.

[0079] The monospecific, bispecific, and trispecific antibodies disclosed herein may be produced by recombinant means. Thus, disclosed herein are nucleic acids encoding the antibodies, expression vectors containing nucleic acids encoding the antibodies, and cells comprising the nucleic acid encoding the antibodies. Methods for recombinant production are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody and usually purification to a pharmaceutically acceptable purity. For the expression of the antibodies as aforementioned in a host cell, nucleic acids encoding the antibody sequences are inserted into expression vectors by standard methods. Expression is performed in appropriate prokaryotic or eukaryotic host cells like CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, or E. coli cells, and the antibody is recovered from the cells (supernatant or cells after lysis).

[0080] Accordingly certain embodiments disclosed herein include a method for the preparation of a monospecific, bispecific, and trispecific antibody, comprising the steps of a) transforming a host cell with at least one expression vector comprising nucleic acid molecules encoding the antibody; b) culturing the host cell under conditions that allow synthesis of the antibody molecule; and c) recovering said antibody molecule from the culture. [0081] The antibodies are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0082] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

[0083] The term "transformation" as used herein refers to process of transfer of a vectors/nucleic acid into a host cell. If cells without formidable cell wall barriers are used as host cells, transfection can be carried out e.g. by the calcium phosphate precipitation method. However, other methods for introducing DNA into cells such as by nuclear injection or by protoplast fusion may also be used. If prokaryotic cells or cells which contain substantial cell wall constructions are used, e.g. one method of transfection is calcium treatment using calcium chloride.

[0084] As used herein, "expression" refers to the process by which a nucleic acid is transcribed into mRNA and/or to the process by which the transcribed mRNA (also referred to as transcript) is subsequently being translated into peptides, polypeptides, or proteins. The transcripts and the encoded polypeptides are collectively referred to as gene product. If the polynucleotide is derived from genomic DNA, expression in a eukaryotic cell may include splicing of the mRNA.

[0085] A "vector" is a nucleic acid molecule, in particular self-replicating, which transfers an inserted nucleic acid molecule into and/or between host cells. The term includes vectors that function primarily for insertion of DNA or RNA into a cell (e.g., chromosomal integration), replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the functions as described.

[0086] An "expression vector" is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide. An "expression system" usually refers to a suitable host cell comprised of an expression vector that can function to yield a desired expression product.

[0087] The term "host cell" as used herein denotes any kind of cellular system which can be engineered to generate the antibodies disclosed herein. In one embodiment HEK293 cells and CHO cells are used as host cells.

[0088] The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, enhancers and polyadenylation signals.

[0089] A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0090] Also disclosed herein are isolated nucleic acid sequences encoding the monospecific, bispecific, or trispecific anti-VEGF antibodies, vectors and host cells comprising the nucleic acids, and recombinant techniques for the production of the antibodies.

[0091] For recombinant production of the antibodies, the nucleic acid encoding it may be isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. In some embodiments, the antibody may be produced by homologous recombination, e.g. as described in US5,204,244, specifically incorporated herein by reference for all it discloses regarding antibody production. DNA encoding the antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, e.g., as described in US5,534,615, specifically incorporated herein by reference for all it discloses regarding protein expression.

[0092] Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., S. marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces. One exemplary E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. co// X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.

[0093] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for monospecific or bispecific human anti-PDGF antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yanowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

[0094] Suitable host cells for the expression of glycosylated monospecific or bispecific human anti-PDGF antibodies are derived from multicellular organisms, including invertebrate cells such as plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), A. albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa califomica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of S. frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts. [0095] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. [Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cellsADHFR (CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human hepatoma cells (Hep G2).

[0096] Host cells are transformed with the above-described expression vectors for monospecific, bispecific, or trispecific anti-VEGF antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0097] The host cells used to produce the monospecific, bispecific, or trispecific anti-VEGF antibodies may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, US4.767.704; US4.657.866; US4.927.762; US4.560.655; or US5, 122,469; WO 90/03430; WO 87/00195; or US Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

[0098] When using recombinant techniques, the antibody can be produced intracellular^, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration.

[0099] The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γ1, γ2, or γ4 heavy chains. Protein G is recommended for all mouse isotypes and for human γ3. The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX™ resin is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS- PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

[00100] Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).

[00101] Also disclosed herein are methods of using the monospecific, bispecific, or trispecific anti-VEGF antibodies for the treatment of ophthalmological disorders. Examples of ophthalmological disorders include, but are not limited to, age-related macular degeneration (AMD), choroidal neovascularization (CNV), cystoid macula edema (CME), myopia-associated choroidal neovascularization, vascular streaks, diabetic macular edema (DME), macular edema, macular edema due to retinal vein occlusion, and angiogenesis in the front of the eye like corneal angiogenesis following e.g. keratitis, corneal transplantation or keratoplasty, corneal angiogenesis due to hypoxia (extensive contact lens wearing), pterygium conjunctivae, subretinal edema and intraretinal edema. Examples of age-related macular degeneration (AMD) include but are not limited to dry or nonexudative AMD, or wet or exudative or neovascular AMD. [0100] Macular degeneration, also referred to as age-related macular degeneration, is the most common cause of vision loss in the United States in those 50 or older, and its prevalence increases with age. AMD is classified as either wet (neovascular) or dry (non-neovascular). The dry form of the disease is most common. It occurs when the central retina has become distorted, pigmented, or most commonly, thinned, a process associated with atrophy of the retinal pigment epithelium and loss of macular photoreceptors. The result is central geographic atrophy. The wet form of the disease is responsible for most severe loss of vision. The wet form is usually associated with aging, but other diseases that can cause wet macular degeneration include severe myopia and some intraocular infections such as histoplasmosis, which may be exacerbated in individuals with AIDS. The wet form is characterized by abnormal blood vessels growing through the retinal pigment epithelium, resulting in hemorrhage, exudation, scarring, or retinal detachment.

[0101] Disclosed herein are sustained release formulations of monospecific, bispecific, or trispecific anti-VEGF antibodies for treatment of ocular disorders. Thus, the monospecific, bispecific, and trispecific anti-VEGF antibodies can be released into the vitreous over a 3 to 6 month period from a sustained release drug delivery system to provide long term treatment of a chronic ocular condition such as dry AMD.

[0102] A hydrogel is a colloidal gel formed as a dispersion in water or other aqueous medium. Thus a hydrogel is formed upon formation of a colloid in which a dispersed phase (the polymer) has combined with a continuous phase (i.e., water) to produce a viscous jellylike product; for example, coagulated silicic acid. A hydrogel is a three-dimensional network of hydrophilic polymer chains that are crosslinked through either chemical or physical bonding. Because of the hydrophilic nature of the polymer chains, hydrogels absorb water and swell (unless they have already absorbed their maximum amount of water). The swelling process is the same as the dissolution of non-crosslinked hydrophilic polymers. By definition, water constitutes at least 0% of the total weight (or volume) of a hydrogel.

[0103] Examples of hydrogels include synthetic polymers such as polyhydroxy ethyl methacrylate, and chemically or physically crosslinked polyvinyl alcohol, polyacrylamide, poly(N- vinyl pyrolidone), polyethylene oxide, and hydrolysed polyacrylonitrile. Examples of hydrogels which are organic polymers include covalent or ionically crosslinked polysaccharide-based hydrogels such as the polyvalent metal salts of alginate, pectin, carboxymethyl cellulose, heparin, hyaluronate and hydrogels from chitin, chitosan, pullulan, gellan and xanthan. The particular hydrogels used in our experiment were a cellulose compound (i.e., hydroxypropylmethylcellulose [HPMC]) and a high molecular weight hyaluronic acid (HA).

[0104] In some embodiments, a hydrogel formulation for intravitreal injection is disclosed using a polymeric hyaluronic acid and the monospecific and/or bispecific human anti-PDGF antibodies disclosed herein. This drug delivery system can provide sustained-release of a low daily dose of the monospecific, bispecific, or trispecific anti-VEGF antibodies over a 3 to 6 month period and prevent of conversion from dry to wet AMD. The drug delivery system can also comprise microsphere encapsulation of the monospecific, bispecific, or trispecific anti- VEGF in the hydrogel. The sustained-release drug delivery system can provide the necessary anti-VEGF blockade in eye to reduce the chance of progression from dry to neovascular AMD. In addition, the low doses released in the eye over a prolonged period of time do not provide a systemic toxic level of the agent.

[0105] The sustained release drug delivery system can also be used to provide a sustained-release anti-VEGF blockade in patients with central retinal vein occlusion that are at risk for neovascularization and in patients with severe non-proliferative diabetic retinopathy that are at risk of progressing to neovascular disease.

[0106] Alternately, the drug delivery system can be a PLGA implant, liposomal encapsulated antibodies optionally entrapped in a cross-linked hyaluronic acid. Additionally, microspheres, microcapsules (ranging from 0.001 to 100 microns) and liposomes with modified surfaces to create an interaction with the hydrogel polymer to modify release.

[0107] Also encompassed herein are particular drug delivery system formulations and methods for administering these drug delivery systems for treating an ocular condition. Intraocular administration can be by implantation or injection into the vitreous cavity (posterior chamber) of the eye. The drug delivery systems within the scope of this disclosure can be biodegradable implants and/or microspheres. The drug delivery systems can be monolithic, that is the active agent is homogenously distributed or dispersed throughout the biodegradable polymer. The therapeutic agent can be released from drug delivery systems made according to the present invention for a period of time between about 2 hours to 12 months or more. An important feature of the drug delivery systems is that they do not include any means (such as a cap, protrusion or suture tab) for fixing the drug delivery system to the intraocular location to which it is administered. [0108] An important characteristic of a drug delivery system within the scope of the present disclosure is that it can be implanted or injected into an intraocular location (such as an anterior sub-Tenon, subconjunctival, intravitreal or suprachoroidal location) to provide sustained release of a therapeutic agent without the occurrence of or the persistence of significant immunogenicity at and adjacent to the site of the intraocular implantation or injection.

[0109] Polylactide (PLA) polymers exist in two chemical forms, poly(L-lactide) and poly(D,L-lactide). The pure poly(L-lactide) is regioregular and therefore is also highly crystalline, therefore degrades in vivo at a very slow rate. The poly(D,L-lactide) is regiorandom which leads to more rapid degradation in vivo. Therefore a PLA polymer which is a mixture of predominantly poly(L-lactide) polymer, the remainder being a poly(D-lactide) polymer will degrade in vivo at a rate slower that a PLA polymer which is predominantly poly(D-lactide) polymer. A PLGA is a copolymer that combines poly(D,L-lactide) with poly(glycolide) in various possible ratios. The higher the glycolide content in a PLGA the faster the polymer degradation.

[0110] In some embodiments, a drug delivery system for intraocular administration (i.e. by intravitreal implantation or injection) comprises, consists of, or consists essentially of at least a 75 weight percent of a PLA and no more than about a 25 weight percent of a poly(D,L-lactide- co-glycolide) polymer.

[0111] Also within the scope are suspensions of microspheres (incorporating an anti- neovascular agent) suspended in a hydrogel (such as a polymeric hyaluronic acid) which can be administered to an intraocular location through a syringe needle. Administration of such a suspension requires that the viscosity of the microsphere suspension at 25°C be less than about 300,000 cP. The viscosity of water at 25°C is about 1.0 cP (cP or cps is centiposie, a measure of viscosity). At 25°C the viscosity of olive oil is 84 cP, of castor oil 986 cP, and of glycerol 1490 cP.

[0112] The antibodies present in the drug delivery systems can be homogeneously dispersed in the biodegradable polymer of the drug delivery system. The selection of the biodegradable polymer used can vary with the desired release kinetics, patient tolerance, the nature of the disease to be treated, and the like. Polymer characteristics that are considered include, but are not limited to, the biocompatibility and biodegradability at the site of implantation, compatibility with the active agent of interest, and processing temperatures. The biodegradable polymer matrix usually comprises at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, or at least about 90 weight percent of the implant. In one variation, the biodegradable polymer matrix comprises about 40% to 50% by weight of the drug delivery system.

[0113] Biodegradable polymers which can be used include, but are not limited to, polymers made of monomers such as organic esters or ethers, which when degraded result in physiologically acceptable degradation products. Anhydrides, amides, orthoesters, or the like, by themselves or in combination with other monomers, may also be used. The polymers are generally condensation polymers. The polymers can be crosslinked or non-crosslinked.

[0114] For the most part, besides carbon and hydrogen, the polymers will include oxygen and nitrogen, particularly oxygen. The oxygen may be present as oxy, e.g., hydroxy or ether, carbonyl, e.g., non-oxo-carbonyl, such as carboxylic acid ester, and the like. The nitrogen can be present as amide, cyano, and amino. An exemplary list of biodegradable polymers that can be used are described in Heller, Biodegradable Polymers in Controlled Drug Delivery, In: "CRC Critical Reviews in Therapeutic Drug Carrier Systems", Vol. 1. CRC Press, Boca Raton, Fla. (1987).

[0115] Of particular interest are polymers of hydroxyaliphatic carboxylic acids, either homo- or copolymers, and polysaccharides. Included among the polyesters of interest are homo- or copolymers of D-lactic acid, L-lactic acid, racemic lactic acid, glycolic acid, caprolactone, and combinations thereof. Copolymers of glycolic and lactic acid are of particular interest, where the rate of biodegradation is controlled by the ratio of glycolic to lactic acid. The percent of each monomer in poly(lactic-co-glycolic)acid (PLGA) copolymer may be 0-100%, about 15-85%, about 25-75%, or about 35-65%. In certain variations, 25/75 PLGA and/or 50/50 PLGA copolymers are used. In other variations, PLGA copolymers are used in conjunction with polylactide polymers.

[0116] Other agents may be employed in a drug delivery system formulation for a variety of purposes. For example, buffering agents and preservatives may be employed. Preservatives which may be used include, but are not limited to, sodium bisulfite, sodium bisulfate, sodium thiosulfate, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric nitrate, methylparaben, polyvinyl alcohol and phenylethyl alcohol. [Examples of buffering agents that may be employed include, but are not limited to, sodium carbonate, sodium borate, sodium phosphate, sodium acetate, sodium bicarbonate, and the like, as approved by the FDA for the desired route of administration. Surfactants which can be used to stabilize particles in a colloid and/or electrolytes such as sodium chloride and potassium chloride can also be included in the formulation. The drug delivery system can also include acid and basic excipients to control pH in the microenvironment as well as at interfaces (diffusional stagnant layer).

[0117] The biodegradable drug delivery systems can also include additional hydrophilic or hydrophobic compounds that accelerate or retard release of the active agent. Additionally, release modulators such as those described in U.S. Pat. No. 5,869,079 can be included in the implants. The amount of release modulator employed will be dependent on the desired release profile, the activity of the modulator, and on the release profile of the glucocorticoid in the absence of modulator. Where the buffering agent or release enhancer or modulator is hydrophilic, it may also act as a release accelerator. Hydrophilic additives act to increase the release rates through faster dissolution of the material surrounding the drug particles, which increases the surface area of the drug exposed, thereby increasing the rate of drug diffusion. Similarly, a hydrophobic buffering agent or enhancer or modulator can dissolve more slowly, slowing the exposure of drug particles, and thereby slowing the rate of drug diffusion.

[0118] A drug delivery system within the scope of the present disclosure can be formulated with particles of an active agent antibody dispersed within a biodegradable polymer. Without being bound by theory, it is believed that the release of the active agent can be achieved by erosion of the biodegradable polymer matrix and by diffusion of the particulate agent into an ocular fluid, e.g., the vitreous, with subsequent dissolution of the polymer matrix and release of the active agent. Factors which influence the release kinetics of active agent from the implant can include such characteristics as the size and shape of the implant, the size of the active agent particles, the solubility of the active agent, the ratio of active agent to polymer(s), the method of manufacture, the surface area exposed, the density of the implant and the erosion rate of the polymer(s).

[0119] The release rate of the active agent can depend at least in part on the rate of degradation of the polymer backbone component or components making up the biodegradable polymer matrix. For example, condensation polymers may be degraded by hydrolysis (among other mechanisms) and therefore any change in the composition of the implant that enhances water uptake by the implant will likely increase the rate of hydrolysis, thereby increasing the rate of polymer degradation and erosion, and thus increasing the rate of active agent release. The release rate of the active agent can also be influenced by the crystallinity of the active agent, the pH in the implant and the pH at interfaces. [0120] The release kinetics of the drug delivery systems of the present invention can be dependent in part on the surface area of the drug delivery systems. A larger surface area exposes more polymer and active agent to ocular fluid, causing faster erosion of the polymer and dissolution of the active agent particles in the fluid.

[0121] Also disclosed herein are pharmaceutical compositions comprising monospecific human anti-VEGF HCAb, bispecific antibodies in which one of the specificities is VEGF, or trispecific antibodies in which one of the specificities is VEGF. Also disclosed is the use of the antibodies described herein for the manufacture of a pharmaceutical composition. Also disclosed are methods of using the disclosed antibodies and pharmaceutical compositions comprising the antibodies for the treatment of ocular disorders

[0122] As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, intraocular, intravitreal, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion).

[0123] A composition disclosed herein can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. To administer the disclosed antibodies by certain routes of administration, it may be necessary to associate the antibodies with, or co-administer the antibodies with, a material to prevent its inactivation. For example, the antibodies may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art.

[0124] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intraocular, intravitreal, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. [0125] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0126] Regardless of the route of administration selected, the disclosed antibodies, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions containing the antibodies, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

[0127] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0128] Suitable doses for the pharmaceutical compositions disclosed herein when delivered intravitreally are in the range of about 0.1 mg to about 50 mg per eye. Additional suitable doses include, but are not limited to, about 0.2 mg to about 40 mg per eye, about 0.3 mg to about 30 mg per eye, about 0.4 mg to about 20 mg per eye, about 0.5 mg to about 15 mg per eye, about 0.5 mg to about 10 mg per eye, about 0.5 mg per eye, about 0.75 mg per eye, about 1 mg per eye, about 1.5 mg per eye, about 2 mg per eye, about 2.5 mg per eye, about 3 mg per eye, about 3.5 mg per eye, about 4 mg per eye, about 4.5 mg per eye, about 5 mg per eye, about 5.5 mg per eye, about 6 mg per eye, about 6.5 mg per eye, about 7 mg per eye, about 7.5 mg per eye, about 8 mg per eye, about 8.5 mg per eye, about 9 mg per eye, about 9.5 mg per eye, about 10 mg per eye, about 11 mg per eye, about 12 mg per eye, about 13 mg per eye, about 14 mg per eye, about 15 mg per eye, about 15 mg per eye, about 17 mg per eye, about 18 mg per eye, about 19 mg per eye, or about 20 mg per eye.

[0129] The composition must be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier preferably is an isotonic buffered saline solution.

[0130] Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.

[0131] The pharmaceutical compositions and drug delivery systems including the monospecific, bispecific, and/or trispecific anti-VEGF antibodies can be injected to an intraocular location by syringe or can be inserted (implanted) into the eye by a variety of methods, including placement by forceps, by trocar, or by other types of applicators, after making an incision in the sclera. In some instances, a trocar or applicator may be used without creating an incision. In a preferred variation, a hand held applicator is used to insert one or more biodegradable implants into the eye. The hand held applicator typically comprises an 18-30 GA stainless steel needle, a lever, an actuator, and a plunger. Suitable devices for inserting an implant or implants into a posterior ocular region or site includes those disclosed in US7,090,681.

[0132] The method of administration generally first involves accessing the target area within the ocular region with the needle, trocar or implantation device. Once within the target area, e.g., the vitreous cavity, a lever on a hand held device can be depressed to cause an actuator to drive a plunger forward. As the plunger moves forward, it can push the implant or implants into the target area (i.e., the vitreous).

[0133] Various techniques may be employed to make implants within the scope of the present disclosure. Useful techniques include phase separation methods, interfacial methods, extrusion methods, compression methods, molding methods, injection molding methods, heat press methods and the like.

[0134] The term "intraocular injection" refers to an injection that is administered by entering the eyeball of the patient. The term "peri-ocular injection" refers to an injection that is administered behind the eye but outside the eye wall. The term "transzonular" refers to an injection administered through the ciliary zonule which is a series of fibers connecting the ciliary body and lens of the eye. The term "intravitreal" refers to an injection administered through an eye of the patient, directly into the inner cavity of the eye.

[0135] Pharmaceutical formulations described herein can be delivered via intraocular intravitreal injection which can be transzonular, or, if desired not transzonular. Intraocular intravitreal injection of this formulation, whether done via transzonular or via direct pars plana (trans-scleral) injection, delivers potent broad spectrum antibiotics directly into the suppurative tissue without requiring the urgent compounding of multiple individual medications or multiple individual injections into the eye.

[0136] Typically, a pharmaceutical composition described above will be intraoculariy administered to a mammalian subject (e.g., humans, cats, dogs, other pets, domestic, wild or farm animals) in need of treatment. The composition is to be injected intravitreally and trans- zonulariy using methods and techniques known to those having ordinary skilled in the art of ophthalmology.

[0137] Typically, the delivery through a typical 27 gauge cannula can be employed utilizing a 1 ml_ TB syringe, with attention to re-suspending the formulation using momentary flicks and shake just prior to injection. The medicinal volume (i.e., dosage) required of this formulation varies based on the type of ocular disorder and anatomic considerations regarding the available volume for the injection being added to a closed intraocular space.

[0138] Additionally, intracameral (that is, anterior chamber) injections are within the scope of the instant disclosure.

[0139] In alternative embodiments, if desired or necessary, the formulations may also be delivered in the form of eye drops or eye sprays, as well as via subconjunctival injection, intraocular intracameral injection, sub-tenon injection, intra-articular injection or intra-lesional injection, particularly, in, but not limited to, some cases when necessary to deliver additional medication when the patients needs warrant.

[0140] The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

EXAMPLES

Example 1. Immunization of llama with VEGF-A [0141] All animal procedures were performed in accordance with the guidelines established by the Institutional Animal Care and Use Committee in a USDA and OLAW certified facility (Abcore, Inc., San Diego, CA).

[0142] A llama was initially immunized subcutaneously (SC) with 500 of purified human VEGF-A and Complete Freund Adjuvant (CFA, Sigma, St. Louis, MO) on day 0 at 8 different sites (62.5 ig per site). The llama was boosted SC with 500 g of purified human VEGF-A and Incomplete Freund Adjuvant (I FA, Sigma, St. Louis, MO) on days 15, 29, 57, and 84 at 8 different sites (62.5 Mg per site). On day 111, the llama was boosted with VEGF-A-KLH (keyhole limpet hemocyanin) at the same dose and manner.

[0143] Production bleeds (500 ml each) were obtained at days 43, 69, 98, and 125. Serum antibody titers were determined and PBMC from each bleed were stored in RNA lysis buffer.

[0144] Antisera from each bleed were tested by ELISA for reactivity and specificity to VEGF-A as they were collected and antisera from previous bleeds were also included. Two independent tests were performed, each with a different binding condition, to confirm the activity. At the end of the immunization protocol, the antiserum titer was positive at greater than 600,000 dilution and absolutely positive at over 10,000 dilution.

Table 2. Anti-serum titer as determined by ELISA

Example 2. Anti-VEGF library construction

[0145] Based on the antisera results, PMBC collected on day 125 were used for VHH library construction. Total RNA was purified from the lysed cells and was used as a template for RT-PCR for construction of a single domain antibody library. The VHH coding DNA was purified after PCR using specific primers. The phage display vector pADL20c (AbDesign Labs, San Diego) was used for cloning. A library of 1.3x10 ⁸ independent clones was obtained. Ten clones were picked randomly and sequenced. All clones had VHH inserts in the correct reading frame.

[0146] The phage display antibody library was screened with antigen-coated plates. After four rounds of panning, 95 clones were randomly picked to select individual positive clones. Over 80% of the clones were positive. Positive lysates were examined for specific binding against other antigens.

[0147] Two clones, LA-C5 and LA-D6, were selected for further analysis (Table 3).

Table 3. Anti-VEGF-A VH

Example 3. Anti-VEGF-A HCAb humanization

[0148] The LA-C5 llama VHH was highly homologous to human germline sequence IGHV3-23. Thus, CDR-1, CDR-1, and CDR-2 were grafted from LA-C5 into IGHV-23 to create PA-C5. Two amino acids from the llama backbone were introduced into PA-C5 at positions 47 (L to W) and 78 (L to Y).

[00102] The humanized anti-VEGF-A VH regions from LA-C5 were fused with a lgG1 hinge region and CD2/CH3 regions to form a HCAb sequence in the GS SYSTEM™ (Lonza, Basel, Switzerland) expression plasmid. The expression plasmid was transfected into a CHO cell line to produce the fully recombinant Hu-C5 HCAb (Table 4). Table 4. Humanized Hu-C5

Example 4. Binding affinity determination of anti-VEGF-A HCAb Hu-C5

[0149] Bio-Layer Interferometry (BLI), a label-free technology was used for measuring the binding kinetics of human VEGF A (R & D systems) with anti-VEGF A HCAb antibody. Affinity measurements were performed with Octet QK ^e equipped with anti-human IgG Fc capture (AHC) biosensor tips (ForteBio®, Menlo Park, CA). The assay was performed at 30°C in 1x PBS buffer (Gibco®, PBS pH7.2). Samples were agitated at 1000 rpm. Prior to analysis, sensors were humidified for 15 min. Purified anti-VEGF A HCAb antibody was tested for its binding capacity with AHC sensor tips. Tips were loaded using 20 Mg/ml of anti-VEGF A HCAb antibody. Loading proceeded for 300 sec resulting in capture levels of between 1.8 and 2 nm. Human VEGF-A antigen was prepared for binding analysis by dilution to concentrations of 100, 150, 250, 350 nM in 1x PBS. Association was initiated and monitored for 200 sec, after which tips were transferred to 1 x PBS buffer without Factor protein (Gibco, PBS pH 7.2), in order to monitor dissociation. Sensor data was collected throughout the experiments, processed, and analyzed using the Octet data analysis software 7 (ForteBio®). The binding results are presented in Table 5.

[0150] Kinitic data anylasis indicated that anti-VEGF-A HCAb Hu-C5 molecule binds to human VEGF A with high binding affinity.

Table 5. Binding Characteristics of Anti-VEGF-A HCAb to Human VEGF-A

"over the limit of detection

Example 5. Cell-based functional characterization of anti-VEGF-A HCAb Hu-C5

[0151] VEGF-dependent calcium mobilization was measured in human umbilical vein endothelial cells (HUVEC). HUVEC were diluted to 1.6x10 ^s cells/ml in EGM-Plus complete medium supplemented with SingleQuots growth factors and supplements (LONZA), and 50 μΙ was added to each well of a 384 well fibronectin plate. The plates were incubated overnight at 37°C and 5% CO2. One hour prior to stimulation, media was then removed and the HUVEC loaded with a calcium sensitive dye, Fluo4 NW (Invitrogen, Inc.) in assay buffer (AB (1L): 880 ml_ H ₂ 0; 20 ml_ HEPES; 100 ml_ 10x HBSS; 10 ml_ FBS; 0.7 g probenecid + 7.5 ml_ 1M NaOH; 40 μΙ per well). The solution was incubated with the cells for 60 min at 37°C and 5% CO ₂ . Cells were washed and replaced with 25 μΙ assay buffer. Inhibition of VEGF-A was determined by adding Hu-C5, bevacizumab, and ranibizumab at concentrations ranging from 7.8 pM to 2.0 nM incubated with 50 pM VEGF-A for 4 hr and then added to the cells, and the calcium response recorded (FIG. 4). The data were analyzed using the average peak fluorescence at each inhibitor concentration tested in quadruplicate. The functional assay indicated that anti-VEGF-A HCAb Hu-C5 molecule fully neutralized VEGF A activity.

Example 6. Characterization of anti-VEGF-A/ANG-2 bispecific antibody

[0152] The structure of a bispecific antibody with specificity for VEGF-A and ANG-2 is depicted in FIG. 5. The bispecific antibody was formed from the ANG-2-specific HCAb A33A8

VH sequence (disclosed in PCT/US2016/044838, which is incorporated by reference for all it discloses regarding A33A8) joined by an immunoglobulin Fc region to the Hu-C5 VH sequence.

(SEQ ID NO:15. QVQLLESGGGLVQPGGSLRLSCAASGRTFSNTGMSVWRQAPGKGLEL

VSAITPSGSGTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAKGGGNY YYISNYD

YvVGQGTLVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV WDV

SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVS NKAL

PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNY

KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKG GGGSG

GGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMHWVRQAPGKGLWWS RI NSDGSSTSYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAREGYSSGGQFDYWG Q GTLVTVSS.

[0153] Examples of PDGF and ANG-2 sequences which can be used in the bispecific antibodies disclosed herein are found in Tables 6 and 7, respectively, and in co-pending applications PCT/US2016/046595 and PCT/US2016/044838, incorporated by reference herein for all they disclose regarding anti-PDGF and anti-ANG-2 antibodies.

Table 6. Sequences of anti-PDGF-BB VH

Table 7. Sequences of anti-ANG-2 VH

[0154] Affinity measurements were performed using a forteBIO OCTET® QK ^e system equipped with anti-hlgG Fc capture (AHC) biosensor tips (forteBIO, Menlo Park, CA). For AHC affinity measurement, purified anti-VEGF/ANG-2 bispecific antibody was tested for its binding capacity with AHC sensor tips. Tips were loaded using 20 Mg/ml anti-VEGF/ANG-2 bispecific antibody. Loading proceeded for 300 sec resulting in capture levels of between 1.8 and 2 nm. VEGF-A (R & D systems) antigen was prepared for binding analysis by dilution to concentrations of 400 nM in 1xPBS. Association was initiated and monitored for 200 sec, after which tips were transferred to 1xPBS buffer without Factor protein (Gibco, PBS pH 7.2), in order to monitor dissociation for 300 sec, and then tips were transferred into second antigen, ANG-2, (R & D systems) and monitored for 200 sec, and then wash in 1 x PBS for 600 SEC (FIG. 6A), This was also done by first AGN-2 followed by VEGF A antigen loading and then ANG-2 (FIG. 6B). Anti- VEGF/ANG-2 bispecific antibody binding activity was then monitored in real time using bio-layer interferometry analysis. Antigen binding affinities were calculated based on the measured association ("Kass∞") and dissociation (" ass∞") constants. Data were processed and analyzed using the OCTET® data analysis software 6.4 (forteBIO).

[0155| The Octet binding curve showed successive binding of both antigens without a corresponding dissociation of the first antigen upon exposure to the second, indicating that the bispecific antibody can simultaneously bind all antigens in a non-competitive association.

Example 7. Trispecific antibody to PDGF BB. VEGF-A. and ANG-2

[0156] A trispecific antibody was generated with antigen-binding regions specific for VEGF-A, PDGF, and ANG-2

[0157] An anti-PDGF BB VH1 (P36C12) disclosed in co-pending application PCT/US2016/046595 was fused with Lucentis LgG1 at N-terminal and anti-ANG-2 VH3 (A33A8) (PCT/US2016/044838) was fused with the same Lucentis lgG1 at C-terminal to form a trispecific sequence (heavy chain) in the GS SYSTEM™ (Lonza, Basel, Switzerland) expression plasmid (FIG. 1) . The heavy chain plasmid along with light chain plasmid were transfected into a CHO cell line to produce fully human recombinant tri-specific antibody, P36-LCNT-A33 (FIG. 8).

[0158] Octet real-time binding of a trispecific antibody to VEGF-A, PDGF BB and ANG-2 is depicted in FIG. 7A-F.

[0159] Affinity measurements were performed using a forteBIO OCTET® QK ^e system equipped with anti-hlgG Fc capture (AHC) biosensor tips (forteBIO, Menlo Park, CA). For AHC affinity measurement, purified anti-PDGF/VEGF/ANG2 trispecific antibody was tested for its binding capacity with AHC sensor tips. Tips were loaded using 20 μα/ml anti- PDGF/VEGF/ANG2 trispecific antibody. Loading proceeded for 300 sec resulting in capture levels of between 1.8 and 2 nm. ANG-2 (R & D systems) antigen was prepared for binding analysis by dilution to concentrations of 400 nM in 1xPBS. Association was initiated and monitored for 200 sec, after which tips were transferred to 1xPBS buffer without Factor protein (Gibco, PBS pH 7.2), in order to monitor dissociation for 300 sec, and then tips were transferred into second antigen (VEGF-A) and monitored for 200 sec, and then wash in 1 x PBS for 300 SEC, and then tips were transferred into third antigen (PDGF BB) (FIG 7A). This was also done by first PDGF BB followed by VEGF-A antigen loading and then ANG-2 (FIG. 7B-F). Anti- PDGF/VEGF/ANG2 trispecific antibody binding activity was then monitored in real time using bio-layer interferometry analysis. Antigen binding affinities were calculated based on the measured association ("Kass∞") and dissociation {" nssoc") constants. Data were processed and analyzed using the OCTET® data analysis software 6.4 (forteBIO). The Octet binding curve showed successive binding of both antigens without a corresponding dissociation of the first antigen upon exposure to the second and third, indicating that the trispecific antibody can simultaneously bind all antigens in a non-competitive association. [0160] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein the terms "about" and "approximately" means within 10 to 15%, preferably within 5 to 10%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0161] The terms "a," "an," "the" and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0162] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims. [0163] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0164] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term "consisting of excludes any element, step, or ingredient not specified in the claims. The transition term "consisting essentially of limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic's). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

[0165] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety.

[0166] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.