TW201840584A - Heavy chain only antibodies to vegf - Google Patents

Abstract

Disclosed herein are monospecific HCAb antibodies with specificity for VEGF, bispecific antibodies with specificities for VEGF-A and PDGF or for VEGF-A and ANG-2, or trispecific antibodies with specificities for VEGF, PDGF, and ANG-2. Methods for using the antibodies are also disclosed.

Description

Heavy chain only antibody against VEGF

The invention described generally relates to antibodies and methods of their use.

Angiogenesis is the formation of new blood vessels from pre-existing vasculature, which is the main cause of blindness in several retinal vascular diseases such as retinopathy of prematurity, proliferative diabetic retinopathy, diabetic macular edema, and age-related macular degeneration Components. Neovascularization of the eye is abnormal or excessive formation of blood vessels in the eye. It has been shown that neovascularization of the eye is associated with diabetic retinopathy and age-related macular degeneration.

Age-related macular degeneration (AMD) is the main cause of blindness in the elderly, and it is recognized that it is in the form of dry and wet AMD. Dry or non-exudative forms include retinal pigment epithelium (RPE) atrophy and overgrowth changes. The dry form is characterized by macular drusen, which is a colored area containing dead cells and metabolites that deform the retina and eventually cause loss of sharp vision. Patients with non-exudative AMD (dry form) can progress to wet or exudative or neovascular AMD, in which pathological choroidal neovascular membrane (CNVM) grows under the retina, leaking fluid and blood, and if not performed Treatment, eventually leads to central blinding oval scars in a relatively short time frame. Choroidal neovascularization (CNV) is the growth of new blood vessels from the choroidal capillary network across the Bruch's membrane / RPE interface into the neural retina, causing retinal detachment, subretinal and intraretinal edema, and scarring.

Diabetes can affect the eyes in many ways. Diabetic retinopathy (Diabetic retinopathy, DR) is a diabetic complication caused by damage to the blood vessels of the photosensitive tissue (retina) behind the eyes. Initially, diabetic retinopathy may cause no symptoms or only mild vision problems. However, eventually diabetic retinopathy can cause blindness. Diabetic macular edema (DME) is a type of diabetes in which the retina expands due to fluid leakage from blood vessels in the macula.

This article discloses heavy chain-only monospecific antibodies (HCAbs) specific for VEGF, bispecific antibodies specific for VEGF and ANG-2 or VEGF and PDGF or specific for VEGF, PDGF and ANG-2 Specific three specific antibodies.

In some embodiments, heavy chain-only antibodies (HCAbs) specific for VEGF are disclosed. In some embodiments, VEGF is VEGF-A. In certain embodiments, the HCAb has the variable heavy chain (VH) region sequence of one of SEQ ID NO: 5, 9, or 13. In other embodiments, the HCAb VH region sequence has at least 85% or 95% identity with the full-length sequence of one of SEQ ID NO: 5, 9 or 13.

In some embodiments, one or more of the complementarity determining regions (CDRs) of the HCAb are selected from SEQ ID NO: 6-8 and 10-12. In certain embodiments, the HCAb CDR has at least 85% or 95% identity with the full-length CDR sequence selected from SEQ ID NOs: 6-8 and 10-12.

In some embodiments, the HCAb specific for VEGF-A comprises the CDR of SEQ ID NO: 6. In some embodiments, the HCAb specific for VEGF-A comprises the CDR of SEQ ID NO: 7. In some embodiments, the HCAb specific for VEGF-A comprises the CDR of SEQ ID NO: 8. In some embodiments, the HCAb specific for VEGF-A comprises the CDR of SEQ ID NO: 10. In some embodiments, the HCAb specific for VEGF-A comprises the CDR of SEQ ID NO: 11. In some embodiments, the HCAb specific for VEGF-A comprises the CDR of SEQ ID NO: 12.

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

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

In some embodiments, bispecific antibodies are provided that have a first specificity for VEGF and a second specificity for PDGF. In some embodiments, the first specificity is exhibited by the HCAb as in any one of claims 1 to 14. In certain embodiments, the second specificity is exhibited by a VH region selected from the group consisting of: 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).

In some embodiments, a bispecific antibody having a first specificity for VEGF and a second specificity for ANG-2 is provided. In some embodiments, the first specificity is exhibited by the HCAb as in any one of claims 1 to 14. In some embodiments, the second specificity is exhibited 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.

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

Also disclosed herein is a method of treating an ophthalmic disorder, which includes administering to a subject in need thereof an HCAb having a VH region disclosed herein that binds VEGF or a bispecific or trispecific antibody disclosed herein.

Also disclosed herein is the use of the HCAb with VH region disclosed herein binding to VEGF or the bispecific or trispecific antibodies disclosed herein for the manufacture of a medicament for the treatment of ophthalmic disorders in individuals in need.

In some embodiments, the ophthalmic condition includes age-related macular degeneration (AMD), choroidal neovascularization (CNV), cystic macular edema (CME), myopic-associated choroidal neovascularization, angioidia, diabetic macular edema (DME), macular edema, retinal vein occlusion, abnormal corneal angiogenesis, pterygium conjunctiva, subretinal edema or intraretinal edema. In some embodiments, abnormal corneal angiogenesis is caused by keratitis, corneal transplantation, keratoplasty, or hypoxia.

Cross-reference to related applications

This application claims the priority rights of US Provisional Patent Application No. 62 / 473,996 titled "Heavy Chain Only Antibodies to VEGF", which was applied on March 20, 2017. The content of the provisional patent application is incorporated by reference in its entirety Incorporated in this article.

This article reveals heavy chain-only monospecific antibodies (HCAb) specific for vascular endothelial growth factor (VEGF), VEGF and angiopoietin-2 (ANG-2) or VEGF and platelet-derived growth factor (PDGF) ) Bispecific antibodies with specificity and trispecific antibodies with specificity for VEGF, PDGF and ANG-2.

The VEGF family in mammals consists of five members: VEFG-A, placenta growth factor (PGF), VEGF-B, VEGF-C, and VEGF-D. All members of the VEGF family are stimulated by dimerization by binding to the tyrosine kinase receptor (VEGFR) on the cell surface and, although the site, time and degree are different, they are activated by transphosphorylation . The VEGF receptor has an extracellular part composed of seven immunoglobulin-like domains, a single transmembrane region, and an intracellular part containing a split tyrosine kinase domain. VEGF-A binds to VEGFR-1 (Flt-1) and VEGFR-2 (KDR / Flk-1). VEGFR-2 seems to mediate almost all known cellular responses to VEGF. The function of VEGFR-1 is not clear, but it is thought to regulate VEGFR-2 signaling. Another function of VEGFR-1 may be to act as a pseudo / bait receptor, preventing VEGF from binding VEGFR-2 (this seems to be particularly important during angiogenesis in the embryo). VEGF-C and VEGF-D, but not VEGF-A, are ligands for the third receptor (VEGFR-3 / Flt4) that mediates lymphangiogenesis. VEGFR-3 is the binding site of the major ligands (VEGF-C and VEGF-D), which mediates the ligand's long-term effects and functions 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; MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMD VYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVYVGARCCLMPWSLPGPHPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR; SEQ ID NO: 1).

Therefore, the present disclosure discloses monospecific and bispecific HCAb antibodies against VEGF and methods of using the disclosed antibodies to treat ophthalmic disorders.

Antibodies for treating diseases are well known in the art. As used herein, the term "antibody" refers to a monomeric or multimeric protein that includes one or more polypeptide chains that include an antigen binding site. Antibodies specifically bind to the antigen and can regulate the biological activity of the antigen. As used herein, the term "antibody" may include "full-length antibody" and "antibody fragment". As used herein, the term "binding site" or "antigen binding site" refers to the region of the antibody molecule to which the ligand actually binds.

Antibody specificity refers to the specific epitope of an antibody that selectively recognizes an antigen. Natural antibodies are, for example, monospecific. As used herein, the term "monospecific" refers to an antibody having one or more binding sites each binding 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.

"Bispecific antibody" refers to an antibody with two different antigen binding specificities. The bispecific antibodies disclosed herein have a first specificity for VEGF and a second specificity for PDGF or ANG-2.

The term "trispecific antibody" refers to an antibody with three different antigen binding specificities. The three specific antibodies disclosed herein are specific for VEGF, PDGF and ANG-2.

As used herein, the term "valency" means that there is a specified number of binding sites in the antibody molecule. Therefore, the terms "bivalent", "tetravalent" and "hexavalent" mean that there are two binding sites, four binding sites and six binding sites in the antibody molecule, respectively. The bispecific antibodies disclosed herein are "bivalent". However, monospecific bivalent antibodies are within the scope of the present invention, where two antigen binding sites bind the same antigen. The antigen binding site of a monospecific bivalent antibody can bind to the same epitope or different epitopes on the antigen. The three specific antibodies disclosed in this article are "trivalent".

In this context, "full-length antibody" means a structure that constitutes the natural biological form of an antibody, including variable and constant regions. For example, in most mammals including humans and mice, full-length antibodies of the IgG class are tetramers and consist of two identical pairs of double immunoglobulin chains, each pair having a light chain and a heavy chain Each light chain contains immunoglobulin domains VL and CL, and each heavy chain contains immunoglobulin domains VH, CH1, CH2, and CH3. In some mammals, such as camels and llamas, IgG antibodies consist of only two heavy chains (HCAbs), each heavy chain containing variable domains connected to the Fc region (CH2 and CH3 domains).

Natural antibody structural units usually contain tetramers. Each tetramer usually consists of two pairs of identical polypeptide chains, each pair having a "light" chain (usually having a molecular weight of about 25 kDa) and a "heavy" chain (usually having a molecular weight of about 50-70 kDa). The light chain and the heavy chain are each composed of two different regions called variable regions and constant regions. For the IgG class of immunoglobulins, the heavy chain is composed of four immunoglobulin domains. The four immunoglobulin domains are connected in a sequence of VH-CH1-CH2-CH3 from N to C terminus, which are called heavy chain variable. Domain, heavy chain constant domain 1, heavy chain constant domain 2 and heavy chain constant domain 3 (also known as VH-Cγ1-Cγ2-Cγ3, respectively called heavy chain variable domain, constant γ1 domain, constant γ2 domain and constant γ3 domain). The IgG light chain is composed of two immunoglobulin domains. The two immunoglobulin domains are connected by VL-CL from N to C terminus, which are called light chain variable domain and light chain constant domain, respectively. The constant region exhibits less sequence diversity and is responsible for the binding of many natural proteins to cause important biochemical events.

The variable region of an antibody contains the antigen binding determinants of the molecule, thus determining the specificity of the antibody for its target antigen. The variable region is so named because it differs most in sequence from other antibodies in the same class. In the variable region, the three loops of each V domain of the heavy chain and the light chain gather to form an antigen binding site. Each loop is called a complementarity determining region (hereinafter referred to as "CDR"), in which the amino acid sequence changes are the most significant. There are a total of six CDRs, three for each heavy and light chain, called VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. The variable region outside the CDR is called the framework (FR) region. Although the FR region is not as variable as the CDR, there are sequence variations in the FR region between different antibodies. In general, this characteristic configuration of antibodies provides a stable framework (FR region) on which the immune system can probe for considerable antigen binding diversity (CDR) to obtain specificity for a wide range of antigens.

The determination of CDR regions in antibodies is well known to those skilled in the art. There are at least two techniques for determining CDRs: (1) methods based on sequence variability between species (Kabat et al. Sequences of Proteins of Immunological Interest, 5th edition, 1991, National Institutes of Health, Bethesda Md.); And (2) A method 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, CDR may refer to a CDR defined by either method or a combination of both methods.

The gene encoding the immunoglobulin locus contains multiple V region sequences and shorter nucleotide sequences called "D" and "J", and they are the V, D and J nucleotide sequences that produce VH diversity combination.

If genetically determined by a constant region, antibodies are divided into multiple categories, also known as isotypes. Human constant light chains are classified as κ (Cκ) and λ (Cλ) light chains. Heavy chains are classified as μ (μ), δ (δ), γ (γ), α (α), or ε (ε), and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG class is most commonly used for therapeutic purposes. In humans, this category includes the subclasses IgG1, IgG2, IgG3, and IgG4. In mice, this category includes subclasses IgG1, IgG2a, IgG2b, IgG3. IgM has subclasses, including (but not limited to) IgM1 and IgM2. IgA has several subclasses, including but not limited to IgA1 and IgA2. Therefore, "isotype" as used herein means any class or subclass of immunoglobulin defined by the chemical and antigenic characteristics of the constant region. Known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD and IgE. The disclosed HCAb antibodies and bispecific antibodies may have constant regions comprising all or part of the aforementioned isotypes.

Antibody fragments are also within the scope of the invention, including (but not limited to) (i) Fab fragments, which contain VL, CL, VH, and CH1 domains; (ii) Fd fragments, which contain VH and CH1 domains; (iii ) Fv fragment, which contains VL and VH domains of a single antibody; (iv) dAb fragment, which contains a single variable region; (v) isolated CDR region; (vi) F (ab ') ₂ fragment, one contains two A bivalent fragment of linked Fab fragments; and (vii) a single chain Fv molecule (scFv), in which the VH domain and the VL domain are connected by a peptide linker that allows the two domains to associate to form an antigen binding site. In certain embodiments, antibodies are produced by recombinant DNA technology. In additional embodiments, antibodies are produced by enzymatic or chemical cleavage of naturally occurring antibodies.

As used herein, "humanized" means an antibody that includes a human framework region (FR) and one or more complementarity determining regions (CDRs) from non-human (usually mouse or rat, but other species are also possible) antibodies. Non-human antibodies that provide CDRs are called "donors", and human immunoglobulins that provide frameworks are called "acceptors." In some embodiments, humanization mainly relies on grafting of donor CDRs onto the recipient (human) VL and VH frameworks. This strategy is called "CDR transplantation". Often it is necessary to "back-mutate" selected residues of the acceptor framework to the corresponding donor residues to regain the affinity lost in the originally transplanted construct. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region of human immunoglobulins, and therefore usually contain human Fc regions. Humanization or other methods to reduce the immunogenicity of the variable regions of non-human antibodies may include surface remodeling. In one embodiment, selection-based methods can be used to humanize and / or affinity mature antibody variable regions, that is, to increase the affinity of the variable regions for their target antigens. Other humanization methods may include transplanting only a portion of the CDR, including but not limited to the methods described in US 6,797,492, all disclosures regarding CDR grafting are incorporated herein by reference. Structure-based methods can be used for humanization and affinity maturation, for example, as described in US 7,117,096, all disclosures regarding humanization and affinity maturation are incorporated herein by reference.

In various embodiments herein, the antibody is a heavy chain only antibody (HCAb). Camelids (camels, dromedaries, and llamas) also contain single chain antibodies that contain only heavy chains in addition to normal heavy chain and light chain antibodies (two light chains and two heavy chains in one antibody) (see figure 1). These are encoded by different classes of VH segments called VHH genes. VH and VHH are scattered in the genome (that is, they appear to be intermingled with each other). The identification of the same D segment in VH and VHH cDNA indicates that VH and VHH share the D segment. Natural VHH-containing antibodies lose the entire CH1 domain of the heavy chain constant region. Although the exon encoding the CH1 domain is present in the genome, it is spliced out due to the loss of functional splice acceptor sequence on the 5 ′ side of the CH1 exon. As a result, the VDJ region was spliced to the CH2 exon. When VHH recombines onto such constant regions (CH2, CH3), antibodies that are used as single-chain antibodies (ie, antibodies that have two heavy chains but no light chain interaction) are produced. Although the binding of the antigen is different from that seen under conventional antibodies, high affinity is achieved in the same way, that is, through high mutations in the variable region and selection of cells expressing such high affinity antibodies.

In an exemplary embodiment, the HCAb is produced by immunizing a transgenic mouse in which the expression of endogenous murine antibodies has been eliminated and a human transgenic gene has 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, In US2011 / 0118444 and US2009 / 0307787, all disclosures regarding heavy chain-only antibodies and their production in transgenic mice are incorporated herein by reference. HCAb mice were immunized, and the resulting presensitized spleen cells were fused with murine myeloma cells to form hybridomas. The resulting HCAb can then be made fully human by replacing the murine CH2 and CH3 regions with human sequences.

In other exemplary embodiments, HCAb are produced by immunizing camelids such as llamas with the desired antigen, and the animal obtains presensitized peripheral blood mononuclear cells (PBMC). RNA isolated from PBMC can be used to generate phage display libraries to isolate antigen-specific VHH sequences.

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) bispecific Fv fragments (Figure 2A); (ii) HCAb with a first specificity associated with a second VH domain with a second specificity (Figure 2B And 2C); (iii) a tetrameric monoclonal antibody having a first specificity associated with a second VH domain having a second specificity, wherein the second VH domain is associated with the first VH domain ( Figure 2D); and (iv) a Fab fragment with a first specificity (VH-CH1 / VL / CL) associated with a second VH domain with a second specificity (Figure 2E). An exemplary Fab fragment is depicted in FIG. 2E, where a second VH sequence with a second specificity is associated with the C-terminus or N-terminus of the first VH domain or the C-terminus or N-terminus of the first CH1 or first CL domain Together. In an additional embodiment also depicted in FIG. 2E, the VH sequence with the second and / or third specificity may be linked to the C-terminal or N-terminal of the first VH domain or the first CH1 or the first CL domain C-terminal or N-terminal association. Also disclosed herein are antibodies in which three antigen-binding domains are joined in a single trispecific molecule. The same method used to generate bispecific antibodies can be used to generate trispecific antibodies.

Bispecific and trispecific antibodies may include linker sequences linking the VEGF-binding antibody disclosed herein to a VH region with a second (or third) specificity, which allows the sequence to fold properly to produce the desired three-dimensional Configuration and antigen binding characteristics. Suitable linkers include (but are not limited to) EPKSCD (SEQ ID NO: 2), ASTKGP (SEQ ID NO: 3) and (GGGGS) _n (SEQ ID NO: 4), where n is between 0 and 8 Integer. In one embodiment, n is 1.

The bispecific antibodies disclosed herein are bivalent and contain a first specificity for VEGF, and the second specificity can include, but is not limited to, PDGF or ANG-2. Bispecific antibodies are within the scope of the present invention, where the first specificity and the second specificity are independently ANG-2, VEGF, or PDGF, and the only limitation is that the first specificity and the second specificity cannot be the same.

Human angiopoietin-1 and -2 (ANG-1 and ANG-2 (UniProtKB-O15123; or abbreviated as ANGPT2 or ANG2)) were found to be ligands of Ties, which are tyramines selectively expressed in the endothelium Acid kinase family. There are four defined members of the Angiopoietin family. Angiopoietin-3 and angiopoietin-4 (ANG-3 and ANG-4) may represent widely different counterparts of the same locus in mice and men. ANG-1 and ANG-2 were initially identified as agonists and antagonists in tissue culture experiments, respectively. All known angiopoietins mainly bind to Tie-2. ANG-1 supports endothelial cell (EC) survival and promotes endothelial integrity, while ANG-2 has the opposite effect and accelerates vascular instability and degradation in the absence of survival factor VEGF or basic fibroblast growth factor. However, many studies on the function of ANG-2 have proposed more complicated situations. ANG-2 may be a complex regulator of vascular remodeling that plays a role in vascular germination and vascular degeneration. In support of such effects of ANG-2, performance analysis revealed that ANG-2 and VEGF are rapidly induced together in an adult environment where angiogenesis is budding, and ANG-2 is induced in the absence of VEGF in a degraded vascular environment. Consistent with environment-dependent effects, ANG-2 specifically binds to the same endothelium-specific receptor Tie-2 activated by ANG-1, but has an environment-dependent effect on its activation.

In the analysis of corneal angiogenesis, ANG-1 and ANG-2 have similar effects, which are used in conjunction with VEGF to promote the growth of new blood vessels. At high concentrations, ANG-2 acts as an apoptosis survival factor for endothelial cells during activation of serum deprived apoptosis via activation of Tie-2 via the PI-3 kinase and Akt pathway. It is believed that the role of ANG-1 is preserved in adults, among which its broad and constitutive expression. In contrast, the performance of ANG-2 is mainly limited to the site of vascular remodeling, which is believed to block the constitutive stabilization or maturation function of ANG-1, allowing blood vessels to recover and maintain a plastic state, which can play a role in germination Greater response. During development, ANG-2 appears at the site where vascular remodeling is present. In adult individuals, ANG-2 performance is limited to vascular remodeling sites and highly vascularized tumors. ANG-2 is required for angiogenesis after birth. The stylized developmental degeneration of the vitreous vasculature in the eye does not exist in ANG-2 knockout mice, and its retinal blood vessels cannot erupt from the central retinal artery. The absence of ANG-2 caused great defects in the patterning and function of lymphatic vessel structure. Gene rescue with ANG-1 corrects lymphatic defects, not angiogenic defects.

PDGF plays an important role in angiogenesis (angiogenesis, the growth of vascular tissue that already exists in blood vessels). PDGF is an effective mitogen that includes fibroblasts, smooth muscle cells, and glial cells between cytoplasmic sources. PDGF is a dimeric glycoprotein composed of two A (-AA) or two B (-BB) subunits or a combination of both (-AB). The A subunit is a sequence of 211 amino acids (UniProtKB-P04085); and the B subunit is a sequence of 241 amino acids (UniProtKB-P01127). Therefore, in various embodiments, antibodies specific for PDGF-AA, PDGF-BB, and / or PDGF-AB or fragments thereof are disclosed. In mice and humans, the PDGF signaling network is composed of four ligands PDGFA-D and two receptors PDGFRα and PDGFRβ (receptor tyrosine kinase). All PDGFs act as secreted homodimers connected by disulfide bonds, but only PDGFA and B can form functional heterodimers. Therefore, in certain embodiments, the PDGF is PDGF-AA, PDGF-BB, or PDGF-AB.

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

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

This article also discloses trispecific antibodies that are specific for VEGF, ANG-2 and PDGF. Trispecific antibodies as disclosed herein include the three VH sequences disclosed herein, where each VH region has a different specificity. One of these VH regions may be part of the heavy chain of the antibody as appropriate. Optionally, one or more linkers are between VH regions and / or heavy chains 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 exhibited by any of SEQ ID NO .: 39, 43, 47, 49-60 (as disclosed in US2010 / 0159587) and 61-65 (as disclosed in US20130259868). The trispecific antibody may be an HCAb or contain heavy and light chains.

An exemplary trispecific antibody has at least 85%, 90%, 95%, or 98% of the full-length heavy chain of SEQ ID NO: 48 and optionally the light chain of SEQ ID NO: 49 (as disclosed in US2010 / 0159587) The amino acid sequence of the sequence identity.

Amino acid sequence variants of human anti-VEGF monospecific, bispecific or trispecific antibodies prepared by introducing appropriate nucleotide changes into antibody DNA or by peptide synthesis are also within the scope of the present invention. Such variants include, for example, the deletion and / or insertion and / or substitution of residues within the amino acid sequence of the antibodies exemplified herein. Any combination of deletion, insertion, and substitution to achieve the final construct, provided that the final construct has the desired characteristics. Amino acid changes can also alter the post-translational process of humanized or mutant antibodies, such as changing the number or location of glycosylation sites.

A useful method for identifying certain residues or regions of antibodies that are in a better position for mutagenesis is called "alanine scanning mutagenesis." Identify residues or a set of target residues (eg charged residues such as Arg, Asp, His, Lys, and Glu) and replace with neutral or negatively charged amino acids (optimally alanine or polyalanine) to affect The interaction of amino acids and antigens. Then, by introducing further or other variants at the substitution site or against the substitution site, the amino acid positions of the other amino acids that show functional sensitivity to the substitution are refined. Therefore, although the site for introducing amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the effectiveness of mutations at both localization points, alanine scans or random mutagenesis are performed on the target codons or regions and the antibody variants displayed are screened for the desired activity.

Amino acid sequence inserts include amino and / or carboxy-terminal fusions ranging in length from one residue to a polypeptide containing one hundred or more residues, and sequences with single or multiple amino acid residues Insert inside. Examples of terminal inserts include anti-VEGF antibodies with N-terminal methionine residues or antibodies fused to epitope tags. Other insertional variants of antibody molecules include fusions of the N-terminus or C-terminus of the antibody with an enzyme or polypeptide that increases the serum half-life of the antibody.

Another type of variant is the amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule that has been removed and inserted with different residues in its place. The most relevant sites for substitution mutagenesis include hypervariable regions, but FR changes are also covered. Conservative substitutions are shown in Table 1 under the heading "Better Substitutions." If such a substitution causes a change in biological activity, a larger change called "exemplary substitution" in Table 1 or as further described below for the amino acid category can be introduced and the product screened. Table 1

A large number of modifications of the biological characteristics of antibodies are achieved by selecting substitutions with significant differences in the following effects: (a) the structure of the antibody backbone in the substitution area, such as a sheet or spiral configuration; (b) at the target site Charge or hydrophobicity of the molecule; or (c) side chain volume. According to the characteristics of common side chains, the naturally occurring residues are divided into groups: (1) Hydrophobicity: leucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr ; (3) Acidity: Asp, Glu; (4) Basicity: Asn, Gin, His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; and (6) Aromatics: Trp, Tyr , Phe.

Non-conservative substitutions replace members who need one of these categories with another category.

Any cysteine residues that are not involved in maintaining the proper configuration of monospecific, bispecific, or trispecific anti-VEGF antibodies can also be substituted, generally by serine to enhance the antioxidant properties of the molecule and prevent Abnormal cross-linking. Conversely, cysteine bonds can be added to the polypeptide to improve its stability (especially if the antibody is an antibody fragment, such as an Fv fragment).

Another type of substitutional variant includes substitution of one or more hypervariable region residues of the parent antibody (eg, humanized or human antibodies). In general, the resulting variant selected for further development will have improved biological properties relative to the parent antibody from which it was produced. One suitable method for producing such substitution variants is the use of affinity maturation presented by phage. Briefly, several hypervariable region sites (eg, 6-7 sites) are mutated to produce all possible amine substitutions at each site. The antibody variants thus produced appear in a monovalent manner from filamentous phage particles as fusions with the gene IIII product of M13 packaged in each particle. Then, as disclosed herein, the phage-displayed variants are screened for biological activity (eg, binding affinity). To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that significantly affect antigen binding. Alternatively or additionally, it is desirable to analyze the crystal structure of the antigen-antibody complex to identify the contact point between the antibody and human VEGF. According to the techniques detailed herein, such contact residues and adjacent residues are candidate substitution residues. Once such variants are generated, the set of variants are screened as described herein, and antibodies with excellent characteristics in one or more corresponding assays can be selected for further development.

Another type of amino acid variant of the polypeptide changes the original glycosylation pattern of the antibody. Alteration means that one or more carbohydrate moieties are found missing in the antibody, and / or one or more glycosylation sites that are not present in the antibody are added.

The glycosylation of antibodies is usually N-linked or O-linked. N-linking means that the carbohydrate moiety is attached to the side chain of an aspartate residue. The tripeptide sequence asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are carbohydrates partially enzymatically linked to the day The recognition sequence of the side chain of aspartic acid. Therefore, the presence of any of these tripeptide sequences in the polypeptide creates potential glycosylation sites. O-linked glycosylation means that one of the sugars N-acetylgalactosamine, galactose, or xylose is attached to a hydroxylamino acid, most commonly serine or threonine, but 5- Hydroxyproline or 5-hydroxylamine acid.

The addition of glycosylation sites to the antibody is preferably achieved by changing the amino acid sequence so that it contains one or more of the above-mentioned tripeptide sequences (for N-linked glycosylation sites). Changes can also be made by adding one or more serine or threonine residues to the original antibody sequence or replacing with one or more serine or threonine residues (for O-linked glycosylation sites point).

Nucleic acid molecules encoding amino acid sequence variants of monospecific, bispecific or trispecific anti-VEGF antibodies are prepared by various methods known in the art. Such methods include (but are not limited to) isolation of oligonucleotides from natural sources (in the case of naturally occurring amino acid sequence variants) or variants or non-variant versions prepared by early preparation of anti-VEGF antibodies Prepared by mediated (or site-directed) mutagenesis, PCR mutagenesis and gated mutagenesis.

Other modifications of monospecific, bispecific or trispecific anti-VEGF antibodies are covered. For example, it may be necessary to modify the antibody in terms of effector function, for example, to enhance the efficacy of the antibody in treating diseases. For example, cysteine residues can be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in this region. The homodimeric antibody thus produced may have improved internalization capacity and / or enhanced complement-mediated cell killing and antibody-dependent cytotoxicity (ADCC). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linking agents. Alternatively, antibodies can be engineered to have dual Fc regions and thus have enhanced complement lysis and ADCC capabilities.

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 the sequence of anti-VEGF HCAb disclosed herein are also within the scope of the present invention .

Exemplary variants or mutant anti-VEGF HCAbs comprise one or more sequences of SEQ ID NO: 5, 9 and 13, or one or more CDRs selected from SEQ ID NO: 6-8 and 10-12, one or Multiple amino acids were substituted with amino acids 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 are added, substituted or deleted , Up to 9, up to 10, up to 12, up to 14, up to 16, up to 18 or up to 20 amino acids, provided that the substituted sequence remains unsubstituted The binding specificity of the sequence may have increased binding specificity. 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 are added, substituted or deleted , Up to 9, up to 10, up to 12, up to 14, up to 16, up to 18 or up to 20 amino acids, provided that the substituted sequence remains unsubstituted The entire length of the sequence is at least 85% identical, at least 90% identical, at least 93% identical, at least 96% identical, or at least 98% identical.

In certain embodiments, the variant or mutant anti-VEGF HCAb includes one or more additions, substitutions, or deletions in the variable region. In some embodiments, the variant or mutant anti-VEGF HCAb includes one or more additions, substitutions, or deletions in one or more complementarity determining regions. In some embodiments, the variant or mutant anti-VEGF HCAb includes one or more additions, substitutions, or deletions in one or more framework regions. In further embodiments, the variant or mutant anti-VEGF HCAb includes one or more additions, substitutions, or deletions in one or more constant regions.

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

In certain embodiments, the variant or mutant anti-VEGF HCAb includes one or more additions, substitutions, or deletions in the framework 1 region. In some embodiments, the variant or mutant anti-VEGF HCAb includes one or more additions, substitutions, or deletions in the framework 2 region. In some embodiments, the variant or mutant anti-VEGF HCAb includes one or more additions, substitutions, or deletions in the framework 3 region. In some embodiments, the variant or mutant anti-VEGF HCAb includes one or more additions, substitutions, or deletions in the framework 4 region. In some embodiments, the variant or mutant anti-VEGF HCAb includes one or more additions, substitutions, or deletions in the hinge region.

Also disclosed herein are immunoconjugates, which comprise conjugates to cytotoxic agents, such as chemotherapeutic agents, toxins (eg, enzyme-active toxins of bacterial, fungal, plant, or animal origin or fragments thereof) or radioisotopes (ie, radioactive conjugates) The antibodies described herein.

Chemotherapeutic agents suitable for producing such immunoconjugates have been described. Enzyme-active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa ), gabatoxin A chain, botulinum toxin, acacia Protein A chain, Modin A chain, α-broom aspergillus, tung protein, caryophyllin, Phytolaca americana protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, Jatropha toxin, croton toxin, sapaonaria officinalis inhibitor, white tree toxin, mitogellin, limited aspergillin, phenomycin, inomycin and trichothecene ( tricothecenes). A variety of radionuclides can be used to generate radioactively bound monospecific or bispecific anti-PDGF antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y, and ¹⁸⁶ Re.

Use a variety of bifunctional protein coupling agents such as the following to make a combination of antibody and cytotoxic agent: N-butadiene imido-3- (2-pyridyl dithiol) propionate (SPDP), imino Thiolane (IT), bifunctional derivatives of imido esters (such as dimethyl adipamide HCL), active esters (such as dibutane diimide suberate), Aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzyl) hexamethylenediamine), dual nitrogen derivatives (such as bis- (p-diazobenzyl) -ethyl Diamine), diisocyanate (such as tolyl 2,6-diisocyanate), and dual reactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylidene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for binding radionucleotide to antibodies .

In another embodiment, the antibody may bind to a "receptor" (such as streptavidin) for pre-targeting, where the antibody-receptor conjugate is administered to the patient, and then removed from the circulation using a scavenger In addition to unbound conjugates, a "ligand" (eg avidin) bound to a cytotoxic agent (eg radionuclide) is administered.

The monospecific, bispecific or trispecific anti-VEGF antibodies disclosed herein can also be formulated in liposomes. Antibody-containing liposomes are prepared by methods known in the art, such as those described in US 4,485,045, US 4,544,545, and US 5,013,556. Particularly useful liposomes can be produced by reverse phase evaporation using a lipid composition comprising phospholipid choline, cholesterol and PEG-derived phospholipid ethanolamine (PEG-PE). The liposomes are squeezed through a filter with a defined pore size to produce liposomes with the desired diameter. Fab 'fragments of antibodies can be bound to liposomes via disulfide exchange reaction.

Covalent modifications of monospecific, bispecific or trispecific anti-VEGF antibodies are also included within the scope of the present invention. Where applicable, it can be made by chemical synthesis or by enzymatic or chemical cleavage of antibodies. By reacting the targeted antibody amino acid residue with an organic derivatizing agent capable of reacting with the selected side chain or N or C-terminal residue, other types of covalent modifications of the antibody are introduced into the molecule. Exemplary covalent modifications of polypeptides are described in US 5,534,615, and all disclosures regarding covalent modifications of polypeptides are specifically incorporated herein by reference. Exemplary covalent modifications of one class of antibodies include linking the antibody to one of a variety of non-protein polymers in the manner described in US 4,640,835, US 4,496,689, US 4,301,144, US 4,670,417, US 4,791,192 or US 4,179,337, For example polyethylene glycol, polypropylene glycol or polyoxyalkylene.

The monospecific, bispecific and trispecific antibodies disclosed herein can be produced recombinantly. Thus, disclosed herein are nucleic acids encoding antibodies, expression vectors containing nucleic acids encoding antibodies, and cells containing nucleic acids encoding antibodies. Methods for recombinant production are generally known in current advanced technology, and include the expression of proteins in prokaryotic and eukaryotic cells, followed by isolation of antibodies and usually purification to a pharmaceutically acceptable purity. To express the antibody as described above in the host cell, the nucleic acid encoding the antibody sequence is inserted into the expression vector by standard methods. Performed in appropriate prokaryotic or eukaryotic host cells such as CHO cells, NS0 cells, SP2 / 0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast or E. coli cells, and antibodies are recovered from the cells (supernatant Or cells after lysis).

Therefore, certain embodiments disclosed herein include a method for preparing monospecific, bispecific and trispecific antibodies, the method comprising the following steps: a) using at least one expression vector comprising a nucleic acid molecule encoding an antibody to host cells Transformation; b) cultivating the host cell under conditions that allow synthesis of the antibody molecule; and c) recovering the antibody molecule from the culture.

By conventional immunoglobulin purification procedures, such as protein A-agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography, the antibody is appropriately separated from the culture medium.

As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such names include offspring. Therefore, the words "transformants" and "transformed cells" include primary individual cells and cultures derived from them, regardless of the number of transfers. It is also understood that due to intentional or unintentional mutations, all progeny cannot be exactly the same in terms of DNA content. This includes mutant offspring that have the same function or biological activity as those screened in the original transformed cells. Under different names intended, it will be obvious from the context.

As used herein, the term "transformation" refers to the process of vector / nucleic acid transfer to a host cell. If cells without a strong cell wall barrier are used as host cells, they can be transfected, for example, by calcium phosphate precipitation. However, other methods such as introducing DNA into cells by nuclear injection or by protoplast fusion can also be used. If prokaryotic cells or cells containing solid cell wall structures are used, for example, one method of transfection is calcium treatment using calcium chloride.

As used herein, "presentation" refers to the process by which nucleic acids are transcribed into mRNA and / or the process by which transcribed mRNA (also called transcripts) is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides are collectively referred to as gene products. If the polynucleotide is derived from genomic DNA, performance in eukaryotic cells may include mRNA splicing.

A "vector" is a nucleic acid molecule that transfers an inserted nucleic acid molecule into and / or between host cells, especially a self-replicating type. The term includes vectors mainly used for inserting DNA or RNA into cells (eg, chromosomal integration), vectors mainly used for DNA or RNA replication, and expression vectors used for transcription and / or translation of DNA or RNA. It also includes carriers that provide more than one of the described functions.

"Expression vector" is a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. "Expression system" generally refers to a suitable host cell composed of an expression vector that can be used to produce the desired expression product.

As used herein, the term "host cell" refers to any type of cellular system that can be engineered to produce the antibodies disclosed herein. In one embodiment, HEK293 cells and CHO cells are used as host cells.

Control sequences suitable for prokaryotes include, for example, promoters, optionally manipulating sequences, and ribosome binding sites. It is known that eukaryotic cells use promoters, enhancers and polyadenylation signals.

A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, if the DNA used for the pre-sequence or secretion leader sequence appears to be a protein involved in the secretion of the polypeptide, it is operably linked to the DNA of the polypeptide; if the promoter or enhancer affects the transcription of the coding sequence, it is operable Linked to the sequence; or if the position of the ribosome binding site facilitates translation, it is operably linked to the coding sequence. Generally, "operably linked" means that the linked DNA sequences are adjacent, and in the case of a secretion leader sequence, adjacent and in reading frame. However, enhancers need not be adjacent. The connection is achieved by connecting at suitable restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used according to conventional practice.

Also disclosed herein are isolated nucleic acid sequences encoding monospecific, bispecific or trispecific anti-VEGF antibodies, vectors and host cells containing nucleic acids, and recombinant techniques for antibody production.

To produce antibodies recombinantly, the nucleic acid encoding them can be isolated and inserted into a replicable vector for further colonization (DNA amplification) or expression. In some embodiments, antibodies can be produced by homologous recombination as described in US 5,204,244, for example, all disclosures regarding antibody production are specifically incorporated herein by reference. It is easy to use conventional procedures (for example, by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of antibodies) to isolate and sequence the DNA encoding the antibodies. Many carriers are available. Vector components generally include (but are not limited to) one or more of the following: signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences, for example, as described in US 5,534,615, regarding proteins All disclosures presented are specifically incorporated herein by reference.

The host cells suitable for colonizing or expressing DNA in the vector herein are the above-mentioned prokaryote, yeast or higher eukaryote cells. Suitable prokaryotic organisms for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae ( Enterobacteriaceae ), such as Escherichia ( Escherichia ), such as E. coli ( E. coli . coli), Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), Proteus (Proteus), the genus Salmonella (Salmonella) (e.g. Salmonella typhimurium ( Salmonella typhimurium)), Serratia (SERRATIA) (e.g. clayey marcescens (S. marcescans)) and the genus Shigella (Shigella) and Bacillus (bacilli), such as Bacillus subtilis (B. subtilis) And Bacillus licheniformis ( B. licheniformis ), Pseudomonas ( Pseudomonas ) (such as P. aeruginosa ( P. aeruginosa )) and Streptomyces ( Streptomyces ). An exemplary E. coli selection host is E. coli 294 (ATCC 31,446), but other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are also suitable. These examples are illustrative, not limiting.

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

Host cells suitable for expressing glycosylated monospecific or bispecific human anti-PDGF antibodies are derived from multicellular organisms, including invertebrate cells, such as plant and insect cells. Many baculovirus strains and variants have been identified and from such sources as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), A. albopictus (mosquito), fruit The corresponding allowable insect host cells are the hosts of Drosophila melanogaster (Drosophila) and Bombyx mori . A variety of virus strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 virus strain of Bombyx mori NPV, and such viruses can be used herein as the present invention The virus in it is especially used to transfect Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco can also be used as hosts.

However, the focus is on vertebrate cells, and the propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of suitable mammalian host cell lines are monkey kidney CV1 cell line transformed from SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cells grown by subculture in suspension culture); young Hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO); mouse podocytes (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer 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 hepatocytes (Hep G2, HB 8065); mouse breast tumors (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human liver cancer cells (Hep G2).

The host cells are transformed with the above-mentioned expression vectors to produce monospecific, bispecific or trispecific anti-VEGF antibodies, and cultured in conventional media as appropriate to induce promoters, select transformants or expand coding sites The genes to be sequenced.

The host cells used to produce monospecific, bispecific or trispecific anti-VEGF antibodies can be cultured in a variety of media. Such as Ham's F10 (Sigma), minimum essential medium ((MEM) (Sigma)), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing host cells . In addition, US 4,767,704, US 4,657,866, US 4,927,762, US 4,560,655 or US 5,122,469, WO 90/03430, WO 87/00195 or US Re. 30,985 can be used as the culture medium for host cells. Any of these media can be supplemented with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffer ( 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 equivalent energy sources . Any other desired supplements may also be included in appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, and the like are those previously used with host cells selected for expression, and will be apparent to those of ordinary skill.

When recombinant techniques are used, antibodies can be produced intracellularly, in the periplasmic space or directly secreted into the culture medium. If the antibody is produced inside the cell, as a first step, for example, by centrifugation or ultrafiltration to remove particulate debris, that is, host cells or lysed fragments.

Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite 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 the isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains. For all mouse isotypes and human gamma 3, protein G is recommended. The matrix to which the affinity ligand is attached is most often agarose, but other matrices can be used. Mechanically stable substrates such as controllable porous glass or poly (styrenedivinyl) benzene allow faster flow rates and shorter processing times than can be achieved with agarose. In the case where the antibody contains a CH3 domain, Bakerbond ABX ™ resin is suitable for purification. Other techniques for protein purification can also be used, such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, silica on chromatography, heparin SEPHAROSE ™ on chromatography, anion or cation exchange resin ( Such as polyaspartic acid columns) upper chromatography, focused chromatography, SDS-PAGE and ammonium sulfate precipitation, depending on the antibody to be recovered.

After any initial purification steps, the mixture containing the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography, using a lysis buffer, with a pH between about 2.5-4.5, preferably in low salt At a concentration (eg about 0-0.25M salt).

Also disclosed herein are methods of using monospecific, bispecific or trispecific anti-VEGF antibodies to treat ophthalmic disorders. Examples of ophthalmic conditions include, but are not limited to, age-related macular degeneration (AMD), choroidal neovascularization (CNV), cystic macular edema (CME), myopic-associated choroidal neovascularization, vascular streaks, diabetic macular Edema (DME), macular edema, macular edema caused by retinal vein occlusion and angiogenesis in the front of the eye, such as, for example, keratitis, corneal transplantation, or corneal angiogenesis after corneal plasty; corneal angiogenesis caused by hypoxia (widely worn Contact lens), pterygium conjunctiva, subretinal edema and intraretinal edema. Examples of age-related macular degeneration (AMD) include but are not limited to dry or non-exudative AMD or wet or exudative or neovascular AMD.

Macular degeneration, also known as age-related macular degeneration, is the most common cause of vision loss in people over the age of 50 in the United States, and its incidence increases with age. AMD is classified as wet (neovascular) or dry (non-neovascular). The dry form of the disease is the most common. It occurs when the central retina has been deformed, colored, or most commonly thinned, a process related to atrophy of retinal pigment epithelial cells and loss of macular photoreceptors. The result is a center-like atrophy. The wet form of the disease causes the worst vision loss. Although the wet form is usually associated with aging, other diseases that can cause wet macular degeneration include severe myopia and some intraocular infections, such as histoplasmosis, which may be aggravated in AIDS individuals. The wet form is characterized by abnormal blood vessels growing through retinal pigment epithelial cells, causing bleeding, exudation, scarring, or retinal detachment.

Disclosed herein are sustained release formulations of monospecific, bispecific, or trispecific anti-VEGF antibodies for use in the treatment of ocular disorders. Therefore, monospecific, bispecific, and trispecific anti-VEGF antibodies can be released from the sustained-release drug delivery system to the vitreous over a period of 3 to 6 months for long-term treatment of chronic eye conditions such as dry AMD.

A hydrogel is a colloidal gel formed as a dispersion in water or other aqueous medium. Therefore, when forming a colloid, a hydrogel is formed in which the dispersed phase (polymer) is combined with the continuous phase (that is, water) to produce a viscous gel-like product; for example, condensed silicic acid. A hydrogel is a three-dimensional network of hydrophilic polymer chains cross-linked via chemical or physical bonds. Because the polymer chain is hydrophilic, the hydrogel absorbs water and swells (unless it has absorbed the maximum amount of water). The swelling process is the same as the dissolution of the non-crosslinked hydrophilic polymer. By definition, water accounts for at least 10% of the total weight (or volume) of the hydrogel.

Examples of hydrogels include synthetic polymers such as polyhydroxyethyl methacrylate, and chemically or physically cross-linked polyvinyl alcohol, polyacrylamide, poly (N-vinylpyrrolidone), polyethylene oxide and Hydrolyzed polyacrylonitrile. Examples of hydrogels as organic polymers include covalently or ionically crosslinked polysaccharide-based hydrogels such as alginate, pectin, carboxymethyl cellulose, heparin, hyaluronate, and chitin derived , Chitosan, pullulan, gellan gum and xanthan gum hydrogel polyvalent metal salt. The specific hydrogels used in the experiments were cellulose compounds (ie hydroxypropyl methyl cellulose [HPMC]) and high molecular weight hyaluronic acid (HA).

In some embodiments, hydrogel formulations for intravitreal injection are disclosed that use polymeric hyaluronic acid and the monospecific and / or bispecific human anti-PDGF antibodies disclosed herein. This drug delivery system can provide sustained release of low-dose monospecific, bispecific, or trispecific anti-VEGF antibodies over a period of 3 to 6 months, and prevents the conversion of autogenous AMD to wet AMD. The drug delivery system may also include microsphere encapsulation of monospecific, bispecific or trispecific anti-VEGF in a hydrogel. A sustained-release drug delivery system can provide the necessary anti-VEGF blockade in the eye to reduce the possibility of self-drying progression to neovascular AMD. In addition, the low doses released in the eye over a sustained period of time do not provide agents with systemic toxicity levels.

Sustained release drug delivery systems can also be used to provide patients at risk of neovascularization with central retinal vein occlusion and patients with severe non-proliferative diabetic retinopathy at risk of progressing to neovascular disease Sustained release of anti-VEGF blockade.

Alternatively, the drug delivery system may be a PLGA implant, liposome-encapsulated antibody optionally clamped in cross-linked hyaluronic acid. In addition, microspheres, microcapsules (in the range of 0.001 to 100 microns) and liposomes have modified surfaces to produce interactions with hydrogel polymers, thereby altering release.

Also included herein are specific drug delivery system formulations and methods for administering these drug delivery systems to treat ocular conditions. The intraocular injection can be implanted or injected into the glass cavity (posterior chamber) of the eye. Drug delivery systems within the scope of the present invention may be biodegradable implants and / or microspheres. The drug delivery system may be monolithic, ie the active agent is evenly distributed or dispersed in the biodegradable polymer. The therapeutic agent can be released from the drug delivery system made according to the present invention for a period of time between about 2 hours and 12 months or more. An important feature of the drug delivery system is that it does not include any means for securing the drug delivery system to the location within the eyeball to which it is administered (such as a cap, protrusion, or suture pull tab).

An important feature of drug delivery systems within the scope of the present invention is that they can be implanted or injected into intraocular locations (such as anterior fascia, subconjunctival, intravitreal or suprachoroidal locations) to provide sustained release of therapeutic agents, However, no significant immunogenicity will occur or continue at or near the implantation or injection site in the eyeball.

Polylactide (PLA) polymers exist in two chemical forms: poly (L-lactide) and poly (D, L-lactide). Pure poly (L-lactide) is stereoregular, and therefore, it is also highly crystalline, so it degrades at a very slow rate in vivo. Poly (D, L-lactide) is stereo-random, which leads to more rapid degradation in vivo. Therefore, as a mixture of the main poly (L-lactide) polymers, the degradation rate ratio of the PLA polymer which is the poly (D-lactide) polymer in vivo is mainly poly (D-lactide) The PLA polymer of the polymer is slow. PLGA is a copolymer that combines poly (D, L-lactide) and poly (glycolide) in various possible ratios. The higher the content of glycolide in PLGA, the faster the polymer degradation.

In some embodiments, a drug delivery system for intraocular administration (ie, by intravitreal implantation or injection) contains at least 75 weight percent PLA and up to about 25 weight percent poly (D, L-lactide -Co-glycolide) polymer, consisting of or consisting essentially of it.

Suspensions of microspheres (with anti-angiogenic agents) suspended in hydrogels (such as polymer hyaluronic acid) are also within the scope, which can be administered into the intraocular position via a syringe needle. Administration of such suspensions requires that the viscosity of the microsphere suspension at 25 ° C is less than about 300,000 cP. The viscosity of water at 25 ° C is about 1.0 cP (cP or cps is centipoise, a measure of viscosity). At 25 ° C, the viscosity of olive oil is 84 cP, the viscosity of castor oil is 986 cP, and the viscosity of glycerin is 1490 cP.

The antibodies present in the drug delivery system can be evenly dispersed in the biodegradable polymer of the drug delivery system. The choice of biodegradable polymer can vary depending on the desired release kinetics, patient tolerance, the nature of the disease to be treated, and similar factors. The polymer characteristics considered include, but are not limited to, biocompatibility and biodegradability at the implantation site, compatibility with the active agent of interest, and processing temperature. The biodegradable polymer matrix generally comprises at least about 10 weight percent, at least about 20 weight percent, at least about 30 weight percent, at least about 40 weight percent, at least about 50 weight percent, at least about 60 weight percent, at least about 70 weight percent , At least about 80 weight percent or at least about 90 weight percent implant. In one variant, the biodegradable polymer matrix contains about 40% to 50% by weight of the drug delivery system.

Biodegradable polymers that can be used include, but are not limited to, polymers made from monomers such as organic esters or ethers, which produce physiologically acceptable degradation products upon degradation. Anhydrides, amides, orthoesters, or the like, alone or in combination with other monomers, can also be used. The polymer is generally a polycondensate. The polymer may be cross-linked or non-cross-linked.

To a large extent, in addition to carbon and hydrogen, the polymer will include oxygen and nitrogen, especially oxygen. Oxygen may be present as an oxygen group, such as a hydroxyl group or an ether, a carbonyl group (eg, a non- pendant carbonyl group, such as a carboxylic acid ester), and the like. Nitrogen can exist as amide, cyano and amine groups. An exemplary list of biodegradable polymers that can be used is described in Heller, Biodegradable Polymers in Controlled Drug Delivery, "CRC Critical Reviews in Therapeutic Drug Carrier Systems", Volume 1. CRC Press, Boca Raton, Fla. (1987) in.

Particular attention is paid to polymers, homopolymers or copolymers of hydroxyaliphatic carboxylic acids, and polysaccharides. The polyesters of interest include homo- or copolymers of D-lactic acid, L-lactic acid, racemic lactic acid, glycolic acid, caprolactone, and combinations thereof. Particular attention is paid to the copolymer of glycolic acid and lactic acid, where the rate of biodegradation is controlled by the ratio of glycolic acid to lactic acid. The percentage of each monomer in the poly (lactic acid-co-glycolic acid) (PLGA) copolymer may be 0-100%, about 15-85%, about 25-75%, or about 35-65%. In some variations, 25/75 PLGA and / or 50/50 PLGA copolymers are used. In other variations, PLGA copolymers are used in combination with polylactide polymers.

Other agents can be used in the formulation of drug delivery systems to achieve a variety of purposes. For example, buffers and preservatives can be used. Preservatives that can be used include, but are not limited to, sodium bisulfite, sodium bisulfate, sodium thiosulfate, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric nitrate, p-hydroxybenzoic acid Methyl ester, polyvinyl alcohol and phenethyl alcohol. Examples of buffers that may be used 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 that can be used to stabilize particles in colloids and / or electrolytes such as sodium chloride and potassium chloride can also be included in the formulation. The drug delivery system may also include acid and base excipients to control the pH in the microenvironment and the interface (diffusion stagnant layer).

Biodegradable drug delivery systems can also include additional hydrophilic or hydrophobic compounds that accelerate or delay the release of the active agent. In addition, release modifiers such as those described in US Patent No. 5,869,079 may be included in the implant. The amount of release modifier used will depend on the desired release characteristics, the activity of the modifier, and the release characteristics of the glucocorticoid in the absence of the modifier. In the case where the buffer or release enhancer or modifier is hydrophilic, it can also act as a release accelerator. Hydrophilic additives are used to increase the release rate through faster dissolution of the substance surrounding the drug particles, which increases the surface area of the exposed drug, thereby increasing the drug diffusion rate. Similarly, hydrophobic buffers or enhancers or modifiers can dissolve more slowly, slowing the exposure of drug particles, thereby slowing the rate of drug diffusion.

Drug delivery systems within the scope of the present invention can be formulated with active agent antibody particles dispersed in a biodegradable polymer. Without being bound by theory, the release of the Xianxin active agent can be achieved by erosion of the biodegradable polymer matrix and diffusion into the intraocular fluid, such as vitreous humor, by microparticles, followed by dissolution of the polymer matrix and release of the active agent. Factors that affect the kinetics of active agent release from the implant can include factors such 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, manufacturing method, exposed surface area, Characteristics of implant density and polymer erosion rate.

The rate of release of the active agent may depend, at least in part, on the rate of degradation of the polymer backbone component of the biodegradable polymer matrix. For example, polycondensates can be degraded by hydrolysis (and other mechanisms), so any change in the implant composition that enhances the absorption of water by the implant will likely increase the rate of hydrolysis, thereby increasing the rate of polymer degradation and erosion, Thus increasing the rate of active agent release. The release rate of the active agent can also be affected by the crystallinity of the active agent, the pH in the implant, and the pH at the interface.

The release kinetics of the drug delivery system of the present invention may depend in part on the surface area of the drug delivery system. The larger surface area exposes more polymer and active agent to the intraocular fluid, causing the polymer to erode faster and the active agent particles in the intraocular fluid to dissolve faster.

Also disclosed herein is a pharmaceutical composition comprising a monospecific human anti-VEGF HCAb, a bispecific antibody with a specificity of VEGF or a trispecific antibody with a specificity of VEGF. The use of the antibodies described herein for the manufacture of pharmaceutical compositions is also disclosed. Also disclosed are methods of using the disclosed antibodies and pharmaceutical compositions containing the antibodies to treat eye disorders.

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

The compositions disclosed herein can be administered by a variety of methods known in the art. As the skilled technician understands, the route and / or mode of administration will vary depending on the desired result. In order to administer the disclosed antibody by certain routes of administration, it may be necessary to associate the antibody with a substance that prevents its inactivation or to co-administer the antibody with this substance. For example, the antibody can be administered to the individual in a suitable carrier, such as liposomes or diluents. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for preparing sterile injectable solutions or dispersions before use. The use of such media and reagents for pharmaceutical active substances is known in the art.

As used herein, the phrases "parenteral administration" and "administered parenterally" mean modes of administration other than enteral and local administration, usually by injection, And including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intraocular, intravitreal, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular , Subarachnoid, intraspine, epidural and intrasternal injection and infusion.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. The presence of microorganisms can be prevented by the above sterilization procedures and by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. There is also a need to include isotonic agents, such as sugar, sodium chloride, and the like in the composition. In addition, the absorption of injectable pharmaceutical forms can be extended by including agents that delay absorption such as aluminum monostearate and gelatin.

Regardless of the route of administration selected, the disclosed antibodies and / or pharmaceutical compositions containing such antibodies that can be used in a suitable hydrated form can be formulated into pharmaceutically acceptable by conventional methods known to those of ordinary skill in the art Accepted dosage form.

The actual dosage level of the active ingredient in the pharmaceutical composition can be varied in order to obtain an amount that is effective for achieving the desired therapeutic response and non-toxic to the patient for a specific patient, composition and mode of administration. The selected dose level will depend on a variety of pharmacokinetic factors, including the activity of the specific composition of the invention used, the route of administration, the time of administration, the excretion rate of the specific compound used, the duration of treatment Other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, condition, overall health and previous medical history of the patient being treated, and similar factors well known in the medical field.

The dosage suitable for the pharmaceutical composition disclosed herein when delivered intravitreally ranges from about 0.1 mg to about 50 mg per eye. Other suitable dosages 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, and about 0.5 mg to about 0.5 mg per eye 15 mg, 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, each 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, each 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, each eye 9.5 mg, 10 mg per eye, 11 mg per eye, 12 mg per eye, 13 mg per eye, 14 mg per eye, 15 mg per eye, approximately 15 mg per eye 15 mg, about 17 mg per eye, about 18 mg per eye, about 19 mg per eye, or about 20 mg per eye.

The composition must be sterile and fluid to the extent that the composition can be delivered by syringe. In addition to water, the carrier is preferably an isotonic buffered physiological saline solution.

The proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. In most cases, preferred compositions include isotonic agents, such as sugars, polyols (such as mannitol or sorbitol), and sodium chloride.

Pharmaceutical compositions and drug delivery systems that include monospecific, bispecific, and / or trispecific anti-VEGF antibodies can be injected into the intraocular location via a syringe, or can be inserted (implanted) into the eye by various methods. The method involves placing an incision in the sclera by forceps, by a trocar or by another type of applicator. In some cases, a trocar or applicator can be used without making an incision. In a preferred variant, a hand-held applicator is used to insert one or more biodegradable implants into the eye. Hand-held applicators usually contain 18-30 GA stainless steel needles, levers, actuators and pistons. Suitable devices for inserting the implant into the posterior eye or site include those disclosed in US 7,090,681.

The method of administration generally first involves using a needle, trocar, or implant device to access the target area within the eye. Once in a target area, such as a glass cavity, the lever on the handheld device can be lowered to cause the actuator driver piston to move forward. When the piston moves forward, it can push the implant into the target area (ie, the vitreous body).

Various techniques can be used to manufacture implants within the scope of the present invention. Applicable technologies include phase separation method, interface method, extrusion method, compression method, molding method, injection molding method, hot pressing method and the like.

The term "intraocular injection" refers to an injection administered by entering the eye of a patient. The term "peri-eye injection" refers to the injection that is administered behind the eye but outside the eye wall. The term "trans-lens ligament" refers to an injection administered through the lens ligament, which is a series of fibers that connect the ciliary body and the lens of the eye. The term "intravitreal" refers to an injection that is directly administered into the lumen of the eye through the patient's eye.

The pharmaceutical formulations described herein can be delivered via intravitreal intravitreal injection, which can be through the lens ligament or, if necessary, without the lens ligament. The intravitreal injection of this formulation, whether through the lens ligament or through the direct flat (transscleral) injection, delivers effective broad-spectrum antibiotics directly to the purulent tissue without the need for multiple individual drug treatments or multiple Individual injections are urgently mixed into the eyes.

Generally, the above pharmaceutical composition will be administered to a mammalian individual in need of treatment (eg, human, cat, dog, other pet, domestic animal, wild animal, or farm animal). The composition is injected intravitreally and through the lens ligament using methods and techniques known to those of ordinary skill in the field of ophthalmology.

In general, delivery via a typical 27 gauge cannula can be used, using a 1 mL TB syringe, taking care to re-suspend the formulation with instantaneous bounce and shock when the injection is about to be used. The medical volume (ie, dosage) required for this formulation varies based on the type of eye disorder and anatomical factors regarding the volume of injection available added to the closed intraocular space.

In addition, injection into the anterior chamber (ie, anterior chamber) is within the scope of the present invention.

In alternative embodiments, the formulation may also be in the form of eye drops or eye sprays as well as via subconjunctival injection, intraocular intracameral injection, anterior subfascial injection, intraarticular injection or lesions, if necessary Intra-injection delivery, especially but not limited to some cases where necessary to deliver other medications when needed by the patient.

The following examples, sequence listings and figures are provided to help understand the present invention, the true scope of which is described in the accompanying patent application. It should be understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. Examples Example 1. Immunization of llamas with VEGF-A

All animal procedures were performed in USDA and OLAW certified equipment (Abcore, Inc., San Diego, CA) according to the guidelines established by the Institutional Animal Care and Use Committee.

The llama was initially subcutaneously (SC) with 500 μg of purified human VEGF-A and complete Freund's adjuvant (CFA, Sigma, St. Louis, MO) on day 0 at 8 different sites (62.5 μg per site) ) Immunization. Llama was treated with 500 μg of purified human VEGF-A and incomplete Freund's adjuvant (IFA, Sigma, St. Louis, MO) on day 15, day 29, day 57 and day 84 in 8 In different parts (62.5 μg per part) SC was strengthened. On day 111, llamas were boosted with VEGF-A-KLH (keyhole-hemocyanin) in the same dose and manner.

Production bleeding (500 ml each) was obtained on Day 43, Day 69, Day 98 and Day 125. Serum antibody titers were measured and PBMC from each hemorrhage was stored in RNA lysis buffer.

When antiserum from each bleeding was collected and also included from the aforementioned bleeding, the reactivity and specificity of the antiserum to VEGF-A was tested by ELISA. Two independent tests were carried out, one each in different combinations to determine activity. At the end of the immunization protocol, the antiserum titer was positive at more than 600,000 dilutions and absolutely positive at more than 10,000 dilutions. Table 2. Antiserum titers as determined by ELISA Example 2. Construction of anti- VEGF library

Based on the antiserum results, PMBC collected on day 125 was used to construct a VHH library. Total RNA was purified from lysed cells and used as a template for RT-PCR to construct a single domain antibody library. The DNA encoding VHH is purified after PCR using specific primers. The phage display vector pADL20c (AbDesign Labs, San Diego) was used for colonization. Get 1.3 × 10 ⁸ independent pure library. Ten pure lines were randomly selected and sequenced. All pure lines have VHH inserts in the correct reading frame.

The phage display antibody library was screened with antigen-coated disks. After four rounds of panning, 95 pure lines were randomly selected to select individual positive pure lines. More than 80% of pure lines are positive. Check the specific binding of positive lysates to other antigens.

Two pure lines 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

The LA-C5 llama VHH is highly homologous to the human germline sequence IGHV3-23. Therefore, CDR-1, CDR-1 and CDR-2 were transplanted from LA-C5 to IGHV-23 to produce PA-C5. Two amino acids from the llama backbone were introduced at positions 47 (L to W) and 78 (L to Y) of PA-C5.

The humanized anti-VEGF-A VH region from LA-C5 was fused with the IgG1 hinge region and CD2 / CH3 region in GS SYSTEM ™ (Lonza, Basel, Switzerland) expression plastids to form HCAb sequences. The expressing plastids were transfected into CHO cell lines to produce fully recombinant Hu-C5 HCAb (Table 4). Table 4. Humanized Hu-C5 Example 4. Determination of the binding affinity of anti- VEGF-A HCAb Hu-C5

Biolayer interferometry (BLI) is a label-free technique used to measure the binding kinetics of human VEGF A (R & D systems) and anti-VEGF A HCAb antibodies. The affinity was measured with Octet QK ^e equipped with an anti-human IgG Fc capture (AHC) biosensor tip (FortéBio®, Menlo Park, CA). The analysis was performed in 1x PBS buffer (Gibco®, PBS pH7.2) at 30 ° C. The sample was agitated at 1000 rpm. Prior to analysis, the sensor was wetted for 15 minutes. The purified anti-VEGF A HCAb antibody was tested for its ability to bind to the tip of the AHC sensor. The tip was loaded with 20 μg / ml anti-VEGF A HCAb antibody. The load is carried out for 300 seconds, so that the capture level is between 1.8 and 2 nm. Human VEGF-A antigen was prepared for binding analysis by diluting to 100 nM, 150 nM, 250 nM, 350 nM in 1x PBS. The association was started and monitored for 200 seconds, and then the tip was transferred to a factor-free protein (Gibco, PBS pH 7.2) in 1x PBS buffer to monitor dissociation. Sensor data was collected throughout the experiment and processed and analyzed using Octet data analysis software 7 (FortéBio®). The combined results are presented in Table 5.

Kinetic data analysis indicates that the 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 * Exceed detection limit example 5. Cell- based functional characterization of anti- VEGF-A HCAb Hu-C5

VEGF-dependent calcium mobilization was measured in human umbilical vein endothelial cells (HUVEC). HUVEC was diluted to 1.6 × 10 ⁵ cells / ml in EGM-Plus complete medium supplemented with SingleQuots growth factor and supplement (LONZA), and 50 μl was added to each well of the 384-well fibronectin plate. The culture plates were incubated overnight at 37 ° C and 5% CO ₂ . One hour before stimulation, the medium was then removed, and HUVEC was loaded with analysis buffer (AB (1L) containing calcium-sensitive dye Fluo4 NW (Invitrogen, Inc.): 880 mL H ₂ O; 20 mL HEPES; 100 mL 10x HBSS; 10 mL FBS; 0.7 g probenecid + 7.5 mL 1M NaOH; 40 μl per well). The solution was incubated with cells at 37 ° C and 5% CO ₂ for 60 minutes. The cells were washed and replaced with 25 μl analysis buffer. The inhibitory effect of VEGF-A was determined by adding Hu-C5, bevacizumab and ranibizumab at concentrations ranging from 7.8 pM to 2.0 nM, and incubated with 50 pM VEGF-A 4 Hours, then added to the cells, and the calcium response was recorded (Figure 4). The data was analyzed in quadruplicate using the average peak fluorescence at each inhibitor concentration tested. Functional analysis indicated that the anti-VEGF-A HCAb Hu-C5 molecule completely neutralized VEGF A activity. Example 6. Characterization of anti- VEGF-A / ANG-2 bispecific antibody

The structure of the bispecific antibody specific for VEGF-A and ANG-2 is depicted in FIG. 5. The bispecific antibody consists of the ANG-2 specific HCAb A33A8 VH sequence (disclosed in PCT / US2016 / 044838, all disclosures regarding A33A8 are incorporated by reference) to the Hu-C5 VH via the immunoglobulin Fc region Sequence formation. (SEQ ID NO:. 15 QVQLLESGGGLVQPGGSLRLSCAASGRTFSNTGMSWVRQAPGKGLEL VSAITPSGSGTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAKGGGNYYYISNYDYWGQGTLVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMHWVRQAPGKGLVWVSRINSDGSSTSYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAREGYSSGGQFDYWGQGTLVTVSS.

Examples of PDGF and ANG-2 sequences that can be used in the bispecific antibodies disclosed herein can be found in Tables 6 and 7 and the co-pending applications PCT / US2016 / 046595 and PCT / US2016 / 044838, regarding anti-PDGF and All disclosures of anti-ANG-2 antibodies are incorporated herein by reference. Table 6. Sequence of anti-PDGF-BB VH Table 7. Sequence of anti-ANG-2 VH

The affinity was measured using the forte BIO OCTET® QK ^e system equipped with an anti-hIgG Fc capture (AHC) biosensor tip (forté BIO, Menlo Park, CA). To measure the affinity of AHC, the purified anti-VEGF / ANG-2 bispecific antibody was tested for its ability to bind to the tip of the AHC sensor. The tip was loaded with 20 μg / ml anti-VEGF / ANG-2 bispecific antibody. The load is carried out for 300 seconds, so that the capture level is between 1.8 and 2 nm. By diluting to a concentration of 400 nM in 1x PBS, VEGF-A (R & D systems) antigen was prepared for binding analysis. Start association and monitor for 200 seconds, then transfer the tip to 1xPBS buffer without factor protein (Gibco, PBS pH 7.2) to monitor dissociation for 300 seconds, then transfer the tip to the second antigen ANG-2 (R & D systems ) And monitoring for 200 seconds, followed by washing in 1 x PBS for 600 seconds (Figure 6A), which was also carried out by loading AGN-2 first, then VEGF A antigen, and then ANG-2 (Figure 6B). The biolayer interferometric analysis was then used to monitor the anti-VEGF / ANG-2 bispecific antibody binding activity in real time. The antigen binding affinity is calculated based on the measured association ("K _assoc ") and dissociation ("K _dissoc ") constants. Use OCTET® data analysis software 6.4 (fortéBIO) to process and analyze data.

The Octet binding curve shows the continuous binding of the two antigens. The first antigen did not dissociate correspondingly when exposed to the second antigen, indicating that the bispecific antibody can bind all antigens when non-competitively contracting. Example 7. specific antibodies against PDGF BB, VEGF-A and ANG-2 ter

Trispecific antibodies are generated using antigen binding regions specific for VEGF-A, PDGF and ANG-2.

The anti-PDGF BB VH1 (P36C12) and Lucentis LgG1 will be fused at the N-terminus and will be resistant to ANG- in the GS SYSTEM ™ (Lonza, Basel, Switzerland) performance plasmid. 2 VH3 (A33A8) (PCT / US2016 / 044838) was fused at the C-terminus with the same Lucentis IgG1 to form a trispecific sequence (heavy chain) (Figure 1). The heavy chain plastid was transfected into the CHO cell line together with the light chain plastid to produce a fully human recombinant trispecific antibody P36-LCNT-A33 (Figure 8).

7A-F depict the Octet immediate binding of trispecific antibodies against VEGF-A, PDGF BB and ANG-2.

The affinity was measured using the forte BIO OCTET® QK ^e system equipped with an anti-hIgG Fc capture (AHC) biosensor tip (forté BIO, Menlo Park, CA). To measure the affinity of AHC, the purified anti-PDGF / VEGF / ANG2 trispecific antibody was tested for its ability to bind to the tip of the AHC sensor. The tip was loaded with 20 μg / ml anti-PDGF / VEGF / ANG2 trispecific antibody. The load is carried out for 300 seconds, so that the capture level is between 1.8 and 2 nm. By diluting to a concentration of 400 nM in 1x PBS, ANG-2 (R & D systems) antigen was prepared for binding analysis. Start association and monitor for 200 seconds, then transfer the tip to 1xPBS buffer without factor protein (Gibco, PBS pH 7.2) to monitor dissociation for 300 seconds, then transfer the tip to the second antigen (VEGF-A) and monitor Seconds, followed by washing in 1 x PBS for 300 seconds, and then the tip was transferred to the third antigen (PDGF BB) (Figure 7A). This was also done by loading PDGF BB first, then VEGF A antigen, and then ANG-2 (Figure 7B-F). The biolayer interferometric analysis is then used to monitor the anti-PDGF / VEGF / ANG2 trispecific antibody binding activity in real time. The antigen binding affinity is calculated based on the measured association ("K _assoc ") and dissociation ("K _dissoc ") constants. Use OCTET® data analysis software 6.4 (fortéBIO) to process and analyze data. The Octet binding curve shows the continuous binding of two antigens. The first antigen does not dissociate correspondingly when exposed to the second and third antigens. This indicates that the trispecific antibody can bind all antigens when it is not competitively contracted.

Unless otherwise indicated, all numerical values used in the specification and patent application to express the amounts, properties (eg, molecular weight, reaction conditions), etc. of the components should be understood to be modified by the term "about" in all cases. As used herein, the terms "about" and "approximately" mean within 10% to 15%, preferably within 5% to 10%. Therefore, unless indicated to the contrary, the numerical parameters set forth in the specification and accompanying patent application are approximate values, which may vary depending on the desired properties sought to be obtained by the present invention. At least and without trying to limit the application of the principle of equivalents to the scope of the patent application, each numerical parameter will be explained at least according to the reported effective number and the application of conventional rounding techniques. Although the numerical ranges and parameters that illustrate the broad scope of the invention are approximate, the numerical values set forth in the specific examples are reported as accurately as possible. However, any numerical value inherently contains certain errors necessarily caused by the standard deviations in the corresponding test measurements.

Unless otherwise indicated herein or the context clearly contradicts, the terms "a (an)" and "the" and similar indicators used in the context of describing the invention (especially in the context of the scope of the patent application below) Treated as both singular and plural. The description of numerical ranges in this document is intended only as a shorthand method of referring individually to individual values within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. Unless otherwise indicated herein or otherwise clearly contradicted by context, all methods described herein can be performed in any suitable order. Unless otherwise claimed, the use of any and all examples or exemplary language (eg, "such as") provided herein is merely to better illustrate the present invention and not to limit the scope of the present invention. The language in the description should not be regarded as indicating any unclaimed elements necessary to implement the present invention.

The grouping of alternative elements or embodiments of the invention disclosed herein should not be considered limiting. Members of each group may be mentioned and claimed individually or in any combination with other members of the group or other elements found herein. It is expected that for reasons of convenience and / or patentability, one or more members of the group may be included in or deleted from the group. When any such inclusion or deletion occurs, it is deemed that the specification contains a modified group and therefore satisfies the written description of all Markush groups used in the scope of the accompanying patent application.

This document describes certain embodiments of the invention, including the best mode known to the inventors for carrying out the invention. Of course, those of ordinary skill in the art after reading the above description will be obvious variations of these described embodiments. The inventor expects skilled artisans to adopt such variants as appropriate, and the inventor intends the invention to be implemented in ways other than those specifically described herein. Therefore, the present invention includes all modifications and equivalents permitted by the applicable law for the subject matter described in the accompanying patent application. Furthermore, unless otherwise indicated herein or otherwise clearly contradicted by context, the present invention covers any combination of all possible variations of the above elements.

The specific embodiments disclosed herein may be further limited by the use of language consisting of or consisting essentially of in the scope of the patent application. When applied to the scope of a patent application, the transitional term "consisting of" excludes any element, step, or component not specified in the scope of the patent application, regardless of each amended application or addition. The transitional term "consisting essentially of" limits the scope of the patent application to specified substances or steps and substances or steps that do not substantially affect basic and novel features. The claimed embodiments of the invention are inherently or explicitly described herein and enable it to proceed.

In addition, throughout this specification, patents and printed publications have been mentioned extensively. The references and printed publications cited above are individually incorporated by reference in their entirety.

Finally, it should be understood that the embodiments of the invention disclosed herein illustrate the principles of the invention. Other modifications that can be used are within the scope of the invention. Thus, by way of example (but not limitation), alternative configurations of the invention can be utilized in accordance with the teachings herein. Therefore, the invention is not limited to what is exactly shown and described.

Figure 1 depicts the HCAb antibody structure.

Figures 2A-G depict several forms of multivalent antibodies, including bispecific single domain VH (Figure 2A); bispecific HCAb antibodies (Figure 2B and 2C); tetrameric antibodies / VH bispecific antibodies (Figure 2D) ; Bispecific Fab / VH antibody (Figure 2E); heavy / light chain bispecific antibody (Figure 2F); and trispecific antibody (Figure 2G). The second and third antigen binding domains are depicted as ellipses. The optional position of the second antigen binding domain is indicated by an asterisk.

Figure 3 depicts the genetic structure of the humanized HCAb disclosed herein.

Figure 4 depicts the effect of Hu-C5 on VEGF-dependent calcium mobilization in human umbilical vein endothelial cells.

Figure 5 depicts the structure of a bispecific anti-VEGF-A / ANG-2 antibody.

Figures 6A-C depict the use of the forteBio OCTET® QK ^e system equipped with anti-hIgG Fc capture biosensor tips and biosensors coated with streptavidin against bispecific anti-VEGF-A / ANG-2 HCAb Different analysis of the combination of affinity.

Figures 7A-F depict six different analyses of Octet immediate binding of trispecific antibodies against VEGF-A, PDGF BB and ANG-2.

Figure 8 depicts an exemplary trispecific antibody.

Claims (29)

A heavy chain only antibody (HCAb) specific for VEGF, wherein the HCAb has the variable heavy chain (VH) region sequence of one of SEQ ID NO: 5, 9 or 13. An HCAb specific for VEGF, wherein the HCAb VH region sequence has at least 85% identity with the sequence of one of SEQ ID NO: 5, 9 or 13. For example, the HCAb in the first or second patent scope, where VEGF is VEGF-A. An HCAb specific for VEGF-A, wherein one or more of the complementarity determining regions (CDRs) are selected from SEQ ID NOs: 6-8 and 10-12. An HCAb specific for VEGF-A, wherein one or more of the CDRs has at least 85% identity with the CDR sequences selected from SEQ ID NOs: 6-8 and 10-12. An HCAb specific for VEGF-A, wherein at least one CDR comprises SEQ ID NO: 6. An HCAb specific for VEGF-A, wherein at least one CDR comprises SEQ ID NO: 7. An HCAb specific for VEGF-A, wherein at least one CDR comprises SEQ ID NO: 8. An HCAb specific for VEGF-A, wherein at least one CDR comprises SEQ ID NO: 10. An HCAb specific for VEGF-A, wherein at least one CDR comprises SEQ ID NO: 11. An HCAb specific for VEGF-A, wherein at least one CDR comprises SEQ ID NO: 12. For example, the HCAb according to any one of claims 1 to 11, wherein at least one amino acid in the VH region is substituted, added or deleted, and the HCAb retains its specificity for VEGF-A. A human or humanized antibody that competes with HCAb LA-C5, LA-D6, and / or Hu-C5 for binding to VEGF-A. A bispecific antibody having a first specificity for VEGF and a second specificity for PDGF. For example, the bispecific antibody according to item 14 of the patent application scope, wherein the first specificity is exhibited by the HCAb according to any one of items 1 to 13 of the patent application scope. A bispecific antibody as claimed in item 14 of the patent scope, wherein the second specificity is exhibited by a VH region selected from the group consisting of: 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). A bispecific antibody having a first specificity for VEGF and a second specificity for ANG-2. For example, the bispecific antibody according to item 17 of the patent application scope, wherein the first specificity is exhibited by the HCAb according to any one of items 1 to 13 of the patent application scope. For example, the bispecific antibody according to item 17 of the patent application scope, wherein the second specificity is exhibited by the VH region selected from the following: 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). For example, the bispecific antibody according to item 17 of the patent application has the amino acid sequence of SEQ ID NO: 15. A trispecific antibody having a first specificity for VEGF, a second specificity for ANG-2, and a third specificity for PDGF. For example, the trispecific antibody of item 21 of the patent application has the heavy chain sequence of SEQ ID NO: 32. For example, the trispecific antibody of item 21 of the patent application includes the light chain sequence of SEQ ID NO: 33 as appropriate. A method for treating an ophthalmological disorder, which comprises administering to a subject in need an HCAb that binds VEGF as described in any one of patent application items 1 to 13 and any one of patent application items 14 to 20 A bispecific antibody or a trispecific antibody according to any one of items 21 to 23 of the patent application. For example, the method of claim 24, wherein the ophthalmic condition includes age-related macular degeneration (AMD), choroidal neovascularization (CNV), cystic macular edema (CME), myopia-related choroidal neovascularization, vascular-like Striae, diabetic macular edema (DME), macular edema, retinal vein occlusion, abnormal corneal angiogenesis, pterygium conjunctiva, subretinal edema, or intraretinal edema. The method of claim 24, wherein the abnormal corneal angiogenesis is caused by keratitis, corneal transplantation, keratoplasty or hypoxia. A HCAb with VEGF binding VH region as in any one of patent application items 1 to 13; a bispecific antibody as in any of patent application items 14 to 20; or as a patent application The use of the trispecific antibody of any one of items 21 to 23 in the range for the manufacture of a medicament for the treatment of an ophthalmological disorder in an individual in need. For the purpose of claim 27, where the ophthalmic condition includes age-related macular degeneration (AMD), choroidal neovascularization (CNV), cystic macular edema (CME), myopia-related choroidal neovascularization, vascular-like Striae, diabetic macular edema (DME), macular edema, retinal vein occlusion, abnormal corneal angiogenesis, pterygium conjunctiva, subretinal edema, or intraretinal edema. The use as claimed in item 27 of the patent scope, wherein the abnormal corneal angiogenesis is caused by keratitis, corneal transplantation, keratoplasty or hypoxia.