CN117545503A

CN117545503A - Methods of treating choroidal neovascularization using anti-ANG 2 x VEGF multispecific antibodies

Info

Publication number: CN117545503A
Application number: CN202280026993.2A
Authority: CN
Inventors: M·穆达; J·卢洛
Original assignee: Abupro Co
Current assignee: Abupro Co
Priority date: 2021-03-30
Filing date: 2022-03-29
Publication date: 2024-02-09

Abstract

The present disclosure relates generally to methods of treating choroidal neovascularization in a subject in need thereof using anti-ANG-2 x VEGF multispecific antibodies.

Description

Methods of treating choroidal neovascularization using anti-ANG 2 x VEGF multispecific antibodies

Cross-reference to related patent applications

The present application claims the benefit and priority of U.S. provisional patent application Ser. No. 63/167,822, filed on 3 months of 2021, and U.S. provisional patent application Ser. No. 63/182,623, filed on 4 months of 2021, the contents of which are incorporated herein by reference in their entirety.

Technical Field

The present technology relates generally to methods of treating choroidal neovascularization in a subject in need thereof using anti-ANG-2 x VEGF multispecific antibodies.

Background

The following description of the background of the invention is provided simply as an aid in understanding the technology and is not admitted to describe or constitute prior art to the technology.

Choroidal Neovascularization (CNV) is characterized by the growth of new blood vessels that originate from the choroid and enter the subretinal pigment epithelium or subretinal space through rupture of the bruch's membrane. The choroid is the vascular layer of the eye, located between the retina and sclera, which is part of the uvea. Bruch's membrane is the innermost layer of the choroid and adjoins the retinal pigment epithelium. Retinal pigment epithelium is adjacent to rod and cone cells of the eye, which illustrates the potential disturbance to vision. CNV may result in vision loss due to intra-retinal or subretinal fluid exudation, hemorrhage, or fibrosis at the macula. The most important causes of CNV are age-related macular degeneration (AMD) and Pathologic Myopia (PM). Other causes such as inflammation, polypoidal chorioretinopathy, or central serous chorioretinopathy are also recognized. For example, AMD is a major cause of infection in the elderly and causes blindness and severe vision impairment in all high-income countries. After age 50, the prevalence of this disease increases exponentially every decade. PM is commonly found in about 2% of the general population. Myopic CNV is the leading cause of severe vision loss and blindness in PM eyes, with 4% -11% of infected eyes suffering from PM. This disease is particularly prevalent in people under the age of 50 in asia. CNV is the most important vision risk factor, often secondary to AMD and PM. Oxidative stress on Retinal Pigment Epithelium (RPE) cells is believed to cause extracellular debris to accumulate, thus forming drusen, and to cause dysfunction of RPE cells by affecting the penetration of nutrients by bruch's membrane.

VEGF is an important pro-angiogenic element, usually produced by the RPE and retinal photoreceptors. In CNV, RPE promotes atypical neovascularization, while VEGF is the primary growth factor responsible for the development and progression of new blood vessels. The idea of inhibiting VEGF activity with anti-VEGF drugs has been studied in the management of ocular neovascular disorders, in particular for the treatment of the consequences, i.e. subretinal and/or intraretinal fluids. However, current anti-VEGF treatments do not induce complete regression of CNV, requiring repeated injections to maintain vision. Furthermore, evidence for resistance to anti-VEGF therapy suggests that alternative drugs are needed to treat CNV.

Thus, there is an urgent need for therapeutic agents that effectively treat CNV in patients in need thereof.

Disclosure of Invention

In one aspect, the present disclosure provides a method for treating Choroidal Neovascularization (CNV) in a subject in need thereof, comprising administering to the subject an effective amount of an anti-ANG-2 x VEGF multispecific antibody, wherein the anti-ANG-2 x VEGF multispecific antibody comprises a heavy chain sequence and a light chain sequence selected from the group consisting of: SEQ ID NO. 1 and SEQ ID NO. 2; SEQ ID NO. 5 and SEQ ID NO. 6; and SEQ ID NO 9 and SEQ ID NO 10.

In one aspect, the present disclosure provides a method for treating Choroidal Neovascularization (CNV) in a subject in need thereof, comprising administering to the subject an effective amount of an anti-ANG-2 x VEGF multispecific antibody, wherein the anti-ANG-2 x VEGF multispecific antibody comprises a first antigen-binding portion that binds a VEGF epitope and a second antigen-binding portion that binds an ANG-2 epitope, wherein the first antigen-binding portion comprises a first heavy chain immunoglobulin variable domain (V _H ) And a first light chain immunoglobulin variable domain (V _L ) And the second antigen binding portion comprises a second V _H And a second V _L The method comprises the steps of carrying out a first treatment on the surface of the Wherein the first V _H Comprising the amino acid sequence of SEQ ID NO. 25 or SEQ ID NO. 44 and a first V _L An amino acid sequence comprising SEQ ID NO. 27; and wherein the second V _H Comprising the amino acid sequence of SEQ ID NO. 26 or SEQ ID NO. 45; and a second V _L Comprising the amino acid sequence of SEQ ID NO. 28. In some embodiments, the anti-ANG-2 xvegf multispecific antibody comprises an immunoglobulin and an scFv. Additionally or alternatively, in some embodiments, the scFv comprises a second antigen binding portion.

In any and all embodiments of the methods disclosed herein, the choroidal neovascularization is caused by age-related macular degeneration (AMD), pathologic Myopia (PM), inflammation, polypoidal choriocaulopathy, or central serous chorioretinopathy.

Additionally or alternatively, in some embodiments, the subject has been diagnosed as having CNV. Examples of signs or symptoms of CNV include, but are not limited to, distortion or waviness of central vision or central vision with gray/black/plaques, retinal blisters or bleeds, loss of brightness or different colors exhibited by each eye, vision deformity, painless vision loss, paracentral or central dark spots, different sizes of objects exhibited by each eye, flashing or blinking in central vision, vision loss due to fluid exudation in or under the retina, bleeding or macular fibrosis. In any and all embodiments of the methods disclosed herein, the subject exhibits an increased level of expression and/or an increased activity of VEGF and/or Ang-2. Additionally or alternatively, in certain embodiments, the subject is a human.

Additionally or alternatively, in some embodiments, the methods of the present technology further comprise administering one or more additional therapies to the subject separately, sequentially or simultaneously. Examples of such additional therapies include laser photocoagulation, photodynamic therapy (PDT), sodium pipadatinib, bevacizumab, ranibizumab, aflibercept, and corticosteroids.

In any and all embodiments of the methods disclosed herein, the anti-ANG-2 x VEGF multispecific antibody is administered by topical, intravitreal, intraocular, subretinal, or subscleral administration. In certain embodiments, the subscleral administration is achieved by implanting a slow release subscleral implant into the subject.

Additionally or alternatively, in some embodiments of the methods disclosed herein, administration of an anti-ANG-2 x VEGF multispecific antibody results in a reduction of neovascularization and/or vascular leakage in the eye of the subject. In some embodiments, the subject does not exhibit ocular inflammation 1 week after administration of the anti-ANG-2 xvegf multispecific antibody.

Drawings

FIG. 1 shows a schematic representation of an anti-ANG-2 XVEGF bispecific antibody of the disclosure.

FIG. 2A shows the binding affinities (KD) of anti-ANG-2 XVEGF bispecific antibody ABP201 (represented by SEQ ID NO:1 and SEQ ID NO: 2) to huVEGF and huANG2, respectively. Figure 2B shows ABP201 is selective for ANG2 and cross-reactive with monkeys, rats, and rabbits.

Figure 3 shows patient preparation for administration of anti-ANG-2 x VEGF bispecific antibodies of the disclosure.

FIG. 4 shows an exemplary experimental design for studying toxicity and Pharmacokinetics (PK) of anti-ANG-2 XVEGF bispecific antibody ABP201 in a rabbit model.

Figure 5 shows the tolerability results of anti ANG-2 x VEGF bispecific antibody ABP201 24 hours post injection. An exacerbation of inflammation was observed in ABP201 group.

Figure 6 shows the tolerability results of anti ANG-2 x VEGF bispecific antibody ABP201 7 days post injection. Inflammation was cleared in ABP201 treated groups.

Figure 7 shows the tolerability results of anti ANG-2 x VEGF bispecific antibody ABP201 14 days post injection. Histopathological data indicated that ABP201 treated groups were not significantly problematic.

Fig. 8 shows an exemplary image demonstrating histopathology of a test rabbit 14 days after injection.

FIG. 9 shows PK results for anti-ANG-2 XVEGF bispecific antibody ABP201 in an in vivo rabbit model.

Fig. 10 shows PK average parameters of anti ANG-2 x VEGF bispecific antibody ABP201 in adult male dutch black-banded rabbits.

FIG. 11 shows PK comparison of anti-ANG-2 XVEGF bispecific antibody ABP201 with Eylea and Eylea-like molecules reported in the literature.

Fig. 12A-12B show the results of a study to evaluate the activity of anti-ANG-2 x VEGF bispecific antibody ABP-201 in eyes using a rat laser induced Choroidal Neovascularization (CNV) disease model. Figure 12A shows lesion volume in ABP-201 treated CNV rats. Figure 12B shows leakage in ABP201 treated CNV rats.

FIG. 13A shows ABP 201%Heavy Chain (HC) and Light Chain (LC) amino acid sequences of SEQ ID NO. 1 and SEQ ID NO. 2). V (V) _H CDR 1-3 and V _L CDR 1-3 amino acid sequences are underlined and V is anti-Ang 2scFv _H And V _L Amino acid sequences are shown in italics. The anti-VEGF HC and anti-Ang-2 scFv are shown as SEQ ID NO 3 and SEQ ID NO 4, respectively.

FIG. 13B shows the Heavy (HC) and Light (LC) chain amino acid sequences of ABP202 (SEQ ID NO:5 and SEQ ID NO: 6). V (V) _H CDR 1-3 and V _L CDR 1-3 amino acid sequences are underlined and V is anti-Ang 2scFv _H And V _L Amino acid sequences are shown in italics. The anti-VEGF HC and anti-Ang-2 scFv are shown as SEQ ID NO:7 and SEQ ID NO:8, respectively.

FIG. 13C shows the Heavy (HC) and Light (LC) amino acid sequences of ABP200 (SEQ ID NO:9 and SEQ ID NO:10, respectively). V (V) _H CDR 1-3 and V _L CDR 1-3 amino acid sequences are underlined and V is anti-Ang 2scFv _H And V _L Amino acid sequences are shown in italics. The anti-VEGF HC and anti-Ang-2 scFv are shown as SEQ ID NO. 11 and SEQ ID NO. 12, respectively.

FIG. 13D shows the CDR regions of an anti-ANG-2 XVEGF bispecific antibody of the disclosure (represented by SEQ ID NOS: 13-24 and 42-43).

FIG. 13E shows the variable heavy chain (V) of the anti-ANG-2 XVEGF bispecific antibodies of the disclosure (represented by SEQ ID NOS: 25-28 and 44-45) _H ) Domain and variable light chain (V _L ) A domain.

FIG. 13F shows the amino acid sequences of the targets VEGF and Ang-2 represented by SEQ ID NO 38-39, respectively.

Fig. 14A shows representative angiography of experimental and control groups. Leakage was assessed by the scoring system described in the experimental methods. Arrows indicate lesions.

Fig. 14B shows representative OCT images of experimental and control groups. The volume is determined by the calculation described in the method. Red arrows indicate lesions.

Fig. 15A shows fluorescein angiography scores in the experimental and control groups.

Fig. 15B shows lesion volumes in the experimental and control groups.

FIG. 16 shows an analysis of the change in fluorescence of a cumulative region of interest (ROI) in vivo over time with anti-ANG-2 XVEGF bispecific antibody ABP201 (represented by SEQ ID NO:1 and SEQ ID NO: 2). Vehicle and aflibercept were used as negative and positive controls, respectively.

FIG. 17 shows the difference in percentage fluorescence of the anti-ANG-2 XVEGF bispecific antibody ABP201 (represented by SEQ ID NO:1 and SEQ ID NO: 2) versus the vehicle at each time point in vivo for the cumulative region of interest (ROI).

Fig. 18A-18D show exemplary images of fluorescence over time for the different treatment groups depicted in fig. 16.

Detailed Description

It should be appreciated that certain aspects, modes, embodiments, variations and features of the present method are described below in various degrees of detail in order to provide a substantial understanding of the present technology. It is to be understood that this disclosure is not limited to particular uses, methods, reagents, compounds, compositions or biological systems, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., sambrook and Russell edition (2001), molecular Cloning: A Laboratory Manual, 3 rd edition; ausubel et al editions (2007), current Protocols in Molecular Biology series; methods in Enzymology (Academic Press, inc., n.y.) series; macPherson et al (1991), PCR 1:A Practical Approach (IRL Press at Oxford University Press); macPherson et al (1995), PCR 2:A Practical Approach; harlow and Lane editions (1999), antibodies, A Laboratory Manual; freshney (2005), culture of Animal Cells: A Manual of Basic Technique, 5 th edition; gait edit (1984), oligonucleotide Synthesis; U.S. Pat. nos. 4,683,195; hames and Higgins editions (1984), nucleic Acid Hybridization; anderson (1999), nucleic Acid Hybridization; hames and Higgins editions (1984), transcription and Translation; immobilized Cells and Enzymes (IRL Press (1986)); perbal (1984), A Practical Guide to Molecular Cloning; miller and Calos editions (1987), gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); makrides edit (2003), gene Transfer and Expression in Mammalian Cells; mayer and Walker editions (1987), immunochemical Methods in Cell and Molecular Biology (Academic Press, london) and Herzenberg et al editions (1996), weir's Handbook of Experimental Immunology. Methods for detecting and measuring the level of polypeptide gene expression products (i.e., the level of gene translation) are well known in the art and include the use of polypeptide detection methods, such as antibody detection and quantification techniques. (see also, strachan & Read, human Molecular Genetics, second edition (John Wiley and Sons, inc., NY, 1999)).

Existing anti-VEGF therapeutics such as bevacizumab (Avastin) can adversely affect the systemic cardiovascular system and induce platelet aggregation, degranulation and thrombosis. In contrast, the anti-ANG-2 x VEGF multispecific antibodies of the present disclosure exhibit rapid systemic clearance following intravitreal delivery and do not cause undesirable inflammatory responses in the treated eye. Furthermore, the anti-ANG-2 x VEGF multispecific antibodies of the present disclosure significantly reduce neovascular lesion formation and/or vascular leakage in an in vivo CNV model at low doses less than 1 week after treatment.

Definition of the definition

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry, and nucleic acid chemistry, and the hybridization described below are those well known and commonly employed in the art.

As used herein, unless otherwise indicated or apparent from the context, the term "about" referring to a number is generally considered to include numbers falling within the range of 1%, 5%, or 10% in either direction (greater than or less than) of the number (excluding the case where such numbers are less than 0% or greater than 100% of the possible values).

As used herein, "administering" a pharmaceutical agent or drug to a subject includes any route of introducing or delivering a compound to a subject to perform its intended function. Administration may be by any suitable route including, but not limited to, oral, intraocular, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intrathecal, or topical administration. Administration includes self-administration and administration via others.

As used herein, the term "antibody" is collectively referred to as an immunoglobulin or immunoglobulin-like molecule, including for example, but not limited to IgA, igD, igE, igG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, e.g., a mammal such as a human, goat, rabbit, and mouse, as well as non-mammalian species, such as a shark immunoglobulin. As used herein, an "antibody" (including intact immunoglobulins) and an "antigen-binding fragment" specifically binds to a molecule of interest (or a group of highly similar molecules of interest), substantially excluding molecules other than the molecule of interest (e.g., having a molecular size of at least 10 greater than other molecules in a biological sample ³ M ^-1 At least 10 a big ⁴ M ^-1 Or at least 10 larger than ⁵ M ^-1 Antibodies and antibody fragments) bind. The term "antibody" also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (such as bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical co., rockford, ill.); kuby, j., immunology, 3 rd edition, w.h. freeman&Co., new York, 1997.

More specifically, an antibody refers to a polypeptide ligand comprising at least a light chain immunoglobulin variable region or a heavy chainImmunoglobulin variable regions and specifically recognize and bind epitopes of antigens. Antibodies consist of heavy and light chains, each of which has a variable region, referred to as the variable heavy chain region (V _H ) And a variable light chain region (V _L )。V _H Region and V _L Together, the regions are responsible for binding to the antigen recognized by the antibody. Typically, immunoglobulins have a heavy (H) chain and a light (L) chain interconnected by disulfide bonds. There are two types of light chains, lambda (lambda) and kappa (kappa). There are five main heavy chain classes (or isotypes) that determine the functional activity of an antibody molecule: igM, igD, igG, igA and IgE. Each heavy and light chain comprises a constant region and a variable region (these regions are also referred to as "domains"). The heavy and light chain variable regions combine to specifically bind antigen. The light and heavy chain variable regions comprise "framework" regions interrupted by three hypervariable regions, also known as "complementarity determining regions" or "CDRs". The scope of framework regions and CDRs has been defined (see Kabat et al, sequences of Proteins of Immunological Interest, u.s. Device of Health and Human Services,1991, which is hereby incorporated by reference). The Kabat database is being maintained online. The sequences of the framework regions of the different light or heavy chains are relatively conserved in the species. The framework regions of antibodies, i.e., the combined framework regions that make up the light and heavy chains, are predominantly in the β -sheet configuration, and the CDRs form loops that connect and in some cases form part of the β -sheet structure. Thus, the framework regions serve to form a scaffold that positions the CDRs in the correct orientation by non-covalent interactions between the chains.

CDRs are primarily responsible for binding to epitopes of the antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus V _H CDR3 is located in the variable domain of the antibody heavy chain in which it resides, while V _L CDR1 is CDR1 from the variable domain of the antibody light chain in which it resides. Antibodies that bind VEGF and/or Ang-2 proteins will have a specific V _H Region and V _L Region sequences, and thus have specific CDR sequences. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. To the greatest extentTube CDRs vary from antibody to antibody, but only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are known as Specificity Determining Residues (SDRs). As used herein, "immunoglobulin-related composition" refers to antibodies (including monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, multispecific antibodies, bispecific antibodies, and the like) and antibody fragments. The antibody or antigen binding fragment thereof specifically binds to the antigen.

As used herein, the term "antibody-related polypeptide" means antigen-binding antibody fragments, including single chain antibodies, which may comprise the variable region alone in combination with all or part of the following polypeptide elements: hinge region, CH of antibody molecule ₁ 、CH ₂ And CH (CH) ₃ A domain. The technology also comprises a variable region, a hinge region, a CH ₁ 、CH ₂ And CH (CH) ₃ Any combination of domains. Antibody-related molecules such as, but not limited to, fab 'and F (ab') ₂ Fd, single chain Fv (scFv), single chain antibody, disulfide-linked Fv (sdFv) and compositions comprising V _L Or V _H Fragments of the domains. Examples include: (i) Fab fragment, from V _L 、V _H 、C _L And CH (CH) ₁ A monovalent fragment of a domain; (ii) F (ab') ₂ A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bond of a hinge region; (iii) From V _H And CH (CH) ₁ Fd fragments of domain composition; (iv) V by antibody single arm _L And V _H Fv fragments consisting of domains; (v) dAb fragment (Ward et al Nature, vol. 341: pages 544-546, 1989), which is defined by V _H Domain composition; and (vi) an isolated Complementarity Determining Region (CDR). Thus, an "antibody fragment" or "antigen-binding fragment" may comprise a portion of a full-length antibody, typically an antigen-binding or variable region thereof. Examples of antibody fragments or antigen-binding fragments include Fab, fab ', F (ab') ₂ And Fv fragments; a double body; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

As used herein, "bispecific antibody" or "BsAb" refers to a binding molecule that can bind simultaneously to a polypeptide having a different junction Antibodies to two targets are constructed, for example, two different target antigens, two different epitopes on the same target antigen, or a hapten and an epitope on the target antigen or target antigen. A variety of different bispecific antibody structures are known in the art. In some embodiments, each antigen binding portion of a bispecific antibody comprises V _H And/or V _L A zone; in some such embodiments, V _H And/or V _L Regions are those found in a particular monoclonal antibody. In some embodiments, the bispecific antibody contains two antigen binding portions, each antigen binding portion comprising V from a different monoclonal antibody _H And/or V _L A zone. In some embodiments, the bispecific antibody contains two antigen binding moieties, wherein one of the two antigen binding moieties comprises a polypeptide having V _H And/or V _L Immunoglobulin molecules having regions containing CDRs from a first monoclonal antibody and another antigen binding portion comprising a polypeptide having V _H And/or V _L Antibody fragments of the region (e.g., fab, F (ab') ₂ Fd, fv, dAB, scFv, etc.), which contain CDRs from the second monoclonal antibody.

As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated immune defense mechanism whereby effector cells of the immune system actively lyse target cells whose membrane surface antigens have been bound by antibodies such as anti-VEGF and/or anti-Ang-2 antibodies.

As used herein, "antigen" refers to a molecule to which an antibody (or antigen binding fragment thereof) can selectively bind. The target antigen may be a protein, carbohydrate, nucleic acid, lipid, hapten or other naturally occurring or synthetic compound. In some embodiments, the target antigen may be a polypeptide (e.g., a VEGF or Ang-2 polypeptide). Antigens may also be administered to animals to generate an immune response in the animals.

The term "antigen binding fragment" refers to a fragment of the entire immunoglobulin structure that has a portion of a polypeptide responsible for binding to an antigen. Examples of antigen binding fragments useful in the present technologyComprising scFv (scFv) ₂ scFvFc, fab, fab 'and F (ab') ₂ But is not limited thereto. Any of the above antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for binding specificity and neutralizing activity in the same manner as the whole antibody.

As used herein, "binding affinity" means the strength of the total non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or antigen peptide). The affinity of molecule X for its partner Y is generally determined by the dissociation constant (K _D ) And (3) representing. Affinity can be measured by standard methods known in the art, including the standard methods described herein. Low affinity complexes contain antibodies that generally tend to dissociate readily from the antigen, while high affinity complexes contain antibodies that generally tend to remain bound to the antigen for a longer period of time.

As used herein, the term "biological sample" means sample material derived from living cells. Biological samples may include tissue, cells, protein or cell membrane extracts and biological fluids (e.g., ascites or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells and fluids present in the subject. Biological samples of the present technology include, but are not limited to, samples taken from the following: breast tissue, kidney tissue, cervix, endometrium, head or neck, gall bladder, parotid gland tissue, prostate, brain, pituitary gland, kidney tissue, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid tissue, heart tissue, lung tissue, bladder, adipose tissue, lymph node tissue, uterus, ovary tissue, adrenal gland tissue, testis tissue, tonsil, thymus, blood, hair, mouth, skin, serum, plasma, CSF, sperm, prostatic fluid, semen, urine, stool, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples may also be obtained from biopsies of internal organs. Biological samples may be obtained from subjects for diagnosis or study, or may be obtained from individuals who are not ill, as controls or for basic studies. Samples may be obtained by standard methods including, for example, venipuncture and surgical biopsy. In certain embodiments, the biological sample is a tissue sample obtained by needle biopsy.

As used herein, the term "CDR grafting" means replacing at least one CDR of a "recipient" antibody with a CDR "graft" from a "donor" antibody having the desired antigen specificity.

As used herein, the term "chimeric antibody" means an antibody in which the Fc constant region (e.g., a mouse Fc constant region) of a monoclonal antibody from one species is replaced with the Fc constant region (e.g., a human Fc constant region) of an antibody from another species using recombinant DNA techniques. See generally, robinson et al, PCT/US86/02269; akira et al, european patent application 184,187; taniguchi, european patent application 171,496; morrison et al, european patent application 173,494; neuberger et al, WO 86/01533; cabill et al, U.S. patent nos. 4,816,567; cabill et al, european patent application 0125,023; better et al, science, volume 240: pages 1041-1043, 1988; liu et al, proc. Natl. Acad. Sci. USA, volume 84: 3439-3443, 1987; liu et al, j.immunol, volume 139: pages 3521-3526, 1987; sun et al, proc.Natl. Acad. Sci.USA, volume 84: pages 214-218, 1987; nishimura et al, cancer Res, volume 47: pages 999-1005, 1987; wood et al, nature, volume 314: page 446-449, 1885; and Shaw et al, j.Natl.cancer Inst., volume 80: pages 1553-1559, 1988.

As used herein, the term "complement-dependent cytotoxicity" or "CDC" generally refers to effector functions of IgG and IgM antibodies that trigger the classical complement pathway when bound to surface antigens, thereby inducing the formation of membrane attack complexes and target cell lysis.

As used herein, the term "conjugation" refers to the association of two molecules by any method known to those skilled in the art. Suitable association types include chemical bonds and physical bonds. Chemical bonds include, for example, covalent bonds and coordinate bonds. Physical bonds include, for example, hydrogen bonding, dipole interactions, van der Waals forces, electrostatic interactions, hydrophobic interactions, and aromatic ring stacking interactions.

As used herein, the term "consensus FR" means the Framework (FR) antibody region in a consensus immunoglobulin sequence. The FR region of the antibody does not contact the antigen.

As used herein, a "control" is an alternative sample in an experiment for comparison purposes. The control may be "positive" or "negative". For example, where the purpose of the experiment is to determine the correlation of therapeutic effects of a therapeutic agent for treating a particular type of disease, positive controls (compounds or compositions known to exhibit the desired therapeutic effect) and negative controls (subjects or samples not receiving therapy or receiving placebo) are typically employed.

As used herein, the term "diabody" refers to small antibody fragments having two antigen binding sites, which fragments comprise a light chain variable domain (V _L ) Linked heavy chain variable domains (V _H )(V _H V _L ). By using a linker that is too short to allow pairing between two domains on the same strand, these domains are forced to pair with the complementary domain of the other strand and create two antigen binding sites. Diabodies are more fully described in, for example, EP 404,097; WO 93/11161; and Hollinger et al, proc Natl Acad Sci USA, volume 90: pages 6444-6448 (1993).

As used herein, the term "effective amount" refers to an amount sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount that results in the prevention or reduction of a disease or disorder described herein or one or more signs or symptoms associated with a disease or disorder described herein. In the case of therapeutic or prophylactic use, the amount of the composition administered to a subject will depend on the composition; the extent, type and severity of the disease; as well as individual characteristics such as general health, age, sex, weight and tolerance to drugs. The skilled artisan will be able to determine the appropriate dosage based on these and other factors. The composition may also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic composition may be administered to a subject having one or more signs or symptoms of the diseases or disorders described herein. As used herein, a "therapeutically effective amount" of a composition refers to the level of the composition at which the physiological effects of the disease or disorder are improved or eliminated. The therapeutically effective amount may be provided in one or more administrations.

As used herein, the term "effector cell" means an immune cell that is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Exemplary immune cells include bone marrow or lymphoid derived cells, for example, lymphocytes (e.g., B cells and T cells, including cytolytic T Cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Effector cells express specific Fc receptors and perform specific immune functions. Effector cells may induce antibody-dependent cell-mediated cytotoxicity (ADCC), such as neutrophils capable of inducing ADCC. For example, fcαr-expressing monocytes, macrophages, neutrophils, eosinophils and lymphocytes are involved in specifically killing target cells and presenting antigens to other components of the immune system, or binding to antigen-presenting cells.

As used herein, the term "epitope" means a protein determinant capable of specific binding to an antibody. Epitopes are typically composed of chemically active surface groupings of molecules such as amino acids or sugar side chains, and typically have specific three-dimensional structural features, as well as specific charge characteristics. Conformational epitopes differ from non-conformational epitopes in that binding to the former is lost but not to the latter in the presence of denaturing solvents. In some embodiments, an "epitope" of VEGF or Ang-2 protein is the region of the protein to which an anti-VEGF X Ang-2 multispecific antibody of the present technology specifically binds. In some embodiments, the epitope is a conformational epitope or a non-conformational epitope. To screen for epitope-binding anti-VEGF X Ang-2 multispecific Antibodies, conventional cross-blocking assays such as those described in Antibodies, A Laboratory Manual, cold Spring Harbor Laboratory, ed Harlow and David Lane (1988) can be performed. This assay can be used to determine whether an anti-VEGF X Ang-2 multispecific antibody binds to the same site or epitope as an anti-VEGF X Ang-2 multispecific antibody of the present technology. Alternatively or in addition, epitope mapping may be performed by methods known in the art. For example, the antibody sequence may be subjected to mutagenesis, such as by alanine scanning, to identify contact residues. In a different approach, peptides corresponding to different regions of VEGF or Ang-2 proteins may be used in competition assays with the test antibodies or with the test antibodies and antibodies having a characteristic or known epitope.

As used herein, "expression" includes one or more of the following: transcription of the gene into a pre-mRNA; splicing and other processing of the pre-mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if desired for proper expression and function.

As used herein, the term "gene" means a DNA fragment that contains all the information used to regulate the biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

As used herein, "homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence that can be aligned for comparison purposes. When a position in the comparison sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matched or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or polypeptide region) that has a certain percentage (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence means that when aligned, the percentage of bases (or amino acids) is the same when comparing two sequences. The alignment and homology or percentage of sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST, using default parameters. Specifically, the programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; chain = two; cut-off value = 60; expected value = 10; matrix = BLOSUM62; description = 50 sequences; ordering = high score; database = non-redundant, genbank+embl+ddbj+pdb+genbank CDS translation+swiss protein+spldate+pir. Details of these procedures are found in the national center for biotechnology information. A biologically equivalent polynucleotide is a polynucleotide that has a particular percentage of homology and encodes a polypeptide having the same or similar biological activity. Two sequences are considered "unrelated" or "non-homologous" if they share less than 40% identity or less than 25% identity with each other.

As used herein, a "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that comprises minimal sequences derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species such as mouse, rat, rabbit or non-human primate (donor antibody) having the desired specificity, affinity and capacity. In some embodiments, fv Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, the humanized antibody may comprise residues that are not present in the recipient antibody or the donor antibody. These modifications are made to further improve antibody properties such as binding affinity. Typically, a humanized antibody will comprise at least one variable domain (e.g., fab ', F (ab') ₂ Or Fv) and typically comprises two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are FR regions of a human immunoglobulin consensus FR sequence, although the FR regions may comprise one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FR is typically no more than 6 in the H chain and no more than 3 in the L chain. Optionally, the humanized antibody may further comprise an immunoglobulin constant region (Fc), typically at least a portion of a constant region of a human immunoglobulin. For details, please see Jones et al, nature, volume 321: pages 522-525 (1986); reichmann et al Nature, volume 332: 323- 329 (1988), presta, curr.op. Struct.biol., volume 2: pages 593-596 (1992). See, e.g., ahmed&Cheung, FEBS Letters, volume 588, phase 2: pages 288-297 (2014).

As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions typically comprise amino acid residues from a "complementarity determining region" or "CDR" (e.g., V _L About residues 24-34 (L1), 50-56 (L2) and 89-97 (L3), and V _H About residues 31-35B (H1), 50-65 (H2) and 95-102 (H3) (Kabat et al, sequences of Proteins of Immunological Interest, 5 th edition, public Health Service, national Institutes of Health, bethesda, MD. (1991)) and/or those from "hypervariable loops" (e.g., V) _L Residues 26-32 (L1), 50-52 (L2) and 91-96 (L3), and V _H Residues 26-32 (H1), residues 52A-55 (H2) and residues 96-101 (H3) (Chothia and Lesk, J. Mol. Biol., volume 196: pages 901-917 (1987)).

As used herein, the term "identical" or percent "identity" when used in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of identical amino acid residues or nucleotides (i.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region (e.g., a nucleotide sequence encoding an antibody described herein or an amino acid sequence of an antibody described herein) when compared and aligned over a comparison window or specified region to obtain maximum correspondence, as measured using a BLAST or BLAST 2.0 sequence comparison algorithm with default parameters described below, or as measured by manual alignment and visual inspection (e.g., NCBI website). Such sequences are then referred to as "substantially identical". The term also refers to or may be applied to the complement of the test sequence. The term also includes sequences having deletions and/or additions, as well as sequences having substitutions. In some embodiments, the identity exists over a region of at least about 25 amino acids or nucleotides in length, or 50-100 amino acids or nucleotides in length.

As used herein, the term "intact antibody" or "intact immunoglobulin" means an antibody having at least two heavy (H) chain polypeptides and two light (L) chain polypeptides connected to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or V _H ) And a heavy chain constant region. The heavy chain constant region consists of three domains CH ₁ 、CH ₂ And CH (CH) ₃ Composition is prepared. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or V _L ) And a light chain constant region. The light chain constant region consists of one domain C _L Composition is prepared. V (V) _H And V _L The regions may be further subdivided into regions of hypervariability termed Complementarity Determining Regions (CDRs) interspersed with regions that are more conserved termed Framework Regions (FR). Each V _H And V _L Consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR (FR) ₁ 、CDR ₁ 、FR ₂ 、CDR ₂ 、FR ₃ 、CDR ₃ 、FR ₄ . The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

As used herein, the term "linker" refers to a functional group (e.g., a chemical or polypeptide) that covalently links two or more polypeptides or nucleic acids to each other. As used herein, "peptide linker" refers to a method for coupling two proteins together (e.g., for coupling V _H And V _L Domain) of a polypeptide. In certain embodiments, the linker comprises (GGGGS) _n Wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more. In certain embodiments, the linker comprises an amino acid having the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 40) or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 41).

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical, except for possible naturally occurring mutations that may be present in minor amounts. For example, a monoclonal antibody may be an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), rather than a method of its production. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically comprise different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including, for example, but not limited to, hybridoma, recombinant, and phage display techniques. For example, monoclonal antibodies used according to the present method can be prepared by the method described by Kohler et al, nature, volume 256: the hybridoma method first described on page 495 (1975) or may be prepared by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "monoclonal antibodies" can also be used, for example, by Clackson et al, nature, volume 352: pages 624-628 (1991) and Marks et al, J.mol.biol., volume 222: the techniques described in pages 581-597 (1991) were isolated from phage antibody libraries.

As used herein, the term "nucleic acid" or "polynucleotide" means any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, but are not limited to, single-stranded and double-stranded DNA, DNA in which a single-stranded region and a double-stranded region are mixed, single-stranded and double-stranded RNA, RNA in which a single-stranded region and a double-stranded region are mixed, and hybrid molecules comprising DNA and RNA that may be single-stranded or more typically double-stranded, or a mixture of a single-stranded region and a double-stranded region. In addition, a polynucleotide refers to a triple-stranded region comprising RNA, or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, as well as DNA or RNA having a backbone modified for stability or other reasons.

As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, compatible with pharmaceutical administration. Pharmaceutically acceptable carriers and formulations thereof are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20 th edition, edited by a. Gennaro, 2000, lippincott, williams & Wilkins, philiadelphia, pa.).

As used herein, the term "polyclonal antibody" means an antibody preparation derived from at least two (2) different antibody-producing cell lines. The use of this term includes preparations of at least two (2) antibodies that contain antibodies that specifically bind to different epitopes or regions of an antigen.

As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer, i.e., a peptide isostere, comprising two or more amino acids linked to each other by peptide bonds or modified peptide bonds. Polypeptides refer to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and long chains, commonly referred to as proteins. The polypeptide may contain amino acids other than the 20 genes encoding the amino acids. Polypeptides include amino acid sequences that are modified by natural processes such as post-translational processing or by chemical modification techniques well known in the art. Such modifications are well described in the basic text and in more detailed monographs, as well as in a number of research literature.

As used herein, the term "recombinant" when used in reference to, for example, a cell, nucleic acid, protein, or vector, means that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or alteration of the native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, recombinant cells express genes not found in the native (non-recombinant) form of the cell, or express native genes that are otherwise abnormally expressed, under expressed, or not expressed at all.

As used herein, the term "separate" therapeutic use refers to the simultaneous or substantially simultaneous administration of at least two active ingredients by different routes.

As used herein, the term "sequential" therapeutic use refers to administration of at least two active ingredients at different times, the route of administration being the same or different. More specifically, sequential use refers to the administration of all of one active ingredient, followed by the start of administration of the other or other active ingredients. Thus, one active ingredient may be administered within minutes, hours or days, followed by another active ingredient or ingredients. In this case there is no concurrent treatment.

As used herein, the term "simultaneous" therapeutic use refers to the simultaneous or substantially simultaneous administration of at least two active ingredients by the same route.

As used herein, the term "single chain antibody" or "single chain Fv (scFv)" refers to the two domains V of the Fv fragment _L And V _H Is described. Single chain antibody molecules may include polymers having multiple individual molecules, such as dimers, trimers, or other polymers. In addition, although F _v Two domains V of the fragment _L And V _H Encoded by different genes, but they can be joined by synthetic linkers using recombinant methods, enabling them to be made into a single protein chain, where V _L And V _H Regions pair to form monovalent molecules (referred to as single chain F _v (scF _v )). Bird et al (1988), science, volume 242: pages 423-426 and Huston et al (1988), proc Natl Acad Sci, volume 85: pages 5879 to 5883. Such single chain antibodies may be prepared by recombinant techniques or enzymatic or chemical cleavage of intact antibodies.

As used herein, "specifically binds" refers to one molecule (e.g., an antibody or antigen binding fragment thereof) recognizing and binding to another molecule (e.g., an antigen), but does not substantially recognize and bind to other molecules. As used herein, the terms "specifically bind," "specifically bind to," or "specifically to" a particular molecule (e.g., polypeptide or epitope on polypeptide) may have about 10 for the molecule to which it binds, e.g., by a molecule ^-4 M、10 ^-5 M、10 ^-6 M、10 ^-7 M、10 ^-8 M、10 ^-9 M、10 ^-10 M、10 ^-11 M or 10- ¹² K of M _D Exhibit the following. The term "specific binding" may also refer to such binding: wherein the molecule (e.g., an antibody or antigen binding fragment thereof) binds to a particular polypeptide (e.g., a VEGF or Ang-2 polypeptide) or an epitope on a particular polypeptide, but does not substantially bind to any other polypeptide or polypeptide epitope.

As used herein, the term "subject," "patient," or "individual" may be an individual organism, vertebrate, mammal, or human. In some embodiments, the subject, patient, or individual is a human.

As used herein, the term "therapeutic agent" is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect in a subject in need thereof.

As used herein, "treating" encompasses treatment of a disease or disorder described herein in a subject, such as a human, and includes: (i) inhibiting the disease or disorder, i.e., arresting its development; (ii) Alleviating the disease or disorder, i.e., causing regression of the disorder; (iii) slowing the progression of the disorder; and/or (iv) inhibit, alleviate or slow the progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that symptoms associated with the disease are, for example, reduced, cured, or in a state of remission.

It should also be understood that the various treatment modes of disorders as described herein are intended to represent "significant" which includes overall treatment but also incomplete treatment, and in which some biologically or medically relevant result is achieved. The treatment may be a single or several administrations of continuous prolonged treatment for chronic diseases or treatment for acute conditions.

Amino acid sequence modifications of the anti-VEGF x Ang-2 multispecific antibodies described herein are contemplated. Such modifications may be made to increase the binding affinity and/or other biological properties of the antibody, e.g., to glycosylate the encoded amino acid, or to disrupt the ability of the antibody to bind to C1q, fc receptors, or to activate the complement system. Amino acid sequence variants of anti-VEGF x Ang-2 multispecific antibodies are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, by peptide synthesis, or by chemical modification. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions is performed to obtain an antibody of interest, so long as the antibody obtained has the desired properties. Modifications also include alterations in the glycosylation pattern of the protein. The substitution mutagenesis sites of most interest include hypervariable regions, but FR alterations are also contemplated.

Conservative amino acid substitutions are amino acid substitutions that change a given amino acid to a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity, and size). "conservative substitutions" are shown in the following table.

One type of substitution variant involves substitution of one or more hypervariable region residues of the parent antibody. A convenient way to generate such substitution variants involves affinity maturation using phage display. Specifically, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus produced are displayed in a monovalent manner from the filamentous phage particles as fusions with the gene III product of M13 packaged within each particle. Phage display variants are then screened for biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that significantly contribute to antigen binding. Alternatively or in addition, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify the point of contact between the antibody and the antigen. Such contact residues and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are produced, the set of variants is subjected to screening as described herein, and antibodies with similar or superior properties in one or more relevant assays may be selected for further development.

VEGF

Vascular Endothelial Growth Factor (VEGF) is typically a subfamily of signaling proteins involved in angiogenesis and vasculogenesis. VEGF-A (UniProt P15692.2, SEQ ID NO: 38) is the most commonly studied and most relevant form of protein. Drugs such as bevacizumab, ranibizumab and albesep Dou Ba are directed against this protein to reduce the disease state associated with angiogenesis. Before the role of VEGF as a mitogen for endothelial cell secretion was identified, it was identified as a vascular permeability factor, highlighting the ability of VEGF to control many different aspects of endothelial cell behavior, including proliferation, migration, specialization and survival (Ruhrberg, 2003, bioEssays, vol.25: pages 1052-1060). VEGF family members include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor (PIGF) and endocrine gland derived VEGF (EG-VEGF). The active form of VEGF is synthesized as a homodimer or heterodimer with other VEGF family members. VEGF-A exists in six isoforms produced by alternative splicing: VEGF121, VEGF 145, VEGF165, VEGF 183, VEGF 189 and VEGF206. The main difference between these isoforms is their bioavailability, with VEGF165 being the major isoform (Podar et al, 2005, blood, volume 105, 4: pages 1383-1395). Modulation of splicing during embryogenesis produces various isoforms in phase-specific and tissue-specific proportions, creating a rich potential for the unique and environmentally dependent behavior of endothelial cells in response to VEGF. VEGF is considered an important stimulator of both normal and disease-associated angiogenesis (Jakeman et al, 1993, endocrinology, volume 133: pages 848-859; kolch et al, 1995, breast Cancer Research and Treatment, volume 36: pages 139-155) and vascular permeability (Connolly et al, 1989, J.biol. Chem., volume 264: pages 20017-20024).

Ang-2

Ang-2 and VEGF act synergistically to promote pathological angiogenesis and metastasis. Upregulation of Ang-2 is an evading mechanism of VEGF pathway inhibition. In human retinal vascular disease, ang2 (but not Ang 1) levels are elevated. In addition to the VEGF family, angiogenin is thought to be involved in vascular development and postnatal angiogenesis. ANG-2 (GenBank AAF21627.2, SEQ ID NO: 39) expression is limited primarily to vascular remodeling sites where it is thought to block the constitutive stabilizing or maturation function of ANG-1, restoring and maintaining the blood vessel in a plastic state that may be more sensitive to germination signals (Hanahan, 1997; holash et al, oncogene, vol. 18: pages 5356-5362 (1999); maisonpierre, 1997). Typically, ang-2 disrupts the angiogenic event. However, in the presence of VEGF, neovascularization occurs. This protein and its associated pathway Tie2 together with VEGF lead to promotion of CNV progression. LC-10 is a major anti-Ang-2 drug that is useful.

Immunoglobulin-related compositions of the present technology

The present technology describes methods and compositions for the production and use of anti-VEGF XAng-2 multispecific immunoglobulin-related compositions (e.g., anti-VEGF XAng-2 multispecific antibodies or antigen-binding fragments thereof). Antibodies and antigen binding fragments of the present technology selectively bind to VEGF and Ang-2 polypeptides. The anti-VEGF x Ang-2 multispecific immunoglobulin-related compositions of the disclosure are useful in the diagnosis or treatment of CNV. anti-VEGF x Ang-2 multispecific immunoglobulin-related compositions within the scope of the present technology include, for example, but are not limited to, monoclonal, chimeric, humanized, bispecific antibodies and diabodies that specifically bind to target polypeptides, homologs, derivatives, or fragments thereof of such polypeptides. The amino acid sequences of the anti-VEGF x Ang-2 multispecific immunoglobulin-related compositions of the present technology are depicted in FIGS. 13A-13E.

In one aspect, the present disclosure provides a multispecific (e.g., bispecific) antibody, or antigen-binding fragment thereof, comprising a first antigen-binding portion that binds to a VEGF epitope and at least a second antigen-binding portion that binds to an Ang-2 epitope, wherein the first antigen-binding portion comprises a first heavy chain immunoglobulin variable domain (V _H ) And a first light chain immunoglobulin variable domain (V _L ) Wherein the second antigen binding portion comprises a second V _H And a second V _L And wherein (a) a first V _H V comprising SEQ ID NO. 13 _H V of the CDR1 sequence of SEQ ID NO. 14 _H -CDR2 sequence selected from SEQV of ID No. 15 or SEQ ID No. 42 _H -CDR3 sequence, and/or first V _L V comprising SEQ ID NO. 16 _L V of CDR1, SEQ ID NO 17 _L V of the CDR2 sequence and SEQ ID NO. 18 _L -CDR3 sequence. Additionally or alternatively, in some embodiments of the multispecific (e.g., bispecific) antibodies or antigen-binding fragments disclosed herein, the second V _H V comprising SEQ ID NO 19 _H V of the CDR1 sequence, SEQ ID NO:20 or SEQ ID NO:43 _H V of the CDR2 sequence and SEQ ID NO. 21 _H -CDR3 sequence, and/or a second V _L V comprising SEQ ID NO. 22 _L V of CDR1, SEQ ID NO. 23 _L V of the CDR2 sequence and SEQ ID NO. 24 _L -CDR3 sequence.

In one aspect, the present disclosure provides a multispecific (e.g., bispecific) antibody, or antigen-binding fragment thereof, comprising a first antigen-binding portion that binds to a VEGF epitope and at least a second antigen-binding portion that binds to an Ang-2 epitope, wherein the first antigen-binding portion comprises a first heavy chain immunoglobulin variable domain (V _H ) And a first light chain immunoglobulin variable domain (V _L ) Wherein the second antigen binding portion comprises a second V _H And a second V _L And wherein the first V _H An amino acid sequence comprising SEQ ID NO. 25 or SEQ ID NO. 44; and/or a first V _L Comprising the amino acid sequence of SEQ ID NO. 27. Additionally or alternatively, in some embodiments of the multispecific (e.g., bispecific) antibodies or antigen-binding fragments disclosed herein, the second V _H Comprising an amino acid sequence selected from any one of SEQ ID NO. 26 or SEQ ID NO. 45; and/or a second V _L Comprising the amino acid sequence of SEQ ID NO. 28.

In any of the above embodiments, the antibody further comprises an Fc domain of any isotype, such as, but not limited to, igG (including IgG1, igG2, igG3, and IgG 4), igA (including IgA1 and IgA 2), igD, igE, or IgM, and IgY. Non-limiting examples of constant region sequences include:

Human IgD constant region, uniprot: p01880 (SEQ ID NO: 29)

APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK

Human IgG1 constant region, uniprot: p01857 (SEQ ID NO: 30)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human IgG2 constant region, uniprot: p01859 (SEQ ID NO: 31)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human IgG3 constant region, uniprot: p01860 (SEQ ID NO: 32)

ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

Human IgM constant region, uniprot: p01871 (SEQ ID NO: 33)

GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

Human IgG4 constant region, uniprot: p01861 (SEQ ID NO: 34)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Human IgA1 constant region, uniprot: p01876 (SEQ ID NO: 35)

ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY human IgA2 constant region, uniprot: p01877 (SEQ ID NO: 36)

ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY

Human igkappa constant region, uniprot: p01834 (SEQ ID NO: 37)

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In some embodiments, the immunoglobulin-related compositions of the present technology comprise a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID NOS 29-36. Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO. 37.

Additionally or alternatively, in some embodiments, the antibody or antigen binding fragment binds to an extracellular region of a VEGF and/or Ang-2 polypeptide. In some embodiments, the VEGF polypeptide has the amino acid sequence of SEQ ID NO. 38. In certain embodiments, the Ang-2 polypeptide has the amino acid sequence of SEQ ID NO. 39. In certain embodiments, the epitope is a conformational epitope or a non-conformational epitope.

In some embodiments, the Heavy Chain (HC) and Light Chain (LC) immunoglobulin variable domain sequences are components of the same polypeptide chain. In other embodiments, the HC and LC immunoglobulin variable domain sequences are components of different polypeptide chains. In certain embodiments, the antibody is a full length antibody.

In some embodiments, the immunoglobulin-related compositions of the present technology specifically bind to at least one VEGF polypeptide and/or at least one Ang-2 polypeptide. In some embodiments, the immunoglobulin-related compositions of the present technology are at about 10 ^-3 M、10 ^-4 M、10 ^-5 M、10 ^-6 M、10 ^-7 M、10 ^-8 M、10 ^-9 M、10 ^-10 M、10 ^-11 M or 10 ^-12 M has a dissociation constant (KD) of binding to at least one VEGF polypeptide and/or of about 10 ^-3 M、10 ^-4 M、10 ^-5 M、10 ^-6 M、10 ^-7 M、10 ^-8 M、10 ^-9 M、10 ^-10 M、10 ^-11 M or 10 ^-12 The dissociation constant (KD) of M binds to at least one Ang-2 polypeptide. In certain embodiments, the immunoglobulin-related composition is a monoclonal antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody. In some embodiments, the antibody comprises a human antibody framework region.

In certain embodiments, the immunoglobulin-related composition comprises an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A, K A, H435A, L234A and L235A. Additionally or alternatively, in some embodiments, the immunoglobulin-related composition comprises an IgG4 constant region comprising an S228P mutation.

In one aspect, the immunoglobulin-related compositions of the present technology comprise Heavy (HC) and Light (LC) chains selected from SEQ ID NOs 1 and 2, SEQ ID NOs 5 and 6, and SEQ ID NOs 9 and 10, respectively.

In one aspect, the present disclosure provides a multispecific (e.g., bispecific) antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently bound to each other, the second polypeptide chain and the third polypeptide chain are covalently bound to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently bound to each other, and wherein: (a) Each of the first polypeptide chain and the fourth polypeptide chain comprises in an N-terminal to C-terminal direction: (i) A light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; (ii) a light chain constant domain of a first immunoglobulin; and (b) each of the second polypeptide chain and the third polypeptide chain comprises in an N-terminal to C-terminal direction: (i) A heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; and (ii) a heavy chain constant domain of a first immunoglobulin; (iii) Comprises an amino acid sequence (GGGGS) ₂ Is a flexible peptide linker of (a); and (iv) a light chain variable domain of a second immunoglobulin linked to a complementary heavy chain variable domain of a second immunoglobulin, or a heavy chain variable domain of a second immunoglobulin linked to a complementary light chain variable domain of a second immunoglobulin, wherein the light chain variable domain and the heavy chain variable domain of the second immunoglobulin are capable of specifically binding to a second epitope and are capable of binding to a second epitope by comprising an amino acid sequence (GGGGS) ₄ Are linked together to form a single-chain variable fragment; and wherein the heavy chain variable domain of the first immunoglobulin comprises any one of SEQ ID NO. 25 or 44 and/or the light chain variable domain of the first immunoglobulin comprises SEQ ID NO. 27.Additionally or alternatively, in some embodiments, the heavy chain variable domain of the second immunoglobulin comprises any one of SEQ ID NO. 26 or 45 and/or the light chain variable domain of the second immunoglobulin comprises SEQ ID NO. 28.

In some aspects, the anti-VEGF x Ang-2 multispecific immunoglobulin-related compositions described herein contain structural modifications to promote rapid binding and cellular uptake and/or slow release. In some aspects, the anti-VEGF x Ang-2 multispecific immunoglobulin-related compositions (e.g., antibodies) of the present technology may contain deletions of the CH2 heavy chain constant region to facilitate rapid binding and cellular uptake and/or slow release. In some aspects, fab fragments are used to promote rapid binding and cellular uptake and/or slow release. In some aspects, the F (ab)' 2 fragment is used to promote rapid binding and cellular uptake and/or slow release.

In one aspect, the present technology provides recombinant nucleic acid sequences encoding any and all embodiments of the immunoglobulin-related compositions described herein. Also disclosed herein are host cells that express any nucleic acid sequence encoding any of the immunoglobulin-related compositions described herein.

The immunoglobulin-related compositions of the present technology (e.g., anti-VEGF x Ang-2 multispecific antibodies) may be bispecific, trispecific, or have greater multispecific. The multispecific antibodies may be specific for different epitopes of one or more VEGF or Ang-2 polypeptides, and for heterologous compositions (such as heterologous polypeptides or solid support materials). See, for example, WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; tutt et al, J.Immunol., volume 147: pages 60-69 (1991); U.S. patent nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648;6,106,835; kostelny et al, j.immunol., volume 148: pages 1547-1553 (1992). In some embodiments, the immunoglobulin-related composition is chimeric. In certain embodiments, the immunoglobulin-related composition is humanized.

The immunoglobulin-related compositions of the present technology may be further recombinantly fused at the N-or C-terminus to a heterologous polypeptide, or chemically conjugated (including covalent and non-covalent conjugation) to a polypeptide or other composition. For example, the immunoglobulin-related compositions of the present technology may be recombinantly fused or conjugated to molecules and effector molecules such as heterologous polypeptides, drugs, or toxins that can be used as markers in detection assays. See, for example, WO 92/08495; WO 91/14438; WO 89/12624; U.S. patent No. 5,314,995; and EP 0 396387.

In any of the above embodiments of the immunoglobulin-related compositions of the present technology, the antibody or antigen-binding fragment may optionally be conjugated to an agent selected from the group consisting of: isotopes, dyes, chromogens, contrast agents, drugs, toxins, cytokines, enzymes, enzyme inhibitors, hormones, hormone antagonists, growth factors, radionuclides, metals, liposomes, nanoparticles, RNA, DNA, or any combination thereof. For chemical or physical bonds, the functional groups on the immunoglobulin-related composition are typically associated with functional groups on the agent. Alternatively, the functional group on the agent associates with a functional group on the immunoglobulin-related composition.

The functional groups on the agent and the functional groups on the immunoglobulin-related composition may be directly associated. For example, a functional group (e.g., a sulfhydryl group) on a pharmaceutical agent may associate with a functional group (e.g., a sulfhydryl group) on an immunoglobulin-related composition to form a disulfide bond. Alternatively, the functional groups may be associated by a cross-linker (i.e., a linker). Some examples of cross-linking agents are described below. The cross-linking agent may be linked to the agent or immunoglobulin-related composition. The number of agents or immunoglobulin-related compositions in the conjugate is also limited by the number of functional groups present on each other. For example, the maximum number of agents associated with the conjugate depends on the number of functional groups present on the immunoglobulin-related composition. Alternatively, the maximum number of immunoglobulin-related compositions associated with an agent depends on the number of functional groups present on the agent.

In yet another embodiment, the conjugate comprises an immunoglobulin-related composition associated with an agent. In one embodiment, the conjugate comprises at least one agent that is chemically bound (e.g., conjugated) to at least one immunoglobulin-related composition. The agent may be chemically bound to the immunoglobulin-related composition by any method known to those of skill in the art. For example, the functional group on the agent may be directly linked to a functional group on the immunoglobulin-related composition. Some examples of suitable functional groups include, for example, amino, carboxyl, mercapto, maleimide, isocyanate, isothiocyanate, and hydroxyl.

The agent may also be chemically bonded to the immunoglobulin-related composition by means of a cross-linking agent such as dialdehydes, carbodiimides, dimaleimides, and the like. The crosslinking agent is available, for example, from Pierce Biotechnology, inc. The Pierce Biotechnology the web site may provide assistance. Additional crosslinking agents include Kreatech Biotechnology, b.v., U.S. patent No. 5,580,990 to Amsterdam, the Netherlands; platinum cross-linking agents described in 5,985,566 and 6,133,038.

Alternatively, the functional groups on the agent and the immunoglobulin-related composition may be the same. The same functional groups are typically crosslinked using the same bifunctional crosslinking agent. Examples of homobifunctional crosslinkers include EGS (i.e., ethylene glycol bis [ succinimidyl succinate ]), DSS (i.e., disuccinimidyl suberate), DMA (i.e., dimethyl diimidinate.2hcl), DTSSP (i.e., 3' -dithiobis [ sulfosuccinimidyl propionate ]), DPDPB (i.e., 1, 4-bis- [3' - (2 ' -pyridyldithio) -propionylamino ] butane), and BMH (i.e., bismaleimidohexane). Such homobifunctional crosslinking agents are also available from Pierce Biotechnology, inc.

In other cases, it may be beneficial to cleave agents from immunoglobulin-related compositions. The website of Pierce Biotechnology, inc. Above may also provide the person skilled in the art with the aid of selecting suitable cross-linking agents which may be cleaved by, for example, enzymes in the cell. Thus, the agent may be separated from the immunoglobulin-related composition. Examples of cleavable linkers include SMPT (i.e., 4-succinimidyloxycarbonyl-methyl- α - [ 2-pyridyldithio ] toluene), sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6- (3- [ 2-pyridyldithio ] -propionamido) hexanoate), LC-SPDP (i.e., succinimidyl 6- (3- [ 2-pyridyldithio ] -propionamido) hexanoate), sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6- (3- [ 2-pyridyldithio ] -propionamido) hexanoate), SPDP (i.e., N-succinimidyl 3- [ 2-pyridyldithio ] -propionamido hexanoate), and AEDP (i.e., 3- [ (2-aminoethyl) dithio ] propionic acid HCl).

In another embodiment, the conjugate comprises at least one agent physically bound to at least one immunoglobulin-related composition. The agent may be physically bound to the immunoglobulin-related composition using any method known to those skilled in the art. For example, the immunoglobulin-related composition and the agent may be mixed together by any method known to those of skill in the art. The order of mixing is not critical. For example, the agent may be physically mixed with the immunoglobulin-related composition by any method known to those of skill in the art. For example, the immunoglobulin-related composition and the agent may be placed in a container and agitated, for example, by shaking the container, to mix the immunoglobulin-related composition and the agent.

The immunoglobulin-related composition may be modified by any method known to those skilled in the art. For example, the immunoglobulin-related composition may be modified by means of a cross-linking agent or functional group, as described above.

Preparation method of anti-VEGF X Ang-2 multispecific antibody of the technology

General overview. Initially, a target polypeptide against which an antibody of the present technology can be raised is selected. For example, antibodies may be raised against full length VEGF or Ang-2 proteins or against a portion of the extracellular domain of VEGF or Ang-2 proteins. Techniques for producing antibodies to such target polypeptides are well known to those skilled in the art. Examples of such techniques include, for example, but are not limited to, those involving display libraries, xenogenic or human mice, hybridomas, and the like. Target polypeptides within the scope of the present technology include any polypeptide derived from VEGF or Ang-2 proteins that contain an extracellular domain capable of eliciting an immune response.

It is to be understood that recombinant engineered antibodies and antibody fragments, e.g., antibody-related polypeptides, directed against VEGF or Ang-2 proteins and fragments thereof are suitable for use in accordance with the present disclosure.

anti-VEGF x Ang-2 antibodies that may be subjected to the techniques set forth herein include monoclonal and polyclonal antibodies as well as antibody fragments, such as Fab, fab ', F (ab') 2, fd, scFv, diabodies, antibody light chains, antibody heavy chains, and/or antibody fragments. Methods have been described for the high yield production of polypeptides containing antibody Fv, such as Fab 'and F (ab') 2 antibody fragments. See U.S. Pat. No. 5,648,237.

Typically, antibodies are obtained from the species of origin. More specifically, nucleic acid or amino acid sequences of variable portions of the light chain, heavy chain, or both of the antibodies of the species of origin specific for the target polypeptide antigen are obtained. The species of origin is any species that can be used to produce antibodies or antibody libraries of the present technology, e.g., rat, mouse, rabbit, chicken, monkey, human, etc.

Phage or phagemid display techniques are techniques that can be used to obtain antibodies of the present technology. Techniques for generating and cloning monoclonal antibodies are well known to those skilled in the art. Expression of the sequences encoding antibodies of the present technology can be performed in E.coli (E.coli).

Because of the degeneracy of the nucleic acid coding sequence, other sequences that encode an amino acid sequence that is substantially identical to the amino acid sequence of a naturally occurring protein may be used in the practice of the present technology. Such sequences include, but are not limited to, nucleic acid sequences, including all or part of the nucleic acid sequences encoding the polypeptides described above, which are altered by substitution of different codons for functionally equivalent amino acid residues within the coding sequence, thereby producing silent changes. It will be appreciated that the nucleotide sequence of an immunoglobulin according to the present technology tolerates up to 25% sequence homology changes, as calculated by standard methods ("Current Methods in Sequence Comparison and Analysis", macromolecule Sequencing and Synthesis, selected Methods and Applications, pages 127-149, 1998, alan r.lists, inc.) provided that such variants form effective antibodies recognizing VEGF or Ang-2 proteins. For example, one or more amino acid residues within a polypeptide sequence may be substituted with another amino acid of similar polarity that serves as a functional equivalent, resulting in a silent change. Substitutions of amino acids within the sequence may be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the present technology are proteins or fragments or derivatives thereof that are differentially modified during or after translation, e.g., by glycosylation, proteolytic cleavage, attachment to an antibody molecule or other cellular ligand, and the like. Furthermore, immunoglobulin encoding nucleic acid sequences may be mutated in vitro or in vivo to create and/or disrupt translation, initiation and/or termination sequences, or to create coding region variations and/or to create new restriction endonuclease sites or disrupt pre-existing sites to facilitate further in vitro modification. Any mutagenesis technique known in the art may be used, including but not limited to in vitro site-directed mutagenesis, j.biol.chem., volume 253: on page 6551, tab linker (Pharmacia) and the like are used.

Preparation of polyclonal antisera and immunogens. Methods of producing antibodies or antibody fragments of the present technology generally comprise immunizing a subject (typically a non-human subject such as a mouse or rabbit) with purified VEGF or Ang-2 protein or fragments thereof, or with cells expressing VEGF or Ang-2 protein or fragments thereof. Suitable immunogenic formulations may contain, for example, recombinantly expressed VEGF or Ang-2 proteins or chemically synthesized VEGF or Ang-2 peptides. The first extracellular domain of VEGF or Ang-2 protein, or a portion or fragment thereof, may be used as an immunogen to generate anti-VEGF or Ang-2 antibodies that bind to VEGF or Ang-2 protein, or a portion or fragment thereof, using standard preparation techniques for polyclonal and monoclonal antibodies.

In some embodiments, the antigenic VEGF or Ang-2 peptide comprises at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acid residues. Depending on the application and according to methods well known to those skilled in the art, longer antigenic peptides are sometimes required instead of shorter antigenic peptides. Multimers of a given epitope are sometimes more efficient than monomers.

If desired, the immunogenicity of VEGF or Ang-2 proteins (or fragments thereof) may be increased by fusion or conjugation to a carrier protein such as Keyhole Limpet Hemocyanin (KLH) or Ovalbumin (OVA). Many such carrier proteins are known in the art. One can also combine VEGF or Ang-2 protein with a conventional adjuvant, such as Freund's complete or incomplete adjuvant, to increase the subject's immune response to the polypeptide. Various adjuvants for increasing the immune response include, but are not limited to, freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as BCG and Corynebacterium parvum (Corynebacterium parvum), or similar immunostimulatory compounds. These techniques are standard in the art.

Alternatively, nanoparticles such as virus-like particles (VLPs) may be used to present antigens such as VEGF or Ang-2 to a host animal. Virus-like particles are multiprotein structures that mimic the organization and conformation of a truly natural virus without infectivity, as they do not carry any viral genetic material (Urakami A et al, clin Vaccine Immunol, volume 24: e00090-17 (2017)). VLPs, when introduced into the host immune system, can elicit an effective immune response, making them attractive foreign antigen carriers. An important advantage of VLP-based antigen presentation platforms is that it can display antigens in a dense, reproducible manner. Thus, antigen-bearing VLPs are able to induce a strong B cell response by effectively effecting cross-linking of B Cell Receptors (BCR). VLPs can be genetically manipulated to improve their properties, such as immunogenicity. These techniques are standard in the art.

Isolation of enough purified protein or polypeptide for which antibodies are raised can be time consuming and sometimes technically challenging. Additional challenges associated with traditional protein-based immunization include concerns about the safety, stability, scalability, and consistency of protein antigens. Nucleic acid (DNA and RNA) based immunization has become a promising alternative. DNA vaccines are typically based on bacterial plasmids encoding polypeptide sequences of candidate antigens such as VEGF or Ang-2. Using a powerful eukaryotic promoter, once the host is inoculated with a plasmid, the encoded antigen can be expressed to give a sufficient level of transgene expression (Galvin T.A. et al, vaccine, vol. 18, 2000: pages 2566-2583). Modern DNA vaccine production relies on DNA synthesis, possibly in one step cloning into a plasmid vector, followed by isolation of the plasmid, which greatly reduces manufacturing time and costs. The resulting plasmid DNA is also highly stable at room temperature, avoiding low temperature transport and resulting in a greatly extended shelf life. These techniques are standard in the art.

Alternatively, a nucleic acid sequence encoding an antigen of interest, such as VEGF or Ang-2, may be synthetically introduced into an mRNA molecule. The mRNA is then delivered to a host animal whose cells will recognize the mRNA sequence and translate it into polypeptide sequences of candidate antigens such as VEGF or Ang-2, thereby triggering an immune response to the foreign antigen. An attractive feature of an mRNA antigen or mRNA vaccine is that the mRNA is a non-infectious, non-integrated platform. There is no potential risk of infection or insertional mutagenesis associated with DNA vaccines. In addition, mRNA is degraded by normal cellular processes and has a controlled in vivo half-life by modification of the design and delivery methods (Kariko, K. Et al, mol Ther, volume 16: pages 1833-1840 (2008); kauffman, K.J. et al, J Control Release, volume 240, pages 227-234 (2016); guan, S. & Rosenecker, J., gene Ther, volume 24, pages 133-143 (2017); thess, A. Et al, mol Ther, volume 23, pages 1456-1464 (2015)). These techniques are standard in the art.

In describing the present technology, an immune response may be described as a "primary" or "secondary" immune response. The primary immune response is also described as a "protective" immune response, meaning an immune response that results in an individual as a result of some degree of initial exposure (e.g., initial "immunization" or "immune priming") to a particular antigen, such as VEGF or Ang-2 protein. In some embodiments, immunization may occur as a result of vaccinating an individual with a vaccine comprising an antigen. For example, the vaccine may be a VEGF or Ang-2 vaccine comprising one or more VEGF or Ang-2 protein derived antigens. The primary immune response may diminish or decay over time, or may even disappear or at least diminish to an undetectable extent. Thus, the present technology also relates to a "secondary" immune response, which is also described herein as a "memory immune response. The term secondary immune response refers to an immune response elicited in an individual after a primary immune response has been generated.

Thus, a secondary immune response is elicited, e.g., an existing immune response that has been attenuated or attenuated (e.g., an immunopotentiating dose), or a previous immune response that has disappeared or can no longer be detected is reconstituted. The secondary or memory immune response may be a humoral (antibody) response or a cellular response. Secondary or memory fluid responses occur upon stimulation of memory B cells that are produced upon first presentation of antigen. Delayed-type hypersensitivity (DTH) reaction is a cellular secondary or memory immune response mediated by cd4+ T cells. The first exposure to antigen triggers the immune system and the re-exposure results in DTH.

After appropriate immunization, anti-VEGF X Ang-2 antibodies may be prepared from the serum of the subject. If desired, antibody molecules directed against VEGF or Ang-2 proteins may be isolated from a mammal (e.g., from blood) and further purified by well known techniques such as polypeptide A chromatography to obtain an IgG fraction.

A monoclonal antibody. In one embodiment of the present technology, the antibody is an anti-VEGF monoclonal antibody or an anti-Ang-2 antibody. For example, in some embodiments, the anti-VEGF or anti-Ang-2 monoclonal antibody may be a human or mouse anti-VEGF or anti-Ang-2 monoclonal antibody. For the preparation of monoclonal antibodies directed against VEGF protein or against the Ang-2 protein or derivatives, fragments, analogs or homologs thereof, any technique that produces antibody molecules by continuous cell line culture may be used. Such techniques include, but are not limited to, hybridoma techniques (see, e.g., kohler & Milstein,1975, nature, volume 256:495-497); three-source hybridoma technology; human B cell hybridoma technology (see, e.g., kozbor et al, 1983, immunol. Today, vol. 4: p. 72) and EBV hybridoma technology to produce human monoclonal antibodies (see, e.g., cole et al, 1985, in: MONOCLONAL ANTIBODIES AND CANCER THERAPY, alan R.Lists, inc., pp. 77-96). Human monoclonal antibodies can be used in the practice of the present technology and can be produced by using human hybridomas (see, e.g., cote et al, 1983, proc. Natl. Acad. Sci. USA, vol. 80: pages 2026-2030) or by transforming human B cells in vitro with EB virus (see, e.g., cole et al, 1985, at MONOCLONAL ANTIBODIES AND CANCER THERAPY, alan R.Lists, inc., pages 77-96). For example, a population of nucleic acids encoding an antibody region may be isolated. PCR using primers derived from sequences encoding conserved regions of antibodies is used to amplify sequences encoding antibody portions from a population, and then reconstruct DNA encoding antibodies or fragments thereof, such as variable domains, from the amplified sequences. Such amplified sequences may also be fused to DNA encoding other proteins (e.g., phage coat or bacterial cell surface proteins) to express and display the fusion polypeptide on phage or bacteria. The amplified sequence may then be expressed and further selected or isolated, for example, based on the affinity of the expressed antibody or fragment thereof for an antigen or epitope present on the VEGF or Ang-2 protein. Alternatively, hybridomas expressing anti-VEGF monoclonal antibodies or anti-Ang-2 monoclonal antibodies can be prepared by immunizing a subject, and then isolating the hybridomas from the spleen of the subject using conventional methods. See, for example, milstein et al (Galfre and Milstein, methods enzymes (1981), vol.73:pages 3-46). Screening hybridomas using standard methods will produce monoclonal antibodies with different specificities (i.e., for different epitopes) and affinities. Selected monoclonal antibodies having the desired properties (e.g., VEGF or Ang-2 binding) can be used when expressed by the hybridoma, which can be conjugated to a molecule such as polyethylene glycol (PEG) to alter the properties, or the cDNA encoding it can be isolated, sequenced and manipulated in a variety of ways. Synthetic dendrons may be added to reactive amino acid side chains, such as lysine, to enhance the immunogenicity of the VEGF or Ang-2 proteins. Furthermore, CPG-dinucleotide technology can be used to enhance the immunogenicity of VEGF or Ang-2 proteins. Other manipulations include substitution or deletion of specific aminoacyl residues that result in instability of the antibody during storage or after administration to a subject, as well as affinity maturation techniques to increase the affinity of the antibody for targeting VEGF or Ang-2 proteins.

Hybridoma technology. In some embodiments, the antibodies of the present technology are anti-VEGF monoclonal antibodies or anti-Ang-2 monoclonal antibodies produced by hybridomas comprising B cells obtained from transgenic non-human animals (e.g., transgenic mice) whose genomes comprise a human heavy chain transgene and a light chain transgene fused to an immortalized cell. Hybridoma techniques include those known in the art and Harlow et al Antibodies A Laboratory Manual, cold Spring Harbor Laboratory, cold Spring Harbor, NY, page 349 (1988); techniques taught by Hammerling et al, monoclonal Antibodies And T-Cell hybrid, pages 563-681 (1981). Other methods for producing hybridomas and monoclonal antibodies are well known to those skilled in the art.

Phage display technology. As described above, antibodies of the present technology can be produced by applying recombinant DNA and phage display techniques. For example, anti-VEGF antibodies or anti-Ang-2 antibodies may be prepared using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying polynucleotide sequences encoding them. Phages with the desired binding properties are selected from a repertoire or a combinatorial antibody library (e.g., human or murine) by direct selection with an antigen (typically an antigen that is bound or captured to a solid surface or bead). The phage used in these methods are typically filamentous phage comprising fd and M13 with Fab, fv, or disulfide stabilized Fv antibody domains that are recombinantly fused to phage gene III or gene VIII proteins. In addition, the method may be adapted for the construction of Fab expression libraries (see, e.g., huse et al, science, vol. 246:1275-1281, 1989) to allow rapid and efficient identification of monoclonal Fab fragments which have the desired specificity for VEGF polypeptides or Ang-2 polypeptides, e.g., polypeptides or derivatives, fragments, analogs or homologs thereof. Other examples of phage display methods that can be used to make antibodies of the present technology include those disclosed in the following: huston et al, proc.Natl. Acad. Sci U.S. A., volume 85: pages 5879-5883, 1988; chaudhary et al proc.Natl. Acad.Sci U.S. A., volume 87: page 1066-1070, 1990; brinkman et al, J.Immunol. Methods, volume 182: pages 41-50, 1995; ames et al, j.immunol methods, volume 184: pages 177-186, 1995; kettleborough et al, eur.j.immunol., volume 24: pages 952-958, 1994; persic et al, gene, volume 187: pages 9-18, 1997; burton et al, advances in Immunology, volume 57: pages 191-280, 1994; PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; WO 96/06213; WO 92/01047 (Medical Research Council et al); WO 97/08320 (Morphosys); WO 92/01047 (CAT/MRC); WO 91/17271 (Affymax); and U.S. Pat. nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743. U.S. Pat. No. 6,753,136 to Lohning has described a method that can be used to display polypeptides on the surface of phage particles by linking the polypeptides via disulfide bonds. As described in the above references, after phage selection, antibody coding regions can be isolated from phage for the production of intact antibodies (including human antibodies or any other desired antigen binding fragments) and expressed in any desired host (including mammalian cells, insect cells, plant cells, yeast, and bacteria). For example, methods known in the art, such as WO 92/22324, may also be employed; mullinax et al, bioTechniques, vol.12: pages 864-869, 1992; and Sawai et al, AJRI, volume 34: pages 26-34, 1995; and Better et al, science, volume 240: techniques for recombinant production of Fab, fab 'and F (ab') 2 fragments by those methods disclosed in 1988, pages 1041-1043.

In general, a hybrid antibody or hybrid antibody fragment cloned into a display vector can be selected against the appropriate antigen in order to identify variants that retain good binding activity, as the antibody or antibody fragment will be present on the surface of a phage or phagemid particle. See, e.g., barbes III et al, phase Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y., 2001). However, other vector formats may be used in this process, such as cloning the library of antibody fragments into a lytic phage vector (modified T7 or λzap system) for selection and/or screening.

Expression of recombinant anti-VEGF XAng-2 antibodies. As described above, antibodies of the present technology can be produced by applying recombinant DNA techniques. Recombinant polynucleotide constructs encoding anti-VEGF X Ang-2 antibodies of the present technology typically comprise expression control sequences, including naturally-associated or heterologous promoter regions, operably linked to the coding sequence of the anti-VEGF X Ang-2 antibody chain. Thus, another aspect of the present technology includes a vector comprising one or more nucleic acid sequences encoding an anti-VEGF x Ang-2 antibody of the present technology. For recombinant expression of one or more of the polypeptides of the present technology, a nucleic acid comprising all or a portion of a nucleotide sequence encoding an anti-VEGF x Ang-2 antibody is inserted into a suitable cloning or expression vector (i.e., a vector containing the necessary elements for transcription and translation of the inserted polypeptide coding sequence) by recombinant DNA techniques well known in the art and as described in detail below. U.S. patent nos. 6,291,160 and 6,680,192 to Lerner et al have described methods for generating different vector populations.

Generally, expression vectors useful in recombinant DNA technology are typically in the form of plasmids. In this disclosure, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the present technology is intended to include such other forms of expression vectors that are technically not plasmids, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which have equivalent functions. Such viral vectors allow for infection of a subject and expression of the construct in the subject. In some embodiments, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Once the vector has been incorporated into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences encoding the anti-VEGF X Ang-2 antibody and collection and purification of the anti-VEGF X Ang-2 antibody (e.g., cross-reactive anti-VEGF X Ang-2 antibody). See, generally, U.S.2002/0199213. These expression vectors are typically replicable in host organisms either as episomes or as part of the host chromosomal DNA. Typically, the expression vector contains a selectable marker (e.g., ampicillin or hygromycin resistance) to allow for detection of those cells transformed with the desired DNA sequence. The vector may also encode a signal peptide, such as pectin lyase, useful for directing secretion of extracellular antibody fragments. See, U.S. patent No. 5,576,195.

Recombinant expression vectors of the present technology comprise a nucleic acid encoding a protein having VEGF x Ang-2 binding properties, the form of which is suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector comprises one or more regulatory sequences based on the host cell selection to be used for expression operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY, volume 185, academic Press, san Diego, calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector may depend on factors such as the choice of host cell to be transformed, the level of expression of the desired polypeptide, and the like. Typical regulatory sequences that may be used as promoters for expression of recombinant polypeptides (e.g., anti-VEGF. Times. Ang-2 antibodies) include, for example, but are not limited to, promoters of 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include promoters derived from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization, and the like. In one embodiment, a polynucleotide encoding an anti-VEGF XAng-2 antibody of the present technology is operably linked to an ara B promoter and is expressible in a host cell. See, U.S. patent 5,028,530. Expression vectors of the present technology can be introduced into host cells to produce polypeptides or peptides encoded by the nucleic acids described herein, including fusion polypeptides (e.g., anti-VEGF x Ang-2 antibodies, etc.).

Another aspect of the technology relates to host cells expressing anti-VEGF X Ang-2 antibodies, which host cells contain nucleic acids encoding one or more anti-VEGF X Ang-2 antibodies. Recombinant expression vectors of the present technology can be designed for expression of anti-VEGF x Ang-2 antibodies in prokaryotic or eukaryotic cells. For example, an anti-VEGF x Ang-2 antibody may be expressed in a bacterial cell such as E.coli, an insect cell (using a baculovirus expression vector), a fungal cell such as a yeast, a yeast cell, or a mammalian cell. Suitable host cells are further discussed in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY, volume 185, academic Press, san Diego, calif. (1990). Alternatively, recombinant expression vectors can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase. Methods for preparing and screening polypeptides having predetermined properties (e.g., anti-VEGF x Ang-2 antibodies) via expression of randomly generated polynucleotide sequences have been previously described. See, U.S. patent No. 5,763,192;5,723,323;5,814,476;5,817,483;5,824,514;5,976,862;6,492,107;6,569,641.

Expression of polypeptides in prokaryotes is most often carried out in E.coli, where vectors containing constitutive or inducible promoters direct expression of fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to the polypeptide encoded therein, typically to the amino terminus of the recombinant polypeptide. Such fusion vectors generally serve three purposes: (i) increasing expression of the recombinant polypeptide; (ii) increasing the solubility of the recombinant polypeptide; and (iii) facilitates purification of the recombinant polypeptide by acting as a ligand in affinity purification. Typically, in fusion expression vectors, proteolytic cleavage sites are introduced at the junction of the fusion moiety and the recombinant polypeptide to separate the recombinant polypeptide from the fusion moiety after purification of the fusion polypeptide. Such enzymes and their cognate recognition sequences include factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; smith and Johnson,1988, gene, 67: pages 31-40), pMAL (New England Biolabs, beverly, mass.) and pRIT5 (Pharmacia, piscataway, N.J.), respectively, fused to a target recombinant polypeptide with glutathione S-transferase (GST), maltose E binding polypeptide or polypeptide A.

Examples of suitable inducible non-fusion E.coli expression vectors include pTrc (Amrann et al (1988), gene, vol. 69: pages 301-315) and pET 11d (Studier et al GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY, vol. 185, academic Press, san Diego, calif. (1990), pages 60-89). U.S. patent No. 6,294,353 to Pack et al; 6,692,935 methods for targeted assembly of different active peptide or protein domains via polypeptide fusion to produce multifunctional polypeptides have been described. One strategy to maximize the expression of recombinant polypeptides, such as anti-VEGF x Ang-2 antibodies, in E.coli is to express polypeptides with an impaired ability to proteolytically cleave the recombinant polypeptide in the host bacterium. See, e.g., gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY, volume 185, academic Press, san Diego, calif. (1990), pages 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into the expression vector such that the individual codons for each amino acid are codons that are preferentially utilized in the expression host, e.g., e.coli (see, e.g., wada et al, 1992, nucleic acids res., volume 20: pages 2111-2118). Such alterations in the nucleic acid sequence of the present technology can be made by standard DNA synthesis techniques.

In another embodiment, the anti-VEGF XAng-2 antibody expression vector is a yeast expression vector. Examples of vectors for expression in the yeast Saccharomyces cerevisiae (Saccharomyces cerevisiae) include pYepSec1 (Baldari et al, 1987, EMBO J., volume 6: pages 229-234), pMFa (Kurjan and Herskowitz, cell, volume 30: pages 933-943, 1982), pJRY88 (Schultz et al, gene, volume 54: pages 113-123, 1987), pYES2 (Invitrogen Corporation, san Diego, calif.) and picZ (Invitrogen Corp, san Diego, calif.). Alternatively, baculovirus expression vectors may be used to express anti-VEGF X Ang-2 antibodies in insect cells. Baculovirus vectors useful for expression of polypeptides (e.g., anti-VEGF x Ang-2 antibodies) in cultured insect cells (e.g., SF9 cells) include pAc series (Smith et al, mol. Cell. Biol., vol. 3: pages 2156-2165, 1983) and pVL series (Lucklow and Summers,1989, virology, vol. 170: pages 31-39).

In yet another embodiment, a mammalian expression vector is used to express a nucleic acid encoding an anti-VEGF x Ang-2 antibody of the technology in mammalian cells. Examples of mammalian expression vectors include, for example, but are not limited to, pCDM8 (Seed, nature, vol. 329:840, 1987) and pMT2PC (Kaufman et al, EMBO J., vol. 6:187-195, 1987). When used in mammalian cells, the control functions of the expression vectors are typically provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma virus, adenovirus 2, cytomegalovirus and simian virus 40. Other suitable expression systems for both prokaryotic and eukaryotic cells useful for expressing anti-VEGF X Ang-2 antibodies of the present technology are described, for example, in Sambrook et al, chapters 16 and 17, MOLECULAR CLONING: A LABORATORY MANUAL, 2 nd edition, cold Spring Harbor Laboratory, cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory element). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include albumin promoters (liver-specific; pinkert et al, genes Dev., vol.1: pp.268-277, 1987), lymphoid-specific promoters (Calame and Eaton, adv. Immunol., vol.43: pp.235-275, 1988), promoters of T-Cell receptors (Wioto and Baltimore, EMBO J., vol.8: pp.729-733, 1989), and immunoglobulins (Banerji et al, 1983, cell, vol.33: pp.729-740; queen and Baltimore, cell, vol.33: pp.741-748, 1983), neuron-specific promoters (e.g., neurofilament promoters; byrne and Ruddle, proc. Natl. Acad. Sci. USA, vol.86: pp.5473-5477, 1989), pancreatic-specific promoters (e.g., pp.729-37, 1983, and European patent application No. 912, 1983, and specific promoters (e.g., european patent application No. 912, 1983, pp.166, 1983). Developmental regulatory promoters are also included, for example, the murine hox promoter (Kessel and Gruss, science, volume 249: pages 374-379, 1990) and the alpha fetoprotein promoter (Campes and Tilghman, genes Dev., volume 3: pages 537-546, 1989).

Another aspect of the present methods relates to host cells into which the recombinant expression vectors of the present technology have been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is to be understood that such terms refer not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

The host cell may be any prokaryotic or eukaryotic cell. For example, an anti-VEGF XAng-2 antibody may be expressed in a bacterial cell such as E.coli, an insect cell, a yeast or a mammalian cell. Mammalian cells are suitable hosts for expression of nucleotide fragments encoding immunoglobulins or fragments thereof. See, winnacker, from Genes To Clones (VCH Publishers, NY, 1987). Many suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, including Chinese Hamster Ovary (CHO) cell lines, various COS cell lines, heLa cells, L cells and myeloma cell lines. In some embodiments, the cell is a non-human cell. Expression vectors for use in these cells may include expression control sequences such as origins of replication, promoters, enhancers, and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Queen et al, immunol. Rev., volume 89: page 49, 1986. Illustrative expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenoviruses, bovine papilloma viruses, and the like. Co et al, J Immunol, volume 148: page 1149, 1992. Other suitable host cells are known to those skilled in the art.

Vector DNA may be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing exogenous nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, biolistics, or virus-based transfection. Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see generally Sambrook et al, molecular Cloning). Methods suitable for transformation or transfection of host cells can be found in Sambrook et al (MOLECULAR CLONING: A LABORATORY MANUAL, 2 nd edition, cold Spring Harbor Laboratory, cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y., 1989) and other laboratory manuals. The vector containing the DNA fragment of interest may be transferred into the host cell by well known methods, depending on the type of cellular host.

Non-limiting examples of suitable vectors include those designed for propagation and amplification or for expression or both. For example, the cloning vector may be selected from the group consisting of pUC series, pBluescript series (Stratagene, laJolla, calif.), pET series (Novagen, madison, wis.), pGEX series (Pharmacia Biotech, uppsala, sweden) and pEX series (Clontech, palo Alto, calif.). Phage vectors such as lambda-GT 10, lambda-GT 11, lambda-ZapII (Stratagene), lambda-EMBL 4 and lambda-NM 1149 can also be used. Non-limiting examples of plant expression vectors include pBI110, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Non-limiting examples of animal expression vectors include pEUK-C1, pMAM, and pMAMneo (Clontech). TOPO cloning systems (Invitrogen, calsbad, calif., carlsbad, calif.) can also be used as recommended by the manufacturer.

In certain embodiments, the vector is a mammalian vector. In certain embodiments, the mammalian vector contains at least one promoter element that mediates initiation of mRNA transcription, antibody coding sequences, and signals required for transcription termination and polyadenylation of the transcript. In certain embodiments, the mammalian vector contains additional elements such as, for example, enhancers, kozak sequences, and intervening sequences flanking the donor and acceptor sites for RNA splicing. In certain embodiments, efficient transcription can be achieved using early and late promoters from SV40, long Terminal Repeat (LTRS) from retroviruses (e.g., RSV, HTLVI, HIVI), and early promoters from Cytomegalovirus (CMV), for example. Cellular elements (e.g., human actin promoter) may also be used. Non-limiting examples of mammalian expression vectors include vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN or pLNCX (Clonetech Labs, palo Alto, calif.), pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or pcDNA3.1/Hygro (+/-) (Invitrogen, calsbad, calif.), PSVL and PMSG (Pharmacia, uppsala, sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Non-limiting examples of mammalian host cells that can be used in combination with such mammalian vectors include human Hela 293, HEK 293, H9 and Jurkat cells, mouse 3T3, NIH3T3 and C127 cells, cos 1, cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese Hamster Ovary (CHO) cells.

In certain embodiments, the vector is a viral vector, such as a retroviral vector, a parvoviral-based vector (e.g., adeno-associated virus (AAV) -based vectors, AAV-adenovirus chimeric vectors, and adenovirus-based vectors), and a lentiviral vector (such as Herpes Simplex Virus (HSV) -based vectors). In certain embodiments, the viral vector is manipulated to cause insufficient replication of the virus. In certain embodiments, the viral vector is manipulated to eliminate toxicity to the host. Such viral vectors can be used, for example, by Sambrook et al Molecular Cloning, a Laboratory Manual, 2 nd edition, cold Spring Harbor Press, cold Spring Harbor, n.y. (1989); and Ausubel et al Current Protocols in Molecular Biology, greene Publishing Associates and John Wiley & Sons, new York, N.Y. (1994).

In certain embodiments, the vectors or polynucleotides described herein can be transferred to cells (e.g., ex vivo cells) by conventional techniques, and the resulting cells can be cultured by conventional techniques to produce the anti-VEGF x Ang-2 antibodies or antigen-binding fragments described herein. Thus, provided herein are cells comprising polynucleotides encoding anti-VEGF x Ang-2 antibodies or antigen-binding fragments thereof operably linked to regulatory expression elements (e.g., promoters) for expression of such sequences in host cells. In certain embodiments, a vector encoding a heavy chain operably linked to a promoter and a vector encoding a light chain operably linked to a promoter may be co-expressed in a cell to express an entire anti-VEGF x Ang-2 antibody or antigen-binding fragment. In certain embodiments, the cell comprises a vector comprising a polynucleotide encoding both the heavy and light chains of an anti-VEGF x Ang-2 antibody or antigen-binding fragment described herein operably linked to a promoter. In certain embodiments, the cell comprises two different vectors, a first vector comprising a polynucleotide encoding a heavy chain operably linked to a promoter and a second vector comprising a polynucleotide encoding a light chain operably linked to a promoter. In certain embodiments, the first cell comprises a first vector comprising a polynucleotide encoding the heavy chain of an anti-VEGF x Ang-2 antibody or antigen-binding fragment described herein, and the second cell comprises a second vector comprising a polynucleotide encoding the light chain of an anti-VEGF x Ang-2 antibody or antigen-binding fragment described herein. In certain embodiments, provided herein are cell mixtures comprising the first cell and the second cell. Examples of cells include, but are not limited to, human cells, human cell lines, E.coli (e.g., E.coli TB-1, TG-2, DH5a, XL-Blue MRF' (Stratagene), SA2821, and Y1090), bacillus subtilis (B.subtilis), pseudomonas aeruginosa (P.aerogenosa), saccharomyces cerevisiae (S.cerevisiae), morchella insolens (N.crassa), insect cells (e.g., sf9, ea 4), and the like.

For stable transfection of mammalian cells, it is well known that, depending on the expression vector and transfection technique used, only a small fraction of cells can integrate the exogenous DNA into their genome. To identify and select these integrants, genes encoding selectable markers (e.g., antibiotic resistance) are typically introduced into the host cells along with the gene of interest. Various selectable markers include those that confer drug resistance, such as G418, hygromycin and methotrexate. The nucleic acid encoding the selectable marker may be introduced into the host cell on the same vector as the nucleic acid encoding the anti-VEGF X Ang-2 antibody, or may be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated a selectable marker gene will survive, while the other cells die).

Host cells, such as cultured prokaryotic or eukaryotic host cells, comprising the anti-VEGF X Ang-2 antibodies of the present technology may be used to produce (i.e., express) recombinant anti-VEGF X Ang-2 antibodies. In one embodiment, the method comprises culturing a host cell (into which has been introduced a recombinant expression vector encoding an anti-VEGF X Ang-2 antibody) in a suitable medium, thereby producing the anti-VEGF X Ang-2 antibody. In another embodiment, the method further comprises the step of isolating the anti-VEGF XAng-2 antibody from the culture medium or host cells. Once expressed, a collection of anti-VEGF X Ang-2 antibodies, e.g., anti-VEGF X Ang-2 antibodies or anti-VEGF X Ang-2 antibody-related polypeptides, are purified from the culture medium and host cells. The anti-VEGF XAng-2 antibodies may be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, and the like. In one embodiment, the anti-VEGF XAng-2 antibodies are produced in the host organism by the method of U.S. Pat. No. 4,816,397 to Boss et al. Typically, the anti-VEGF XAng-2 antibody chain is expressed with a signal sequence and is thus released into the culture medium. However, if the anti-VEGF X Ang-2 antibody chain is not naturally secreted by the host cell, the anti-VEGF X Ang-2 antibody chain may be released by treatment with a mild detergent. Purification of recombinant polypeptides is well known in the art and includes ammonium sulfate precipitation, affinity chromatography purification techniques, column chromatography, ion exchange purification techniques, gel electrophoresis, and the like (see generally, scope, protein Purification (Springer-Verlag, n.y., 1982).

Polynucleotides encoding anti-VEGF X Ang-2 antibodies, for example, anti-VEGF X Ang-2 antibody coding sequences, may be incorporated into a transgene for introduction into the genome of the transgenic animal and subsequent expression in the milk of the transgenic animal. See, for example, U.S. patent nos. 5,741,957, 5,304,489 and 5,849,992. Suitable transgenes include coding sequences for light and/or heavy chains operably linked to promoters and enhancers from breast-specific genes (such as casein or β -lactoglobulin). To produce a transgenic animal, the transgene may be microinjected into a fertilized oocyte, or it may be incorporated into the genome of an embryonic stem cell, and the nucleus of such cell transferred into an enucleated oocyte.

A single chain antibody. In one embodiment, the anti-VEGF XAng-2 antibody of the present technology is a single chain anti-VEGF XAng-2 antibody. In accordance with the present technology, the techniques may be adapted to produce single chain antibodies specific for VEGF or Ang-2 proteins (see, e.g., U.S. Pat. No. 4,946,778). Examples of techniques that can be used to produce single chain Fv and antibodies of the present technology include U.S. Pat. nos. 4,946,778 and 5,258,498; huston et al, methods in Enzymology, volume 203: 46-88, 1991; shu, l. Et al, proc.Natl. Acad.Sci.USA, volume 90: pages 7995-7999, 1993; and Skerra et al, science, volume 240: pages 1038-1040, and those described in 1988.

Chimeric antibodies and humanized antibodies. In one embodiment, the anti-VEGF XAng-2 antibody of the present technology is a chimeric anti-VEGF XAng-2 antibody. In one embodiment, the anti-VEGF XAng-2 antibody of the present technology is a humanized anti-VEGF XAng-2 antibody. In one embodiment of the present technology, the donor and acceptor antibodies are monoclonal antibodies from different species. For example, the recipient antibody is a human antibody (to minimize its antigenicity in humans), in which case the resulting CDR-grafted antibody is referred to as a "humanized" antibody.

Recombinant anti-VEGF x Ang-2 antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions can be prepared using standard recombinant DNA techniques and are within the scope of the present technology. For some uses, including in vivo use of the anti-VEGF X Ang-2 antibodies of the present technology in humans and use of these agents in vitro detection assays, chimeric or humanized anti-VEGF X Ang-2 antibodies may be used. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art. Such useful methods include, for example, but are not limited to, the methods described in the following: international application No. PCT/US86/02269; U.S. Pat. nos. 5,225,539; european patent No. 184387; european patent No. 171496; european patent No. 173494; PCT International publication No. WO 86/01533; U.S. Pat. nos. 4,816,567;5,225,539; european patent No. 125023; better et al, 1988, science, volume 240: pages 1041-1043; liu et al, 1987, proc.Natl.Acad.Sci.USA, volume 84: pages 3439-3443; liu et al, 1987, j.immunol., volume 139: pages 3521-3526; sun et al, 1987, proc. Natl. Acad. Sci. USA, volume 84: pages 214-218; nishimura et al, 1987, cancer res., volume 47: pages 999-1005; wood et al, 1985, nature, volume 314: pages 446-449; shaw et al, 1988, j.Natl.cancer Inst., volume 80: pages 1553-1559; morrison (1985), science, volume 229: pages 1202-1207; oi et al (1986), bioTechniques, volume 4: page 214; jones et al, 1986, nature, volume 321: pages 552-525; verhoeye et al, 1988, science, volume 239: page 1534; morrison, science, volume 229: page 1202, 1985; oi et al, bioTechniques, volume 4: page 214, 1986; gilles et al, j.immunol methods, volume 125: pages 191-202, 1989; U.S. patent No. 5,807,715; and Beidler et al, 1988, J.Immunol., volume 141: pages 4053-4060. For example, antibodies can be humanized using a variety of techniques including CDR grafting (EP 0 239 400; WO 91/09967; U.S. Pat. No. 5,530,101;5,585,089;5,859,205;6,248,516; EP 460167), veneering or resurfacing (EP 0 592 106;EP 0 519 596;Padlan E.A, molecular Immunology, volume 28: pages 489-498, 1991; studnicka et al, protein Engineering, volume 7: pages 805-814, 1994; roguska et al, PNAS, volume 91: pages 969-973, 1994) and chain shuffling (U.S. Pat. No. 5,565,332). In one embodiment, the cDNA encoding the murine anti-VEGF XAng-2 monoclonal antibody is digested with a specially selected restriction enzyme to remove the sequence encoding the Fc constant region and replaced with an equivalent portion of the cDNA encoding the human Fc constant region (see, robinson et al, PCT/US86/02269; akira et al, european patent application 184,187; taniguchi, european patent application 171,496; morrison et al, european patent application 173,494; neuberger et al, WO 86/01533; cabilly et al, U.S. Pat. No. 4,816,567; cabilly et al, european patent application 125,023; better et al (1988), science, volume 240: pages 1041-1043; liu et al (1987), proc.Natl.Acad.Sci.USA, volume 84: pages 3439-3443, liu et al (1987), J Immunol, volume 139: pages 3521-3526, sun et al (1987), proc.Natl.Acad.Sci.USA, volume 84: pages 214-218, nishimura et al (1987), cancer Res, volume 47: pages 999-1005, wood et al (1985), nature, volume 314: pages 446-449, and Shaw et al (1988), J.Natl.cancer Inst, volumes 80: pages 1553-1559, U.S. Pat. No. 6,180,370, U.S. Pat. No. 6,300,064, 6,696,248, 6,706,828,422).

In one embodiment, the present technology provides for the construction of humanized anti-VEGF x Ang-2 antibodies that are unlikely to induce a human anti-mouse antibody (hereinafter "HAMA") response while still having potent antibody effector function. As used herein, the terms "human" and "humanized" in connection with an antibody refer to any antibody that is expected to elicit a therapeutically tolerable weak immunogenic response in a human subject. In one embodiment, the present technology provides humanized anti-VEGF X Ang-2 antibodies, heavy chain and light chain immunoglobulins.

CDR antibodies. In some embodiments, the anti-VEGF XAng-2 antibodies of the present technology are anti-VEGF XAng-2 CDR antibodies. Typically, the donor and acceptor antibodies used to generate the anti-VEGF XAng-2 CDR antibodies are monoclonal antibodies from different species; typically the receptor antibody is a human antibody (toMinimizing its antigenicity in humans), in which case the resulting CDR-grafted antibody is referred to as a "humanized" antibody. The graft may be a single V of recipient antibody _H Or V _L Within a single CDR (or even a portion of a single CDR), or may be V _H And V _L A plurality of CDRs (or portions thereof) within one or both of (i) and (ii). Typically, all three CDRs in all variable domains of the recipient antibody will be replaced by corresponding donor CDRs, but only the necessary number of substitutions is required to allow the resulting CDR-grafted antibody to bind well to the VEGF x Ang-2 protein. U.S. Pat. No. 5,585,089 to Queen et al; U.S. Pat. nos. 5,693,761; U.S. Pat. No. 5,693,762 and Winter, U.S. Pat. No. 5,225,539 and EP 0682040 teach methods for producing CDR-grafted and humanized antibodies. Winter et al, U.S. Pat. No. 4,816,397;6,291,158;6,291,159;6,291,161;6,545,142; EP 0368684; EP0451216 and EP 0126694 teach that they can be used for the preparation of V _H And V _L A method of producing a polypeptide.

Either or both of the heavy and light chain variable regions are produced by grafting CDRs from the species of origin into the hybrid framework regions after selection of appropriate framework region candidates from the same family and/or members of the same family. The assembly of hybrid antibodies or hybrid antibody fragments having hybrid variable chain regions in accordance with any of the above aspects may be accomplished using conventional methods known to those skilled in the art. For example, DNA sequences encoding the hybrid variable domains described herein (i.e., based on the framework of the target species and CDRs from the species of origin) can be generated by oligonucleotide synthesis and/or PCR. Nucleic acids encoding CDR regions can also be isolated from antibodies of the species of origin using suitable restriction enzymes and ligated into the framework of the species of interest by ligation with suitable ligases. Alternatively, the framework regions of the variable chains of antibodies of the species of origin can be altered by site-directed mutagenesis.

Since hybrids are constructed from multiple candidate selections corresponding to each framework region, there are many sequence combinations that can be constructed according to the principles described herein. Thus, a hybrid library can be assembled, the members of which have different combinations of the individual framework regions. Such libraries may be electronic database collections of sequences or physical collections of hybrids.

This process typically does not alter the FR of the grafted CDRs flanking the recipient antibody. However, those skilled in the art can sometimes increase the antigen binding affinity of the resulting anti-VEGF XAng-2 CDR-grafted antibodies by replacing certain residues of a given FR to make the FR more similar to the corresponding FR of the donor antibody. Suitable substitution positions include amino acid residues adjacent to or capable of interacting with the CDRs (see, e.g., US 5,585,089, especially columns 12-16). Alternatively, one skilled in the art can start with a donor FR and modify it to more resemble an acceptor FR or a human consensus FR. Techniques for making these modifications are known in the art. In particular, if the resulting FR corresponds to a human consensus FR at that location, or has at least 90% or greater identity to such consensus FR, doing so may not significantly increase the antigenicity of the resulting modified anti-VEGF x Ang-2 CDR-grafted antibody compared to the same antibody with a fully human FR.

Bispecific antibody (BsAb). Bispecific antibodies are antibodies that can bind simultaneously to two targets having different structures, e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or an epitope on a target antigen. For example, bsabs may be prepared by combining heavy and/or light chains that recognize different epitopes of the same or different antigens. In some embodiments, the bispecific binding agent binds one antigen (or epitope) on one of its two binding arms (one VH/VL pair) and a different antigen (or epitope) on its second arm (a different VH/VL pair) by molecular function. According to this definition, a bispecific binding agent has two different antigen binding arms (in both the specificity and CDR sequences), and is monovalent for each antigen to which it binds.

Multispecific antibodies such as bispecific antibodies (bsabs) and bispecific antibody fragments (BsFab) have at least one arm that specifically binds to, for example, VEGF x Ang-2 and at least one other arm that specifically binds to a second target antigen.

A variety of bispecific fusion proteins can be produced using molecular engineering. For example, bsabs have been constructed that utilize an intact immunoglobulin framework (e.g., igG), a single chain variable fragment (scFv), or a combination thereof. In some embodiments, the bispecific fusion protein is bivalent, comprising, for example, an scFv with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen. In some embodiments, the bispecific fusion protein is bivalent, comprising, for example, an scFv having a single binding site for one antigen and another scFv fragment having a single binding site for a second antigen. In other embodiments, the bispecific fusion protein is tetravalent, comprising, for example, an immunoglobulin (e.g., igG) having two binding sites for one antigen and two identical scFv for a second antigen. BsAb consisting of two scFv units in tandem has been shown to be a clinically successful form of bispecific antibody.

More recently methods for producing bsabs have included engineered recombinant monoclonal antibodies that have additional cysteine residues and therefore have been more cross-linked than the more common immunoglobulin isotype. See, e.g., fitzGerald et al, protein eng., volume 10, phase 10: pages 1221-1225 (1997). Another approach is to engineer recombinant fusion proteins to link two or more different single chain antibodies or antibody fragments with the desired dual specificity. See, e.g., coloma et al, nature Biotech, volume 15: pages 159-163 (1997). A variety of bispecific fusion proteins can be produced using molecular engineering.

Bispecific fusion proteins linking two or more different single chain antibodies or antibody fragments are produced in a similar manner. Recombinant methods can be used to produce a variety of fusion proteins. In some particular embodiments, a BsAb according to the present technology comprises an immunoglobulin comprising a heavy chain and a light chain and an scFv. In some specific embodiments, the scFv is linked to the C-terminus of the heavy chain of any VEGF XAng-2 immunoglobulin disclosed herein. In some specific embodiments, the scFv is linked to the C-terminus of any VEGF XAng-2 immunoglobulin light chain disclosed herein. In various embodiments In a variant, the scFv is linked to the heavy or light chain via a linker sequence. Introduction of appropriate linker sequences required for the in-frame attachment of heavy chain Fd to scFv into V by PCR reaction _L And V _κ A domain. The DNA fragment encoding the scFv is then ligated into a staging vector containing the DNA sequence encoding the CH1 domain. The resulting scFv-CH1 construct was excised and ligated into a vector containing a V encoding VEGF X Ang-2 antibody _H In a vector of the DNA sequence of the region. The resulting vector may be used to transfect an appropriate host cell, such as a mammalian cell, to express the bispecific fusion protein.

In some embodiments, the length of the linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more amino acids. In some embodiments, the linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility (e.g., first and/or second antigen binding sites) to the polypeptide. In some embodiments, linkers are employed in the bsabs described herein based on imparting specific properties to the BsAb such as, for example, an increase in stability. In some embodiments, a BsAb of the present technology comprises G ₄ S joint. In some specific embodiments, a BsAb of the present technology comprises (G ₄ S) _n A linker, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or greater.

Fc modification. In some embodiments, an anti-VEGF x Ang-2 antibody of the present technology comprises an Fc region variant, wherein the Fc region variant comprises at least one amino acid modification relative to a wild-type Fc region (or parent Fc region) such that the molecule has an altered affinity for an Fc receptor (e.g., fcγr), provided that it is based on the polypeptide described by Sondermann et al, nature, volume 406: crystallographic and structural analysis of Fc-Fc receptor interactions, disclosed on pages 267-273 (2000), the Fc region variants have no substitution at the position of direct contact with the Fc receptor. Examples of positions within the Fc region that are in direct contact with Fc receptors such as FcgammaR include amino acids 234-239 (hinge region), amino acids 265-269 (B/C loop), amino acids 297-299 (C7E loop) and amino acids 327-332 (F/G) loop.

In some embodiments, the anti-VEGF x Ang-2 antibodies of the present technology have altered affinity for activating and/or inhibiting receptors, have an Fc variant region comprising one or more amino acid modifications, wherein the one or more amino acid modifications is substitution of N297 with alanine or substitution of K322 with alanine. Additionally or alternatively, in some embodiments, the Fc region of the VEGF x Ang-2 antibodies disclosed herein comprises two amino acid substitutions Leu234Ala and Leu235Ala (so-called LALA mutations) to eliminate fcyriia binding. LALA mutations are commonly used to attenuate cytokine induction from T cells, thereby reducing antibody toxicity (Wines BD et al, J Immunol, volume 164: pages 5313-5318 (2000)).

Glycosylation modification. In some embodiments, the anti-VEGF XAng-2 antibodies of the present technology have an Fc region with a glycosylation variant compared to the parent Fc region. In some embodiments, the glycosylation variant comprises the absence of fucose; in some embodiments, the glycosylation variant is caused by expression in GnT 1-deficient CHO cells.

In some embodiments, the antibodies of the present technology may have modified glycosylation sites relative to an appropriate reference antibody that binds to an antigen of interest (e.g., VEGF x Ang-2) without altering the function of the antibody, e.g., binding activity to the antigen. As used herein, a "glycosylation site" includes any particular amino acid sequence in an antibody in which oligosaccharides (i.e., carbohydrates containing two or more monosaccharides linked together) will be specifically and covalently linked.

Oligosaccharide side chains are typically attached to the main chain of the antibody via N-or O-bonds. N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxy amino acid, e.g., serine, threonine.

In some embodiments, the carbohydrate content of the immunoglobulin-related compositions disclosed herein is altered by the addition or deletion of glycosylation sites. Methods for altering the carbohydrate content of antibodies are well known in the art and are included in the present technology, see, for example, U.S. Pat. nos. 6,218,149; EP 0359096B1; U.S. patent publication No. US2002/0028486; international patent application publication WO 03/035835; U.S. patent publication No. 2003/015614; U.S. Pat. nos. 6,218,149; U.S. patent No. 6,472,511; all of these patents are incorporated by reference in their entirety. In some embodiments, the carbohydrate content of the antibody (or related portion or component thereof) is altered by deleting one or more endogenous carbohydrate portions of the antibody. In some particular embodiments, the present technology includes deleting the glycosylation site of the Fc region of the antibody by changing position 297 from asparagine to alanine.

The engineered glycoforms can be used for a variety of purposes including, but not limited to, enhancing or reducing effector function. The engineered glycoforms may be produced by any method known to those of skill in the art, for example, by using engineered or variant expression strains, by co-expression with one or more enzymes such as N-acetylglucosamine transferase III (GnTIII), by expression of molecules comprising the Fc region in or from various organisms or by altering the carbohydrates after the molecules comprising the Fc region have been expressed. Methods for generating engineered glycoforms are known in the art and include, but are not limited to, those described in: umana et al, 1999, nat. Biotechnol., vol.17: pages 176-180; davies et al, 2001, biotechnol. Bioeng., volume 74: pages 288-294; shields et al, 2002, J.biol. Chem., volume 277: pages 26733-26740; shinkawa et al, 2003, j.biol. Chem., volume 278: pages 3466-3473; U.S. Pat. nos. 6,602,684; U.S. patent application Ser. No. 10/277,370; U.S. patent application Ser. No. 10/113,929; international patent application publication WO 00/61739A1; WO 01/292246A1; WO 02/311140A1; those in WO 02/30954A 1; pots ^TM Technology (Biowa, inc., princeton, n.j.); glycomab ^TM Glycosylation technology (GLYCART biotechnology AG, zurich, switzerland); each of these patents is incorporated by reference in its entiretyIncorporated herein. See, for example, international patent application publication WO 00/061739; U.S. patent application publication No. 2003/015614; okazaki et al, 2004, JMB, volume 336: pages 1239-1249.

A fusion protein. In one embodiment, the anti-VEGF XAng-2 antibodies of the present technology are fusion proteins. The anti-VEGF X Ang-2 antibodies of the present technology may be used as an antigen tag when fused to a second protein. Examples of domains that can be fused to polypeptides include not only heterologous signal sequences, but also other heterologous functional regions. Fusion need not be direct, but may occur through a linker sequence. In addition, fusion proteins of the present technology can also be engineered to improve the characteristics of anti-VEGF X Ang-2 antibodies. For example, additional amino acids, particularly regions of charged amino acids, may be added to the N-terminus of the anti-VEGF x Ang-2 antibody to improve stability and persistence during purification from the host cell or during subsequent handling and storage. In addition, peptide moieties may be added to the anti-VEGF x Ang-2 antibodies to facilitate purification. Such regions may be removed prior to final preparation of the anti-VEGF x Ang-2 antibody. The addition of peptide moieties to facilitate processing of polypeptides is well known and conventional in the art. The anti-VEGF x Ang-2 antibodies of the present technology may be fused to a marker sequence such as a peptide that facilitates purification of the fusion polypeptide. In selected embodiments, the tagged amino acid sequence is a hexahistidine peptide, such as the tag provided in the pQE vector (QIAGEN, inc., chatsworth, calif), among others, many of which are commercially available. For example, as in Gentz et al, proc.Natl. Acad. Sci. USA, volume 86: hexahistidine can be conveniently used to purify fusion proteins as described in pages 821-824, 1989. Another peptide tag "HA" tag that can be used for purification corresponds to an epitope derived from influenza hemagglutinin protein. Wilson et al, cell, volume 37: page 767, 1984.

Thus, polynucleotides or polypeptides of the present technology can be used to engineer any of these fusion proteins described above. Furthermore, in some embodiments, the fusion proteins described herein exhibit an extended in vivo half-life.

Fusion proteins having disulfide-linked dimer structures (due to IgG) can bind and neutralize other molecules more effectively than monomeric secreted proteins or protein fragments alone. Fountoulakis et al, J.biochem., volume 270: pages 3958-3964, 1995.

Similarly, EP-A-O464 533 (Canadian counterpart patent 2045869) discloses fusion proteins comprising parts of the constant region of an immunoglobulin molecule together with another human protein or a fragment thereof. In many cases, the Fc portion of the fusion protein is beneficial for therapy and diagnosis, thus resulting in, for example, improved pharmacokinetic properties. See EP-A0232 262. Alternatively, it may be desirable to delete or modify the Fc portion after the fusion protein has been expressed, detected and purified. For example, if the fusion protein is used as an antigen for immunization, the Fc portion may hinder therapy and diagnosis. In drug discovery, for example, human proteins such as hIL-5 have been fused to Fc portions for high throughput screening assays to identify antagonists of hIL-5. Bennett et al, j.molecular Recognition, volume 8: pages 52-58, 1995; johanson et al, j.biol.chem., volume 270: pages 9459-9471, 1995.

A labeled anti-VEGF x Ang-2 antibody. In one embodiment, the anti-VEGF XAng-2 antibodies of the present technology are conjugated to a labeling moiety, i.e., a detectable moiety. The particular label or detectable group conjugated to the anti-VEGF X Ang-2 antibody is not a critical aspect of the present technology, so long as it does not significantly interfere with the specific binding of the anti-VEGF X Ang-2 antibody of the present technology to VEGF or Ang-2 protein. The detectable group may be any material having a detectable physical or chemical property. Such detectable labels are well developed in the field of immunoassays and imaging. In general, almost any label that can be used in such methods can be applied to the present technology. Thus, a label is any composition that is detectable spectroscopically, photochemically, biochemically, immunochemically, electrically, optically or chemically. Labels useful in practicing the present technology include magnetic beads (e.g., dynabeads ^TM ) Fluorescent dyes (e.g., fluorescein isothiocyanate, texas red, rhodamine, etc.), radiolabels (e.g., ³ H、 ¹⁴ C、 ³⁵ S、 ¹²⁵ I、 ¹²¹ I、 ¹³¹ I、 ¹¹² In、 ⁹⁹ mTc), other imaging agents such as microbubbles (for ultrasound imaging), and, ¹⁸ F、 ¹¹ C、 ¹⁵ O、 ⁸⁹ Zr (for positron emission tomography), ^99m TC、 ¹¹¹ In (for single photon emission tomography), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used In ELISA), and thermal labels, such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents describing the use of such tags include U.S. Pat. nos. 3,817,837;3,850,752;3,939,350;3,996,345;4,277,437;4,275,149; and 4,366,241, each of which is incorporated by reference herein in its entirety for all purposes. See also Handbook of Fluorescent Probes and Research Chemicals (6 th edition, molecular Probes, inc., eugene, OR.).

The labels may be coupled directly or indirectly to the desired components of the assay according to methods well known in the art. As noted above, a variety of labels can be used, and the choice of label depends on factors such as the sensitivity desired, the ease of conjugation with the compound, stability requirements, available instrumentation and handling regulations.

The nonradioactive labels are typically linked by indirect means. Typically, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand is then bound to an anti-ligand (e.g., streptavidin) molecule which itself may be detected or covalently bound to a signaling system such as a detectable enzyme, fluorescent compound, or chemiluminescent compound. A variety of ligands and anti-ligands may be used. When the ligand has a natural anti-ligand such as biotin, thyroxine and cortisol, it may be used in combination with a labeled, naturally occurring anti-ligand. Alternatively, any hapten or antigenic compound can be used in combination with an antibody, such as an anti-VEGF x Ang-2 antibody.

The molecule may also be conjugated directly to the signal generating compound, for example by conjugation to an enzyme or fluorophore. Enzymes of interest as labels are mainly hydrolases (in particular phosphatases, esterases and glycosidases), or oxidoreductases (in particular peroxidases). Fluorescent compounds used as labeling moieties include, but are not limited to, for example, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like. Chemiluminescent compounds useful as labeling moieties include, but are not limited to, for example, luciferin and 2, 3-dihydro phthalazinedione, such as luminol. For an overview of the various labels or signal generation systems that can be used, see U.S. patent No. 4,391,904.

Means for detecting the label are well known to those skilled in the art. Thus, for example, where the label is a radiolabel, the means for detecting comprises a scintillation counter or film in autoradiography. When the label is a fluorescent label, it can be detected by exciting the fluorescent dye with light of an appropriate wavelength and detecting the resulting fluorescence. Fluorescence can be visually detected through the film by using an electron detector such as a Charge Coupled Device (CCD) or photomultiplier tube, or the like. Similarly, enzyme labels can be detected by providing an appropriate substrate for the enzyme and detecting the resulting reaction product. Finally, a simple colorimetric label may be detected simply by observing the color associated with the label. Thus, in various oiled paper assays, the conjugated gold is typically pink, while the various conjugated beads are bead colored.

Some assay formats do not require the use of labeled components. For example, agglutination assays can be used to detect the presence of target antibodies, such as anti-VEGF x Ang-2 antibodies. In this case, the antigen-coated particles are agglutinated by the sample containing the target antibody. In this form, no labeling of any components is required and the presence of the target antibody can be detected by simple visual inspection.

Identification and characterization of anti-VEGF X Ang-2 antibodies of the present technology

Methods for identifying and/or screening anti-VEGF X Ang-2 antibodies of the present technology. Methods that can be used to identify and screen antibodies against VEGF or Ang-2 polypeptides to obtain antibodies having the desired specificity for VEGF or Ang-2 proteins (e.g., those antibodies that bind to the extracellular domain of VEGF or Ang-2 proteins, such as polypeptides comprising the amino acid sequences of SEQ ID NO:38 and SEQ ID NO: 39) include any immunologically mediated technique known in the art. The components of the immune response may be detected in vitro by various methods well known to those of ordinary skill in the art. For example, (1) cytotoxic T lymphocytes can be incubated with radiolabeled target cells and lysis of these target cells detected by radioactive release; (2) Helper T lymphocytes can be incubated with antigen and antigen presenting cells and cytokine synthesis and secretion measured by standard methods (Windhagen A et al, immunity, vol.2:373-380, 1995); (3) Antigen presenting cells can be incubated with whole protein antigen and the antigen presented on MHC can be detected by T lymphocyte activation assay or biophysical methods (Harding et al, proc. Natl. Acad. Sci., vol 86: pages 4230-4234, 1989); (4) Mast cells can be incubated with an agent that cross-links their Fc-epsilon receptor and histamine release measured by an enzyme immunoassay (Siraganian et al, TIPS, vol. 4: pages 432-437, 1983); (5) enzyme-linked immunosorbent assay (ELISA).

Similarly, the products of an immune response in a model organism (e.g., a mouse) or human subject can also be detected by various methods well known to those of ordinary skill in the art. For example, (1) antibodies produced in response to vaccination can be readily detected by standard methods currently used in clinical laboratories, such as ELISA; (2) Migration of immune cells to the site of inflammation can be detected by scraping the skin surface and placing a sterile container to capture the migrating cells on the scraping site (Peters et al Blood, volume 72: pages 1310-1315, 1988); (3) Proliferation of Peripheral Blood Mononuclear Cells (PBMC) in response to mitogenic or mixed lymphocyte responses may be used ³ H-thymidine for measurement; (4) Phagocytic capacity of granulocytes, macrophages and other phagocytes in PBMC can be measured by placing PBMC in wells together with labeled particles (Peters et al, blood, volume 72: pages 1310-1315, 1988); (5) Differentiation of immune system cells can be measured by labelling PBMCs with antibodies to CD molecules such as CD4 and CD8 and measuring the fraction of PBMCs expressing these markers.

In one embodiment, the anti-VEGF XAng-2 antibodies of the present technology are selected using the display of VEGF or Ang-2 peptides on the surface of replicable genetic packages. See, for example, U.S. patent No. 5,514,548;5,837,500;5,871,907;5,885,793;5,969,108;6,225,447;6,291,650;6,492,160; EP 585 287; EP 605522; EP 616640; EP 1024191; EP 589 877; EP 774 511; EP 844 306. Methods have been described that can be used to generate/select filamentous phage particles containing a phagemid genome encoding a binding molecule with the desired specificity. See, e.g., EP 774 511; US 5871907; US 5969108; US 6225447; US 6291650; US 6492160.

In some embodiments, the anti-VEGF XAng-2 antibodies of the present technology are selected using the display of VEGF or Ang-2 peptides on the surface of yeast host cells. Kieke et al, protein eng., october 1997; volume 10, 11 th phase: methods that can be used to isolate scFv polypeptides by yeast surface display have been described on pages 1303-1310.

In some embodiments, ribosome display is used to select anti-VEGF XAng-2 antibodies of the present technology. Mattheakis et al, proc. Natl. Acad. Sci. USA, volume 91: pages 9022-9026, 1994; and Hanes et al, proc.Natl. Acad. Sci. USA, volume 94: methods that can be used to identify ligands in peptide libraries using ribosome display have been described in 1997 on pages 4937-4942.

In certain embodiments, the anti-VEGF XAng-2 antibodies of the present technology are selected using tRNA display of VEGF or Ang-2 peptides. Merryman et al chem.biol., volume 9: methods that can be used to select ligands in vitro using tRNA display have been described on pages 741-746, 2002.

In one embodiment, RNA display is used to select anti-VEGF XAng-2 antibodies of the present technology. Roberts et al, proc.Natl. Acad. Sci.USA, volume 94: pages 12297-12302, 1997; and Nemoto et al, FEBS lett, volume 414: methods that can be used to select peptides and proteins using RNA display libraries have been described in 1997 on pages 405-408. Frankel et al, curr.opin.struct.biol., volume 13: methods that can be used to select peptides and proteins using non-natural RNA display libraries have been described in 2003 on pages 506-512.

In some embodiments, the anti-VEGF XAng-2 antibodies of the present technology are expressed in the periplasm of gram-negative bacteria and mixed with a labeled VEGF or Ang-2 protein. See WO 02/34886. In clones expressing recombinant polypeptides with affinity for VEGF or Ang-2 protein, the concentration of labeled VEGF or Ang-2 protein bound to anti-VEGF X Ang-2 antibodies is increased and cells are allowed to separate from the rest of the library, as in Harvey et al, proc. Natl. Acad. Sci. Volume 22: pages 9193-9198, 2004 and U.S. patent publication No. 2004/0058403.

After selection of the desired anti-VEGF x Ang-2 antibody, it is contemplated that the antibody may be produced in large amounts by any technique known to those skilled in the art, such as prokaryotic or eukaryotic cell expression, and the like. anti-VEGF x Ang-2 antibodies, such as, but not limited to, anti-VEGF x Ang-2 hybrid antibodies or fragments, can be produced by constructing an expression vector encoding the antibody heavy chain, wherein the CDRs and, if necessary, the minimal portion of the variable region framework required to preserve the binding specificity of the original species antibody (as engineered according to the techniques described herein) are derived from the species of origin antibody and the remainder of the antibody is derived from the target species immunoglobulin, which can be manipulated as described herein, thereby producing a vector for expressing the hybrid antibody heavy chain.

Measurement of VEGF or Ang-2 binding. In some embodiments, the VEGF XAng-2 binding assay refers to an assay format in which VEGF and/or Ang-2 protein and anti-VEGF XAng-2 antibody are mixed under conditions suitable for binding between VEGF and/or Ang-2 protein and anti-VEGF XAng-2 antibody and assessing the amount of binding between VEGF and/or Ang-2 protein and anti-VEGF XAng-2 antibody. The amount of binding is compared to a suitable control, which may be the amount of binding in the absence of VEGF and/or Ang-2 protein, the amount of binding in the presence of a non-specific immunoglobulin composition, or both. The amount of binding may be assessed by any suitable method. Binding assays include, for example, ELISA, radioimmunoassay, scintillation proximity assay, fluorescent energy transfer assay, liquid chromatography, membrane filtration assay, and the like. Biophysical assays for direct measurement of binding of VEGF and/or Ang-2 proteins to anti-VEGF X Ang-2 antibodies are for example nuclear magnetic resonance, fluorescence polarization, surface plasmon resonance (BIACORE chip) and the like. Specific binding is determined by standard assays known in the art, such as radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectrometry, and the like. Candidate anti-VEGF X Ang-2 antibodies may be used as anti-VEGF X Ang-2 antibodies in the present technology if the specific binding of the candidate anti-VEGF X Ang-2 antibody is at least 1% greater than that observed in the absence of the candidate anti-VEGF X Ang-2 antibody.

Therapeutic method

The following discussion is presented by way of example only and is not intended to be limiting.

One aspect of the present technology includes a method of treating a disease or disorder characterized by the growth of new blood vessels into the subretinal pigment epithelium or subretinal space. Additionally or alternatively, in some embodiments, the present technology includes a method of treating CNV. In one aspect, the present disclosure provides a method for inhibiting VEGF and/or Ang-2-induced angiogenesis in an eye, the method comprising administering to a subject a therapeutically effective amount of at least one VEGF x Ang-2 antibody of the present technology, and wherein the subject has a disease or disorder characterized by neovascular growth into the subretinal pigment epithelium or subretinal space.

In some embodiments, the subject is diagnosed with, suspected of having, or at risk of having a disease or disorder characterized by elevated levels of VEGF and/or Ang-2 expression and/or increased activity. Additionally or alternatively, in some embodiments, the subject is diagnosed as having CNV.

In therapeutic applications, a composition or medicament comprising an anti-VEGF x Ang-2 antibody disclosed herein is administered to a subject suspected of having or already having such a disease or disorder (such as a subject diagnosed with a disease or disorder characterized by elevated levels of VEGF and/or Ang-2 expression and/or increased activity and/or a subject diagnosed with CNV) in an amount sufficient to cure or at least partially arrest the symptoms of the disease, including its complications and intermediate pathological phenotypes during the course of the disease.

A subject suffering from a disease or disorder characterized by elevated levels of expression and/or increased activity of VEGF and/or Ang-2, or alternatively, a subject diagnosed with CNV, may be identified by any one or a combination of diagnostic or prognostic assays known in the art. For example, typical symptoms of CNV include, but are not limited to: distortion or waving of central vision or central vision with gray/black/plaques, retinal blisters or bleeds, loss of brightness of color or different colors exhibited by each eye, vision deformity (i.e., distorted vision, straight lines appear curved, skewed or irregular), painless vision loss, paracenter or central dark spots (i.e., islands of relative or absolute vision loss at or near the center of vision), different sizes of objects exhibited by each eye, sparkle or blink in central vision.

In some embodiments, a subject with a disease or disorder characterized by elevated levels of VEGF and/or Ang-2 expression and/or increased activity and/or a subject with CNV treated with an anti-VEGF x Ang-2 antibody will exhibit an improvement or elimination of one or more of the following symptoms: distortion or waving of central vision or central vision with gray/black/plaques, retinal blisters or bleeds, loss of brightness of color or different colors exhibited by each eye, vision deformity (i.e., distorted vision, straight lines appear curved, skewed or irregular), painless vision loss, paracenter or central dark spots (i.e., islands of relative or absolute vision loss at or near the center of vision), different sizes of objects exhibited by each eye, sparkle or blink in central vision.

For therapeutic use, a composition comprising an anti-VEGF x Ang-2 antibody disclosed herein is administered to a subject. In some embodiments, the anti-VEGF x Ang-2 antibody is administered once, twice, three times, four times, or five times daily. In some embodiments, the anti-VEGF x Ang-2 antibody is administered more than five times per day. Additionally or alternatively, in some embodiments, the anti-VEGF x Ang-2 antibody is administered daily, every other day, every third day, every fourth day, every fifth day, or every sixth day. In some embodiments, the anti-VEGF x Ang-2 antibody is administered weekly, biweekly, tricyclically, or monthly. In some embodiments, the anti-VEGF x Ang-2 antibody is administered for a period of one week, two weeks, three weeks, four weeks, or five weeks. In some embodiments, the anti-VEGF x Ang-2 antibody is administered for six weeks or more. In some embodiments, the anti-VEGF x Ang-2 antibody is administered for twelve weeks or more. In some embodiments, the anti-VEGF x Ang-2 antibody is administered for a period of time less than one year. In some embodiments, the anti-VEGF x Ang-2 antibody is administered for a period of time exceeding one year. In some embodiments, the anti-VEGF x Ang-2 antibody is administered throughout the life of the subject.

In some embodiments of the methods of the present technology, the anti-VEGF X Ang-2 antibody is administered daily for a period of 1 week or more. In some embodiments of the methods of the present technology, the anti-VEGF x Ang-2 antibody is administered daily for a period of 2 weeks or more. In some embodiments of the methods of the present technology, the anti-VEGF X Ang-2 antibody is administered daily for 3 weeks or more. In some embodiments of the methods of the present technology, the anti-VEGF x Ang-2 antibody is administered daily for a period of 4 weeks or more. In some embodiments of the methods of the present technology, the anti-VEGF X Ang-2 antibody is administered daily for a period of 6 weeks or more. In some embodiments of the methods of the present technology, the anti-VEGF X Ang-2 antibody is administered daily for 12 weeks or longer. In some embodiments, the anti-VEGF x Ang-2 antibody is administered daily throughout the life of the subject.

Determination of biological Effect of anti-VEGF XAng-2 antibodies

In various embodiments, suitable in vitro or in vivo assays are performed to determine whether the effects of a particular anti-VEGF x Ang-2 antibody, and administration thereof, are suitable for treatment. In various embodiments, in vitro assays may be performed with representative animal models to determine whether a given anti-VEGF x Ang-2 antibody exerts a desired effect on reducing or eliminating signs and/or symptoms of CNV. Compounds for treatment may be tested in suitable animal model systems including, but not limited to, rats, mice, chickens, cattle, monkeys, rabbits, etc., prior to testing in human subjects. Similarly, for in vivo testing, any of the animal model systems known in the art may be used prior to administration to a human subject. In some embodiments, the in vitro or in vivo test is directed against the biological function of one or more anti-VEGF x Ang-2 antibodies.

Animal models of CNV can be generated using techniques known in the art. Such models can be used to demonstrate the biological effects of anti-VEGF x Ang-2 antibodies in the prevention and treatment of conditions resulting from disruption of a particular gene, and to determine the biological effects of compositions comprising a therapeutically effective amount of one or more anti-VEGF x Ang-2 antibodies disclosed herein in a given context.

Mode of administration and effective dosage

Any method known to those of skill in the art for contacting a cell, organ or tissue with one or more anti-VEGF x Ang-2 antibodies disclosed herein may be employed. Suitable methods include in vitro, ex vivo or in vivo methods. In vivo methods generally comprise administering one or more anti-VEGF x Ang-2 antibodies to a mammal, suitably a human. When used for treatment in vivo, one or more anti-VEGF x Ang-2 antibodies described herein are administered to a subject in an effective amount (i.e., an amount having a desired therapeutic effect). The dose and dosage regimen will depend on the extent of the disease state of the subject, the characteristics of the particular anti-VEGF x Ang-2 antibody used (e.g., its therapeutic index), and the subject's medical history.

The effective amount can be determined by methods familiar to physicians and clinicians during preclinical and clinical trials. An effective amount of one or more anti-VEGF x Ang-2 antibodies disclosed herein that can be used in these methods can be administered to a mammal in need thereof by any of a variety of well-known methods for administering pharmaceutical compounds. The anti-VEGF x Ang-2 antibodies may be administered systemically or locally.

One or more anti-VEGF x Ang-2 antibodies disclosed herein may be administered by any route that allows contact with RPE cells. Administration may be, for example, ocular, parenteral (e.g., subcutaneous, intramuscular, or intravenous), topical, transdermal, intravitreal, retroorbital, subretinal, subscleral, oral, sublingual, or buccal modes of administration. Some of the foregoing exemplary modes of administration may be achieved by injection. However, in some embodiments, injection is avoided by using a slow release implant near the retina (e.g., the sub-scleral route) or by administering drops to the conjunctiva. One or more anti-VEGF x Ang-2 antibodies of the present technology may be topically administered to the eye of a patient suffering from CNV. Topical administration includes intravitreal, topical ocular, transdermal patch, subcutaneous, parenteral, intraocular, subconjunctival or retrobulbar or sub-tenon injection, transscleral (including iontophoresis), posterior juxtascleral delivery, or sustained release biodegradable polymers or liposomes. One or more anti-VEGF x Ang-2 antibodies may also be delivered in an ocular lavage solution. The concentration may range from about 0.001 μm to about 100 μm, preferably from about 0.01 μm to about 5 μm.

The compositions of the present technology can be topically applied to the eyes of a patient suffering from CNV. One or more anti-VEGF x Ang-2 antibodies of the present technology may be incorporated into various types of ophthalmic formulations for delivery to the eye (e.g., topically, intracamerally, juxtasclerally, or by implants). One or more anti-VEGF x Ang-2 antibodies of the present technology may be combined with an ophthalmically acceptable preservative, surfactant, viscosity enhancing agent, gelling agent, penetration enhancing agent, buffer, sodium chloride, and water to form an aqueous sterile ophthalmic suspension or solution or pre-formed gel or in situ formed gel.

In some embodiments of the methods of the present disclosure, the anti-VEGF x Ang-2 antibody composition is administered by the subretinal route. In some embodiments, the anti-VEGF x Ang-2 antibody composition is administered by subretinal injection or infusion. In some embodiments, the anti-VEGF x Ang-2 antibody composition is administered by subretinal injection, the injection comprising a volume of between 50 μl and 1000 μl. In some embodiments, the anti-VEGF x Ang-2 antibody composition is administered by subretinal injection, the injection comprising a volume of between 50 μl and 300 μl. In some embodiments, the anti-VEGF XAng-2 antibody composition is administered by subretinal injection comprising a volume of 100 μL or up to 100 μL (e.g., 25 μL-100 μL, 50 μL-100 μL, 75 μL-100 μL). In some embodiments, the subretinal injection comprises a two-step injection.

The anti-VEGF x Ang-2 antibodies described herein may be incorporated into a pharmaceutical composition for administration to a subject alone or in combination to treat or prevent CNV. Such compositions typically comprise an active agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds may also be incorporated into the compositions.

The pharmaceutical compositions are generally formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, intraperitoneal, or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoresis, and transmucosal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine tetraacetic acid; buffers such as acetate, citrate or phosphate; and agents for modulating tonicity, such as sodium chloride or dextrose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be packaged in ampules, disposable syringes or multiple dose vials made of glass or plastic. For the convenience of the patient or treating physician, the administration formulation may be provided in the form of a kit containing all the equipment (e.g., drug vials, diluent vials, syringes and needles) necessary for the treatment process (e.g., 7 day treatment).

Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL ^TM (BASF, parippany, n.j.) or Phosphate Buffered Saline (PBS). In some embodiments, the composition is sterile and should be fluid to the extent that easy injectability exists. It should be stable under the conditions of preparation and storage; and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

Pharmaceutical compositions having an anti-VEGF x Ang-2 antibody disclosed herein may comprise a carrier, which may be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. For example, proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like). Glutathione and other antioxidants may be included to prevent oxidation. In many cases, it will be advantageous to include isotonic agents, for example, sugars, polyalcohols (such as mannitol, sorbitol) or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by: the desired amount of active compound is incorporated with one or a combination of the ingredients listed above into an appropriate solvent as desired, and then filter sterilized. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, typical methods of preparation include vacuum drying and freeze-drying which techniques may yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The therapeutic agent may be formulated in a carrier system. The carrier may be a colloidal system. The colloidal system may be a liposome, phospholipid bilayer vehicle. In one embodiment, the therapeutic agent is encapsulated in a liposome while maintaining the structural integrity of the agent. Those skilled in the art will appreciate that there are a variety of methods for preparing liposomes. (see, lichtenberg et al, methods biochem. Anal., volume 33: pages 337-462 (1988); anselem et al, liposome Technology, CRC Press (1993)). Liposome formulations delay clearance and increase cellular uptake (see, reddy, ann. Pharmacothers., volume 34, stages 7-8: pages 915-923 (2000)). The active agent may also be loaded into particles prepared from pharmaceutically acceptable ingredients including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable or gastroretentive polymers or liposomes. Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles, and viral vector systems.

The carrier may also be a polymer, such as a biodegradable, biocompatible polymer matrix. In one embodiment, the therapeutic agent may be embedded in the polymer matrix while maintaining the structural integrity of the agent. The polymer may be natural, such as a polypeptide, protein or polysaccharide; or synthetic, such as poly-alpha-hydroxy acids. Examples include carriers made of, for example, collagen, fibronectin, elastin, cellulose acetate, nitrocellulose, polysaccharides, fibrin, gelatin, and combinations thereof. In one embodiment, the polymer is polylactic acid (PLA) or co-lactic acid/glycolic acid (PGLA). The polymer matrix can be prepared and isolated in a variety of forms and sizes, including microspheres and nanospheres. The polymer formulation may result in an extended duration of therapeutic effect. (see, reddy, ann. Pharmacothers., volume 34, stages 7-8: pages 915-923 (2000)). Polymeric formulations of human growth hormone (hGH) have been used in clinical trials. (see, kozarich and Rich, chemical Biology, volume 2: pages 548-552 (1998)).

Examples of polymeric microsphere sustained release formulations are described in PCT publication WO 99/15154 (Tracy et al), U.S. Pat. Nos. 5,674,534 and 5,716,644 (both to Zale et al), PCT publication WO 96/40073 (Zale et al) and PCT publication WO 00/38651 (Shah et al). U.S. patent nos. 5,674,534 and 5,716,644 and PCT publication WO 96/40073 describe polymer matrices containing erythropoietin particles that are stable against aggregation with salts.

In some embodiments, the therapeutic compound is prepared with carriers that will protect the therapeutic compound from rapid elimination from the body, such as controlled release formulations including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid may be used. Such formulations may be prepared using known techniques. These materials are also commercially available from, for example, alza Corporation and Nova Pharmaceuticals, inc. Liposomal suspensions (including liposomes targeted to specific cells with monoclonal antibodies to cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These materials may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

Therapeutic compounds may also be formulated to enhance intracellular delivery. For example, liposome delivery systems are known in the art, see, e.g., chonn and Cullis, "Recent Advances in Liposome Drug Delivery Systems", current Opinion in Biotechnology, volume 6: pages 698-708 (1995); weiner, "Liposomes for Protein Delivery: selecting Manufacture and Development Processes", immunomethods, volume 4, phase 3: pages 201 to 209 (1994) and Gregorian dis, "Engineering Liposomes for Drug Delivery: progress and Problems", trends Biotechnol., volume 13, 12: pages 527 to 537 (1995). Mizguchi et al, cancer lett, volume 100: methods for delivering proteins to cells both in vivo and in vitro using membrane fusion liposomes are described on pages 63-69 (1996).

The dose, toxicity and therapeutic efficacy of any therapeutic agent can be determined by standard pharmaceutical procedures in cell culture or experimental animals, for example, for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio of LD50/ED 50. Compounds exhibiting a high therapeutic index are advantageous. While compounds exhibiting toxic side effects may be used, care should be taken to design delivery systems that target such compounds to the affected tissue site in order to minimize potential damage to uninfected cells and thereby reduce side effects.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds may be in a range including circulating concentrations of ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any of the compounds used in these methods, a therapeutically effective dose can be estimated initially from the cell culture assay. The dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of test compound that achieves half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to accurately determine useful doses in humans. The level in plasma can be measured, for example, by high performance liquid chromatography.

In general, an effective amount of an anti-VEGF X Ang-2 antibody disclosed herein sufficient to achieve a therapeutic or prophylactic effect will range from about 0.000001mg per kilogram of body weight per day to about 10,000mg per kilogram of body weight per day. Suitably, the dosage range is from about 0.0001mg per kg of body weight per day to about 100mg per kg of body weight per day. For example, the dosage may be 1mg/kg body weight or 10mg/kg body weight per day, every two or three days, or in the range of 1mg/kg-10mg/kg per week, every two or three weeks. In one embodiment, the single dose of therapeutic compound ranges from 0.001 micrograms to 10,000 micrograms per kg body weight. In one embodiment, the concentration of the one or more anti-VEGF x Ang-2 antibodies in the carrier ranges from 0.2 micrograms to 2000 micrograms per milliliter delivered. Exemplary treatment regimens require administration once daily or once weekly. In therapeutic applications, it is sometimes desirable to administer relatively high doses at relatively short intervals until the progression of the disease is reduced or terminated, or until the subject exhibits a partial or complete improvement in the symptoms of the disease. Thereafter, a prophylactic regimen can be administered to the patient.

In one placeIn some embodiments, a therapeutically effective amount of an anti-VEGF X Ang-2 antibody may be defined as an inhibitor concentration of 10 at the target tissue ^-32 To 10 ^-6 Molar concentration, e.g. about 10 ^-7 Molar concentration. The concentration may be delivered by a systemic dose of 0.001mg/kg to 100mg/kg or an equivalent dose calculated as body surface area. The dosage regimen will be optimized to maintain therapeutic concentrations at the target tissue, such as by daily or weekly administration.

The skilled artisan will appreciate that certain factors may affect the dosage and time of administration required to effectively treat a subject, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Furthermore, treating a subject with a therapeutically effective amount of a therapeutic composition described herein can include a single treatment or a series of treatments.

The mammal treated according to the method of the invention may be any mammal, including for example farm animals such as sheep, pigs, cattle and horses; pet animals such as dogs and cats; experimental animals such as rats, mice and rabbits. In some embodiments, the mammal is a human.

Combination therapy

In some embodiments, one or more antibodies of the anti-VEGF x Ang-2 multispecific antibodies disclosed herein may be used in combination with one or more additional therapies to prevent or treat CNV. Additional therapies include laser photocoagulation, photodynamic therapy (PDT), sodium pipadatanib, bevacizumab, ranibizumab, aflibercept and corticosteroids.

In some embodiments, an anti-VEGF x Ang-2 multispecific antibody disclosed herein may be administered separately, sequentially or simultaneously with at least one additional therapy selected from laser photocoagulation, photodynamic therapy (PDT), pegaptanib sodium, bevacizumab, ranibizumab, albesipu, and corticosteroids.

In any case, the multiple therapies may be administered in any order or even simultaneously. If administered simultaneously, the multiple therapies may be provided in a single, unified form or in multiple forms (as a single bolus or as two separate boluses, for example only). One of the therapies may be administered in multiple doses, or both may be administered as multiple doses. If not administered simultaneously, the time of administration between doses may vary from more than zero weeks to at least four weeks. Furthermore, the combination methods, compositions and formulations are not limited to the use of only two agents.

Kit for detecting a substance in a sample

The present disclosure also provides kits comprising one or more of the anti-VEGF x Ang-2 multispecific antibodies disclosed herein and instructions for using the antibodies to prevent and/or treat CNV. Optionally, the above components of the kits of the present technology are packaged in suitable containers and labeled for the prevention and/or treatment of CNV.

The above components may be stored in unit-dose or multi-dose containers (e.g., sealed ampoules, vials, bottles, syringes, and test tubes), as an aqueous solution, preferably a sterile solution, or as a lyophilized formulation for reconstitution, preferably a sterile formulation. The kit may further comprise a second container containing a diluent suitable for diluting the pharmaceutical composition to a higher volume. Suitable diluents include, but are not limited to, pharmaceutically acceptable excipients for pharmaceutical compositions and saline solutions. Furthermore, the kit may comprise instructions for diluting the pharmaceutical composition and/or instructions for administering the pharmaceutical composition, whether diluted or not. The container may be made of a variety of materials such as glass or plastic and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The kit may further comprise further containers containing pharmaceutically acceptable buffers, such as phosphate buffered saline, ringer's solution and dextrose solution. The kit may also include other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, culture media for one or more of the appropriate hosts. The kit may optionally include instructions, typically included in commercial packaging of therapeutic or diagnostic products, that include information regarding, for example, indications, usage, dosages, manufacturers, administration, contraindications, and/or warnings regarding the use of such therapeutic or diagnostic products.

The kit may also comprise, for example, buffers, preservatives or stabilizers. The kit may also contain a control sample or a series of control samples, which can be assayed and compared to the test sample. Each component of the kit may be packaged in a separate container, and all of the individual containers may be in a single package, along with instructions for interpreting the results of the assays performed using the kit. The kits of the present technology may comprise written products on or in the kit containers. The written product describes how the reagents contained in the kit are used. In certain embodiments, the use of reagents may be in accordance with methods of the present technology.

Examples

The present technology is further illustrated by the following examples, which should not be construed as limiting in any way.

Example 1: pre-intravitreal injection risk management instructions

(1) Applying a local anesthetic; (2) Applying 5% or 10% povidone-iodine drops and/or periocular povidone-iodine eyelid formulations; (3) inserting a sterile speculum to separate the eyelid; (4) Povidone-iodine was reapplied at the injection site immediately prior to injection. See fig. 3.

Example 2: ABP201 toxicity and PK study design in rabbits

And (5) processing. Test article: the test article is provided in a ready-to-use form. The vehicle contained 10mM sodium citrate, 0.02% polysorbate 80 and 5.8% sucrose, pH 7.2. Administration: on day 0, rabbits received a single 50 μl dose (4 mg/mL ABP 201) or vehicle per eye via the intravitreal route. Fig. 4 shows the experimental design for studying toxicity and Pharmacokinetics (PK) of anti ANG-2 x VEGF bispecific antibody ABP201 in a rabbit model. Toxicity and PK results are shown in figures 5-11.

No inflammation was detected in ABP201 treated group and cleared 7 days after treatment. Comparing fig. 6-7 to fig. 5.

Group 1 (vehicle): there are 5-10 monocytes in the vitreous of two of the four eyes (right eye of each rabbit), which are located primarily near the inner surface of the retina. In addition, no other abnormalities were observed in vehicle treated rabbits.

Group 2 (OU ABP 201): no abnormalities were found in the vitreous of both eyes of one rabbit in this group. One eye (left eye) of the other rabbit in the group showed moderate diffuse vitreous mononuclear cell infiltration and its optic nerve showed moderate focal mononuclear cell infiltration. The vitreous of the other eye (right eye) has some (5-10) monocytes.

The overall histopathology between the vehicle-treated group and the ABP 201-treated group was comparable. Fig. 7-8.

Example 3: assessing ABP-201 activity in eyes using CNV disease model

The right eye of BN rats received a single 5. Mu.L intravitreal injection of three concentrations (0.0385. Mu.g/. Mu.L-low, 0.385. Mu.g/. Mu.L-medium, 3.85. Mu.g/. Mu.L-high) of ABP-201, vehicle (10 mM sodium citrate, 0.02% polysorbate 80, 5.8% sucrose) or Abelmoschus @0.2. Mu.g/. Mu.L). Four to five lesions of 200 μm size (100 ms,200 mw) were burned in two discs from the optic disc of each OD eye with a 532nm laser connected to a Micron III camera (Phoenix Labs) immediately followed by injection. Fundus imaging and optical coherence tomography (OCT; bioptigen) verify the presence, size and correct targeting of lesions. On days 7 and 15, fluorescein angiography and OCT assess lesion leakage and volume. Data are expressed as mean ± SEM and analyzed using one-way ANOVA. On study day 15, animals were sacrificed. Leakage data was assessed by expert readers and scored using custom ImageJ macros based on an established 0 (no leakage) -4 (very high leakage) experience scale. Lesion data was established by manually looking up a central OCT scan for each lesion and measuring lesion bottom (b) and height (h) size using calipers in biopteninsument software. Thereafter Using a semi-ellipsoidal formula (v=hb ² Pi/12) to estimate lesion volume. Data are expressed as mean ± SEM and analyzed using one-way ANOVA in Graphpad Prism. />

Fig. 14A-14B show representative angiographic and OCT images of experimental and control groups, respectively. On day 7, all three doses of ABP-201 caused a highly significant decrease in lesion volume (27%, 27% and 40%, all p < 0.01%, respectively) and leakage (33%, 35% and 37%, all p <0.0002, respectively) compared to vehicle. On day 15, medium and high doses of ABP-201 caused a significant reduction in lesion volume (26% and 27% respectively, both p < 0.05), and all three doses caused a significant reduction in leakage (28%, 33% and 36% respectively, all p < 0.05). ABP-201 performs similar to or better than aflibercept for both lesion volume and leakage. See fig. 12A-12B; fig. 15A-15B.

These results indicate that ANG-2 x VEGF bispecific antibodies of the disclosure are useful for reducing neovascularization and vascular leakage in laser induced CNV in vivo.

Example 4: tolerability and pharmacokinetic studies of novel compounds following Intravitreal (IVT) delivery in rabbits Study of

Animal health and acclimatization: animals were allowed to acclimate to the study environment for a period of at least 1 week prior to anesthesia. At the end of the acclimation period, the laboratory animal technician performs a physical examination of each animal to determine whether it is suitable for participation in the study. The examination includes skin and outer ear, eyes, abdomen, neural behavior and general physical condition. Animals with well-established health conditions were released for study participation.

Randomization and study identification: animals were assigned to study groups according to the Powered Research Standard Operating Program (SOP).

Test formulations and dosing: the test article is provided in a ready-to-use form and is intravitreally administered based on the experimental design.

Intravitreal injection: on day 0, animals were distended with 1.0% topiramate HCl and buprenorphine was given 0.01mg/kg-0.05mg/kg SQ. Rabbits were then sedated (ketamine/xylazine) for injection and eyes were aseptically treated with topical 5% povidone-iodine solution and then rinsed with sterile eyewash. Then, 0.5% procaine HCL and 10% phenylephrine HCL were topically applied. The conjunctiva was gently grasped with a colibri forceps and an injection was made 2mm-3mm posterior to the upper edge (through the ciliary body plane) using a 30G needle, with the needle pointed slightly posteriorly to avoid contact with the lens. After dispensing the syringe contents, the needle is slowly withdrawn. Both eyes received injections as shown in the experimental design table above. After injection, 1 drop of neomycin polymyxin B sulfate bacitracin ophthalmic solution or ofloxacin was topically applied to the ocular surface to allow the animal to wake up from anesthesia normally.

Eye examination: the veterinary ophthalmologist uses a slit-lamp biopsy microscope and indirect ophthalmoscope to perform a complete ocular examination of all animals to assess ocular surface morphology and anterior segment inflammation prior to dosing as a baseline and on additional days as indicated in the experimental design table. All animals must undergo normal ocular examination to be considered for participation in the study. Scoring was performed using the Hackett and McDonald eye grading system. Animals were not sedated at the time of examination. See, hackett, r.b. and McDonald, t.o., ophthalmic Toxicology and Assessing Ocular Irritation, dermotology, fifth edition, editions: marzulli and H.I. Maibach, washington, D.C.: hemisphere Publishing Corporation,1996, pages 299-305 and 557-566.

Tonometry: intraocular pressure (IOP) was measured in both eyes at the time points shown in the study design sheet and in all surviving animals. The baseline measurements were performed on awake animals using a Tonovet probe (icae Tonometer, espoo, finland) without the use of local anesthetics. The tip of the Tonovet probe is directed to gently contact the central cornea. The average IOP shown on the display was recorded and three measurements were made.

Blood collection: at the times indicated in the experimental design table, approximately 1mL of whole blood was aspirated by cardiac puncture (or other suitable vein/artery) into 1.3mL (Sarsredt reference number 41.1392.105 or similar product) of plastic red-top evacuated blood collection tube (without anticoagulant or serum separation gel) for serum collection. After collection, the tube was gently mixed by inverting the tube 3-5 times. The blood sample is stored at room temperature for a period of at least 30 minutes but less than 60 minutes prior to treatment. The samples were centrifuged in a refrigerated centrifuge at 10,000Xg for 10 minutes at 4 ℃. Immediately after centrifugation, the clarified serum was transferred to a pre-labeled 2mL frozen polypropylene tube and stored frozen at-80 ℃ until shipment to analytical analysis. If the erythrocytes were inadvertently aspirated into the serum, the sample was immediately re-centrifuged. Each aliquot is marked with the following information: study number, animal number, group number, matrix, time point, and date of collection.

Euthanasia/tissue collection: after the final examination, animals were sedated with 50/10mg/kg IM ketamine/xylazine and blood was collected on the days shown in the experimental design table. The animals were then euthanized by IV administration of excess sodium pentobarbital, followed by auscultation to ensure death.

Eyes designated for PK analysis: after euthanasia, both eyes are enucleated. Aqueous humor from both eyes was removed by syringe No. 27 or 30, transferred to pre-weighed polypropylene tubing, and weighed to determine tissue weight. The samples were then snap frozen by immersing them in liquid nitrogen. The eye tissue was dissected in a frozen state according to Powered Research SOP. All samples were placed in separate vials and weighed. The samples were stored at-80 ℃ until homogenized. List of tissues collected: serum and aqueous humor (2 mL polypropylene spiral cap tube); vitreous humor (7 mL pre-cell homogenization tube); retina/choroid/RPE (2 mL pre-cell lys homogenizing tube)

Histological: immediately after euthanasia and confirmation of death, both eyes of animals designated for histology were enucleated and fixed in Davidson solution for 24 hours, then with alcohol, as previously described. The central portion of each sphere (including the optic nerve) was stained with hematoxylin and eosin and examined using an optical microscope.

FIGS. 16-18 show analysis of the fluorescence of a cumulative region of interest (ROI) in vivo over time with anti-ANG-2 XVEGF bispecific antibody ABP201 (represented by SEQ ID NO:1 and SEQ ID NO: 2). Vehicle and aflibercept were used as negative and positive controls, respectively.

ABP-201 bispecific antibodies perform comparable to aflibercept. These results indicate that ANG-2 x VEGF bispecific antibodies of the disclosure are useful for treating CNV.

Equivalents (Eq.)

The present technology is not limited to the specific embodiments described in this application, which are intended as single illustrations of various aspects of the technology. It will be apparent to those skilled in the art that many modifications and variations can be made to the present technology without departing from the spirit and scope of the technology. Functionally equivalent methods and apparatus within the scope of the technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that the present technology is not limited to particular methods, reagents, compound compositions, or biological systems, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Furthermore, where features or aspects of the present disclosure are described in terms of markush groups, those skilled in the art will recognize that the present disclosure is thereby also described in terms of any individual member or subgroup of members of the markush group.

As will be understood by those skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be readily identified as sufficiently descriptive and capable of decomposing the same range into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each of the ranges discussed herein can be readily broken down into a lower third, a middle third, an upper third, and the like. As will also be appreciated by those of skill in the art, all language such as "at most", "at least", "greater than", "less than", and the like, include the recited numbers and refer to ranges that may be subsequently broken down into subranges as described above. Finally, as will be appreciated by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 units refers to a group having 1, 2, or 3 units. Similarly, a group having 1-5 units refers to a group having 1, 2, 3, 4, or 5 units, and so forth.

All patents, patent applications, provisional applications, and publications mentioned or cited herein are incorporated by reference in their entirety, including all figures and tables, so long as they do not inconsistent with the explicit teachings of this specification.

Claims

1. A method for treating Choroidal Neovascularization (CNV) in a subject in need thereof, said method comprising administering to said subject an effective amount of an anti-ANG-2 x VEGF multispecific antibody, wherein said anti-ANG-2 x VEGF multispecific antibody comprises a heavy chain sequence and a light chain sequence selected from the group consisting of seq id nos: SEQ ID NO. 1 and SEQ ID NO. 2; SEQ ID NO. 5 and SEQ ID NO. 6; and

SEQ ID NO 9 and SEQ ID NO 10.

2. A method for treating Choroidal Neovascularization (CNV) in a subject in need thereof, said method comprising administering to said subject an effective amount of an anti-ANG-2 XVEGF multispecific antibody, wherein said anti-ANG-2 XVEGF multispecific antibody comprises a first antigen-binding portion that binds to a VEGF epitope and a second antigen-binding portion that binds to an ANG-2 epitope,

wherein the first antigen binding portion comprises a first heavy chain immunoglobulin variable domain (V _H ) And a first light chain immunoglobulin variable domain (V _L ) And the second antigen binding portion comprises a second V _H And a second V _L ；

Wherein the first V _H Comprising SEQ ID25 or SEQ ID NO. 44 and said first V _L An amino acid sequence comprising SEQ ID NO. 27; and is also provided with

Wherein the second V _H Comprising the amino acid sequence of SEQ ID NO. 26 or SEQ ID NO. 45; and the second V _L Comprising the amino acid sequence of SEQ ID NO. 28.

3. The method of claim 2, wherein the anti-ANG-2 x VEGF multispecific antibody comprises an immunoglobulin and an scFv.

4. The method of claim 3, wherein the scFv comprises the second antigen binding portion.

5. The method of any one of claims 1-4, wherein the choroidal neovascularization is caused by age-related macular degeneration (AMD), pathologic Myopia (PM), inflammation, polypoidal chorioretinopathy, or central serous chorioretinopathy.

6. The method of any one of claims 1 to 5, wherein the subject has been diagnosed as having CNV.

7. The method of claim 6, wherein the sign or symptom of CNV comprises one or more of: distortion or waving of central vision or appearance of gray/black/plaques on central vision, bleeds or bleeds on the retina, loss of brightness of color or different color appearance on each eye, vision deformity, painless vision loss, paracenter or central dark spots, different size of objects present on each eye, glints or glints in central vision, vision loss due to fluid exudation in or under the retina, bleeding or macular fibrosis.

8. The method of any one of claims 1 to 7, wherein the subject exhibits an increased expression level and/or an increased activity of VEGF and/or Ang-2.

9. The method of any one of claims 1 to 8, further comprising administering one or more additional therapies to the subject separately, sequentially or simultaneously.

10. The method of claim 9, wherein the one or more additional therapies are selected from laser photocoagulation, photodynamic therapy (PDT), sodium pipadatanib, bevacizumab, ranibizumab, aflibercept, and corticosteroids.

11. The method of any one of claims 1 to 10, wherein the anti-ANG-2 x VEGF multispecific antibody is administered by topical, intravitreal, intraocular, subretinal or subscleral administration.

12. The method of claim 11, wherein sub-scleral administration is achieved by implanting a slow release sub-scleral implant into the subject.

13. The method of any one of claims 1 to 12, wherein administration of the anti-ANG-2 x VEGF multispecific antibody results in a reduction of neovascular lesion formation and/or vascular leakage in the subject.

14. The method of any one of claims 1 to 13, wherein the subject is a human.

15. The method of any one of claims 1 to 14, wherein the subject does not exhibit ocular inflammation 1 week after administration of the anti-ANG-2 x VEGF multispecific antibody.