WO2022179432A1

WO2022179432A1 - Antibody fusion proteins targeting il-6 receptor and angiogenic factors

Info

Publication number: WO2022179432A1
Application number: PCT/CN2022/076673
Authority: WO
Inventors: Jinzhong Zhang; Wei Yong SHEN; Charles Semba; John Zhenze HU; Zhen Qin XIA
Original assignee: Eluminex Biosciences (Suzhou) Limited
Priority date: 2021-02-27
Filing date: 2022-02-17
Publication date: 2022-09-01
Also published as: AU2022227047A1; KR20230150858A; EP4298127A1; TW202246359A; CA3211803A1; CN116897165A; JP2024507397A

Abstract

An antibody fusion protein comprises an antibody against IL-6 receptor or an antigen-binding fragment thereof linked to a VEGF binding unit comprising a plurality of Ig-like domains of a first VEGF receptor, a second human VEGF receptor, and/or a third human VEGF receptor. The antibody fusion protein is useful in treating or controlling an ocular disease, condition, or disorder that has etiology in aberrant angiogenesis or inflammation.

Description

ANTIBODY FUSION PROTEINS TARGETING IL-6 RECEPTOR AND ANGIOGENIC FACTORS

BACKGROUND OF THE INVENTION

The present invention relates to antibody fusion proteins targeting interleukin-6 receptor ( “IL-6R” ) and angiogenic factors. In particular, the present invention relates to antibody fusion proteins targeting IL-6R and members of the family of vascular endothelial growth factors ( “VEGF family members” ) . More particular, the present invention relates to such fusion proteins, their uses, and processes for production.

The major cellular components of the mammalian vascular system are the endothelium, smooth muscle cells, and pericytes. Endothelial cells form the lining of the inner surface of all blood vessels in the mammal and constitute a non-thrombogenic interface between blood and tissue. Therefore, the proliferation of endothelial cells is an important component for the development of new capillaries and blood vessels which, in turn, is a necessary process for the growth and/or regeneration of mammalian tissues.

A family of secreted polypeptides has been shown to play an extremely important role in promoting endothelial cell proliferation and angiogenesis. A pathological feature of uncontrolled angiogenesis caused by VEGF over-expression is increased vascular permeability, which results in fluid leakage into, and swelling of, the surrounding tissues. In mammals, this family consists of five related growth factors having highly conserved receptor-binding structure: vascular endothelial growth factors A-D ( “VEGF-A, ” “VEGF-B, ” “VEGF-C, ” and “VEGF-D” ) and placental growth factor ( “PlGF” ) . In this disclosure, this family of growth factors is also referred to as the VEGF family.

The cytokine interleukin-6 ( “IL-6” ) plays an important role in host defense against environmental stress such as infection and injury. Under physiological conditions, IL-6 is barely detectable, but its levels can increase more than 100,000-fold during early phase of inflammation. However, not all occurrences of IL-6 stimulation are beneficial. Dysregulated, persistent production of IL-6 has been implicated in the development of various autoimmune, chronic inflammatory diseases. There has been evidence that unchecked production of IL-6 in inflamed tissues induces excess production of VEGF-A and VEGF-C.

The VEGF-family growth factors act through a family of cognate receptor tyrosine kinases, which exist only on the surface of vascular endothelial cells, to stimulate formation of blood vessels: VEGF receptor-1 ( “VEGFR-1, ” also known as “flt-1” ) , VEGF receptor-2 ( “VEGFR-2, ” also known as “KDR” in humans and “flk-1” in mice) , VEGF receptor-3 ( “VEGFR-3, ” also known as “flt-4” ) .

VEGF-A (also sometimes simply referred to as VEGF) has emerged as the most important member of this family of growth factors. Human VEGF-A is expressed in a variety of tissues as multiple homodimeric forms (121, 145, 165, 183, 189 and 206 amino acids per monomer) , wherein each form arises as a result of alternative splicing of a single RNA transcript.

Since VEGFs promote vascular endothelial cell proliferation and angiogenesis, they may be useful for the therapeutic treatment of numerous conditions in which a growth-promoting activity on the vascular endothelial cells is beneficially important; for example, in treatment of ulcers, vascular injuries, and myocardial infarction.

In contrast, however, while vascular endothelial proliferation is desirable under certain circumstances, vascular endothelial proliferation and angiogenesis are also undesirable components of a variety of diseases and disorders including tumor growth and metastasis, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic retinopathy, retrolental fibroplasia, neovascular glaucoma, neovascular age-related macular degeneration, hemangiomas, immune rejection of transplanted corneal tissue and other tissues, and chronic inflammation. In individuals suffering from any of these disorders, one would want to inhibit, or at least substantially reduce, the endothelial proliferating activity of VEGFs.

Each of flt-1, KDR, and flt-4 tyrosine kinase receptors has seven extracellular immunoglobulin-like ( “Ig-like” ) domains that are available for ligand binding, a transmembrane domain that serves to anchor the receptor on the surface of cells in which it is expressed and an intracellular catalytic tyrosine kinase domain. Flt-1 binds VEGF-A, VEGF-B, and PlGF. KDR binds VEGF-A, VEGF-C, and VEGF-D. Flt-4 binds VEGF-C and VEGF-D.

In view of the role of the growth factors of the VEGF family in vascular endothelial proliferation and angiogenesis, and the role that these processes play in many different diseases and disorders, treatments have been devised that target the control of these growth factors. However, anti-VEGF therapy alone has not been able to block completely the progression of angiogenic diseases.

Therefore, it is desirable to have a pharmacological means for more completely reducing or inhibiting one or more of the biological activities of these growth factors in patients whose pathological conditions are rooted in aberrant angiogenesis. It is also desirable to have a pharmacological means for improved treatment or control of pathological conditions that are rooted in aberrant angiogenesis.

SUMMARY OF THE INVENTION

As used herein, the term “control” also includes reduction, alleviation, amelioration, or prevention.

In general, the present invention provides antibody fusion or chimeric proteins, methods of producing and compositions comprising the same, and methods for treating or controlling at least a pathological condition in a subject, which condition has etiology in aberrant angiogenesis. In this disclosure the terms “fusion protein” and “chimeric protein” are used interchangeably.

In one aspect, the present invention provides antibody fusion or chimeric proteins or antigen-binding fragments or antigen-binding domains thereof that are capable of binding substantially to IL-6R, including either the membrane-bound or the soluble form of IL-6R, and one or more VEGF family members; thereby, concurrently reducing or inhibiting both IL-6 and VEGF family member signaling transduction.

In another aspect, an antibody fusion or chimeric protein of the present invention comprises an antibody against IL-6R or an antigen-binding fragment or domain thereof linked to a VEGF binding unit that comprises an Ig-like domain selected from the group consisting of Ig-like domains of a first VEGF receptor, a second VEGF receptor, a third VEGF receptor, and combinations thereof. A VEGF binding unit, as used herein, comprises a polypeptide that binds to, or substantially to, a VEGF family member. Thus, in one aspect, an antibody fusion protein of the present invention may be viewed at least as a bispecific construct that can bind to two different ligands. In some embodiments, said VEGF binding unit comprises a plurality of Ig-like domains of one or more VEGF receptors.

In still another aspect, said IL-6R is human IL-6R.

In still another aspect, an antibody fusion or chimeric protein of the present invention comprises an antibody against IL-6R or an antigen-binding fragment or domain thereof; wherein at least one of: (1) a light chain or an antigen-binding fragment or domain thereof and (2) a heavy chain or an antigen-binding fragment or domain thereof is linked to a VEGF binding unit that comprises a plurality of Ig-like domains selected from the group consisting of those of a first VEGF receptor, a second VEGF receptor, a third VEGF receptor, and combinations thereof.

In still another aspect, said VEGF binding unit comprises: (1) (a) Ig-like domain 2, or substantially Ig-like domain 2, of human VEGFR-1 (or flt-1) ; and (b) Ig-like domain 3, or substantially Ig-like domain 3, of human VEGFR-2 (or KDR) ; or (2) at least an Ig-like domain selected from the group consisting of Ig-

like domains

1, 2, and 3, or substantially Ig-

like domains

1, 2, and 3, of human VEGFR-3 (or flt-4) .

In yet another aspect, each of the light chains or an antigen-binding fragment or domain thereof and the heavy chains or an antigen-binding fragment or domain thereof of the antibody against IL-6R or an antigen-binding thereof is linked to a VEGF binding unit comprising: (a) Ig-like domain 2, or substantially Ig-like domain 2, of human VEGFR-1 (or flt-1) ; and (b) Ig-like domain 3, or substantially Ig-like domain 3, of human VEGFR-2 (or KDR) .

In yet another aspect, each of the light chains or an antigen-binding fragment or domain thereof of the antibody against IL-6R or an antigen-binding thereof is linked to a first VEGF binding unit comprising: (a) Ig-like domain 2, or substantially Ig-like domain 2, of human VEGFR-1 (or flt-1) ; and (b) Ig-like domain 3, or substantially Ig-like domain 3, of human VEGFR-2 (or KDR) ; and each of the heavy chains or an antigen-binding fragment or domain thereof of the antibody against IL-6R or an antigen-binding thereof is linked to a second VEGF binding unit comprising: (a) Ig-

like domains

1 and 2, or substantially Ig-

like domains

1 and 2, of human VEGFR-3; or (b) Ig-

like domains

2 and 3, or substantially Ig-

like domains

2 and 3, of human VEGFR-3; or Ig-

like domains

1, 2, and 3, or substantially Ig-

like domains

1, 2, and 3, of human VEGFR-3.

In yet another aspect, each of the heavy chains or an antigen-binding fragment or domain thereof of the antibody against IL-6R is linked to a VEGF binding unit selected from the group consisting of: (1) a first VEGF binding unit comprising (a) Ig-like domain 2, or substantially Ig-like domain 2, of human VEGFR-1 (or flt-1) ; and (b) Ig-like domain 3, or substantially Ig-like domain 3, of human VEGFR-2 (or KDR) ; (2) a second VEGF binding unit comprising Ig-

like domains

1 and 2, or substantially Ig-

like domains

1 and 2, of human VEGFR-3 (or flt-4) ; and (3) combinations of said first and second VEGF binding units.

In still another aspect, the present invention provides an isolated nucleic acid molecule encoding at least one of said antibody fusion or chimeric protein.

In still another aspect, the present invention provides a vector that comprises said nucleic acid molecule, including an expression vector comprising said nucleic molecule operatively linked to an expression control sequence. As used herein, the phrase “operatively linked” refers to components of a construct being placed in a functional relationship with each another and each component retaining its function. A nucleic acid is “operatively linked” when it is placed in a functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretory leader is “operatively linked” to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer is operatively linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to facilitate translation.

In still another aspect, the present invention provides a host-vector system for the production of said antibody fusion or chimeric protein that comprises the expression vector in a suitable host cell.

In another aspect, the present invention provides a method of producing an antibody fusion or chimeric protein, which method comprises: (a) growing cells of the host- vector system under conditions permitting production of the antibody fusion or chimeric protein; and (b) recovering the antibody fusion or chimeric protein so produced.

In still another aspect, the present invention provides a method for treating or controlling at least a disease, condition, or disorder, in a subject, which has etiology in a condition selected from the group consisting of aberrant angiogenesis, inflammation, and combinations thereof.

In yet another aspect, the present invention provides a method for treating or controlling at least a disease, condition, or disorder, in a subject, which has etiology in aberrant angiogenesis and/or inflammation. In certain embodiments, such disease, condition, or disorder is an ocular disease, condition, or disorder. In certain other embodiments, such disease, condition, or disorder involves tumor growth and metastasis. In other embodiments, such disease, condition, or disorder is rheumatoid arthritis, psoriasis, or atherosclerosis.

Other features and advantages of the present invention will become apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a schematic diagram of a first and a second embodiment of an antibody fusion protein of the present invention, comprising an antibody against human IL-6R, each of the light chain and the heavy chain of which is linked through a flexible linker to a VEGF binding unit comprising Ig-like domain 2 of VEGFR-1 and Ig-like domain 3 of VEGFR-2.

Figure 2 shows a schematic diagram of a third and a fourth embodiment of an antibody fusion protein of the present invention, comprising an antibody against human IL-6R, the light chain of which is linked through a flexible linker to a first VEGF binding unit comprising Ig-like domain 2 of VEGFR-1 and Ig-like domain 3 of VEGFR-2, and the heavy chain of which is linked through a flexible linker to a second VEGF binding unit comprising Ig-

like domains

2 and 3 of VEGFR-3.

Figure 3 shows a schematic diagram of a fifth embodiment of an antibody fusion protein of the present invention, comprising scFvs of an antibody against human IL-6R, each variable domain of the heavy chains of which is linked to a Fc domain which is in turn linked through a flexible linker to a VEGF binding unit comprising Ig-like domain 2 of VEGFR-1 and Ig-like domain 3 of VEGFR-2.

Figure 4 shows a schematic diagram of a sixth embodiment of an antibody fusion protein of the present invention, comprising scFvs of an antibody against human IL-6R, each variable domain of the heavy chains of which is linked through a flexible linker to a VEGF binding unit comprising Ig-like domain 2 of VEGFR-1 and Ig-like domain 3 of VEGFR-2, which in turn is linked through a flexible linker to a Fc domain.

Figure 5 shows a schematic diagram of a seventh embodiment of an antibody fusion protein of the present invention, comprising scFvs of an antibody against human IL-6R, each variable domain of the heavy chains of which is linked through a flexible linker to a first VEGF binding unit comprising Ig-like domain 2 of VEGFR-1 and Ig-like domain 3 of VEGFR-2, which in turn is linked through a flexible linker to a Fc domain, which in turn is linked through a flexible linker to a second VEGF binding unit comprising Ig-

like domains

1 and 2 of VEGFR-3.

Figure 6 shows a schematic diagram of an eighth embodiment of an antibody fusion protein of the present invention, comprising scFvs of an antibody against human IL-6R, each variable domain of the heavy chains of which is linked through a flexible linker to a first VEGF binding unit comprising Ig-like domain 2 of VEGFR-1 and Ig-like domain 3 of VEGFR-2, and a second VEGF binding unit comprising Ig-

like domains

1 and 2 of VEGFR-3, which in turn is linked through a flexible linker to a Fc domain.

Figure 7A-D show molecular designs of six antibody fusion proteins (B781401-781406) of the present invention.

Figure 8 shows molecular designs of fusion proteins EB-DJ1, EB-vvA, EB-vvB, and EB-vvC of the present invention.

Figure 9 shows an example of SEC-HPLC chromatograms for the purification of B781401 that was expressed in HEK293 cells.

Figure 10 shows an example of SEC-HPLC chromatograms for the purification of B781401 that was expressed in CHO cells

Figure 11 shows a comparison of the binding affinities of B781401-781406 to that of aflibercept for recombinant human VEGF-A ₁₆₅ using enzyme linked immunosorbent assay (ELISA) .

Figure 12 shows a comparison of the ELISA binding affinities of B781401-781406 to that of aflibercept for recombinant human VEGF-B ₁₆₇.

Figure 13 shows a comparison of the ELISA binding affinities of B781401-781406 to that of aflibercept for recombinant human PlGF.

Figure 14 shows a comparison of the ELISA binding affinities of B781401-781406 to that of tocilizumab for recombinant humanIL-6R.

Figure 15 shows a comparison of the effect of B781401-781406 to that of aflibercept on inhibiting VEGF-A ₁₆₅-mediated VEGFR-2 signaling in VEGFR-2-NFAT-RE luciferase reporter cells.

Figure 16A-B show a comparison of the effect of B781401-781406 to that of tocilizumab on inhibiting IL-6R signaling.

Figure 17 shows a comparison of the effect of B781401-781406 to that of siltuximab and tocilizumab on inhibiting IL-6R signaling.

Figure 18 shows a comparison of the ELISA binding affinities of EB-DJ1, EB-vvA, EB-vvB, and EB-vvC to those of aflibercept, B781403, and B781405, for recombinant human VEGF-A ₁₆₅.

Figure 19 shows a comparison of the ELISA binding affinities of EB-DJ1, EB-vvA, EB-vvB, and EB-vvC to those of aflibercept, B781402, B781403, and B781405, for recombinant human VEGF-B ₁₆₇.

Figure 20 shows a comparison of the ELISA binding affinities of EB-DJ1, EB-vvA, EB-vvB, and EB-vvC to those of aflibercept, B781403, and B781405, for recombinant human PlGF.

Figure 21 shows a comparison of the ELISA binding affinities of EB-DJ1, EB-vvA, EB-vvB, and EB-vvC to those of tocilizumab, B781402, B781403, and B781405, for recombinant humanIL-6R.

DETAILED DESCRIPTION OF THE INVENTION

In general, the present invention provides an antibody fusion or chimeric protein or an antigen-binding fragment or domain thereof that is capable of binding substantially to IL-6R and one or more VEGF family members; thereby, reducing or inhibiting both IL-6 and VEGF family member signaling transduction. In this disclosure, the term “antibody fusion protein” is sometimes used in place of “antibody fusion or chimeric protein. ” In one aspect, an antibody fusion protein or an antigen-binding fragment thereof is capable of binding substantially to IL-6R and one or more VEGF family members; thereby, reducing or inhibiting signaling transduction of IL-6 and VEGF family members.

In another aspect, an antibody fusion or chimeric protein of the present invention comprises an antibody against IL-6R or an antigen-binding fragment or domain thereof linked to a VEGF binding unit that comprises a plurality of Ig-like domains selected from the group consisting of those of a first VEGF receptor, a second VEGF receptor, and a third VEGF receptor.

An antibody fusion protein of the present invention has advantages in that it can substantially bind, and thereby inhibit the action of, both membrane-bound IL-6R and soluble IL-6R. It can inhibit all three signaling pathways including classic, trans-signaling and trans-presentation pathways while an antibody fusion protein including an IL-6 antibody can only inhibit the free form of IL-6.

In still another aspect, said IL-6R is human IL-6R.

In yet another aspect, said VEGF receptors are human VEGF receptors.

In still another aspect, an antibody fusion or chimeric protein of the present invention comprises an antibody against IL-6R or an antigen-binding fragment or domain thereof; wherein one of: (1) at least a light chain or an antigen-binding fragment or domain thereof and (2) a heavy chain or an antigen-binding fragment or domain thereof is linked to a VEGF binding unit that comprises an Ig-like domain selected from the group consisting of those of a first VEGF receptor, a second VEGF receptor, and a third VEGF receptor. In certain embodiments, such VEGF binding unit comprises a plurality of Ig-like domains selected from the group consisting of those of a first VEGF receptor, a second VEGF receptor, and a third VEGF receptor. In one embodiment, an antibody fusion or chimeric protein of the present invention comprises an antibody against IL-6R or an antigen-binding fragment or domain thereof; wherein each of a light chain or an antigen-binding fragment or domain thereof and a heavy chain or an antigen-binding fragment or domain thereof is linked to a VEGF binding unit that comprises a plurality of Ig-like domains selected from the group consisting of those of a first VEGF receptor, a second VEGF receptor, and a third VEGF receptor.

like domains

1, 2, 3, and combinations thereof of VEGFR-3 (or flt4) , or the group consisting of domains substantially the same as said Ig-like domains of VEGFR-3. In one embodiment, the VEGF binding unit comprises: (a) Ig-

like domains

1 and 2, or substantially Ig-

like domains

1 and 2; or (b) Ig-

like domains

2 and 3, or substantially Ig-

like domains

2 and 3; or (c) Ig-

like domains

1, 2 and 3, or substantially Ig-

like domains

1, 2, and 3 of human VEGFR-3.

In still another aspect, the light chains or an antigen-binding fragment or domain thereof of the antibody against IL-6R or an antigen-binding thereof is linked to a first VEGF binding unit comprising: (a) Ig-like domain 2, or substantially Ig-like domain 2, of human VEGFR-1 (or flt-1) ; and (b) Ig-like domain 3, or substantially Ig-like domain 3, of human VEGFR-2 (or KDR) ; and the heavy chains or an antigen-binding fragment or domain thereof of the antibody against IL-6R or an antigen-binding thereof is linked to a second VEGF binding unit comprising Ig-

like domains

1 and 2, or substantially Ig-

like domains

1 and 2, of human VEGFR-3 (or flt-4) . In one embodiment, the heavy chains or an antigen-binding fragment or domain thereof of the antibody against IL-6R or an antigen-binding thereof is linked to a second VEGF binding unit comprising Ig-

like domains

2 and 3, or substantially Ig-

like domains

2 and 3, of human VEGFR-3 (or flt-4) .

As disclosed herein, an “antigen-binding fragment or domain” of a light chain or a heavy chain of an antibody refers to a fragment or portion of such antibody that comprises a complementarity determining region ( “CDR” ) of a variable domain of such antibody. In one embodiment, an antigen-binding fragment or domain of such antibody comprises a variable domain of the light chain or the heavy chain of such antibody. An antigen-binding fragment or domain of a heavy chain may further comprise a Fc domain. Other non-limiting examples of antibody fragments or domains include single-domain antibody (sdAb, also known as nanobody) , minibody, diabody, tribody, scFv-Fc antibody, (Fab’) ₂, and others known in the art. Non-limiting examples of sdAbs are nanobodies against human IL-6R as disclosed in US Patent 10,618,964; more specifically, vobarilizumab, of which an amino acid sequence is listed below as SEQ ID NO: 73.

In one aspect, an antibody fusion protein of the present invention comprises: (1) a heavy chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 34, 38, 42, 46, 78, 82, 86, 90, 94, and 98; (1) a light chain having an amino acid sequence selected from the group consisting of SEQ ID NO: 36, 40, 44, 48, 80, 84, 88, 92, 96, and 100.

In embodiment 5, an antibody fusion protein of the present invention comprises a pair or a dimer of IL-6R single chain variable fragments ( “IL-6R scFvs” ) , each of which is linked sequentially to a Fc domain of IgG1 and a VEGF binding unit that comprises: (a) Ig-like domain 2, or substantially Ig-like domain 2, of human VEGFR-1 (or flt-1) ; and (b) Ig-like domain 3, or substantially Ig-like domain 3, of human VEGFR-2 (or KDR) . The amino acid sequence of this embodiment is listed in SEQ ID NO: 60. The nucleic acid sequence of this embodiment is listed in SEQ ID NO: 59. In one aspect, said IgG1 is human IgG1.

In embodiment 6, an antibody fusion protein of the present invention comprises a pair or dimer of IL-6R scFvs; wherein the carboxy terminus (C-terminus) of each IL-6R scFv is linked to a VEGF binding unit that comprises: (a) Ig-like domain 2, or substantially Ig-like domain 2, of human VEGFR-1 (or flt-1) ; and (b) Ig-like domain 3, or substantially Ig-like domain 3, of human VEGFR-2 (or KDR) ; and the carboxy terminus of each VEGF binding unit is linked to a Fc domain of IgG1. The amino acid sequence of this embodiment is listed in SEQ ID NO: 62. The nucleic acid sequence of this embodiment is listed in SEQ ID NO: 61. In one aspect, said IgG1 is human IgG1.

In embodiment 7, an antibody fusion protein of the present invention comprises a pair or dimer of IL-6R scFvs; wherein the carboxy terminus of each IL-6R scFv is linked to a first VEGF binding unit that comprises: (a) Ig-like domain 2, or substantially Ig-like domain 2, of human VEGFR-1 (or flt-1) ; and (b) Ig-like domain 3, or substantially Ig-like domain 3, of human VEGFR-2 (or KDR) ; the carboxy terminus of each first VEGF binding unit is linked to a Fc domain of IgG1; and the carboxy terminus of the Fc domain is linked to a second VEGF binding unit that comprises Ig-

like domains

1 and 2, or substantially Ig-

like domains

1 and 2, of human VEGFR-3 (or flt-4) . The amino acid sequence of this embodiment is listed in SEQ ID NO: 64. The nucleic acid sequence of this embodiment is listed in SEQ ID NO: 63.

In embodiment 8, an antibody fusion protein of the present invention comprises a pair or dimer of IL-6R scFvs; wherein the carboxy terminus of each IL-6R scFv is linked to a first VEGF binding unit that comprises: (a) Ig-like domain 2, or substantially Ig-like domain 2, of human VEGFR-1 (or flt-1) ; and (b) Ig-like domain 3, or substantially Ig-like domain 3, of human VEGFR-2 (or KDR) ; the carboxy terminus of each first VEGF binding unit is linked to a second VEGF binding unit that comprises Ig-

like domains

1 and 2, or substantially Ig-

like domains

1 and 2, of human VEGFR-3 (or flt-4) ; and the carboxy terminus of the second VEGF binding unit is linked to a Fc domain of IgG1. The amino acid sequence of this embodiment is listed in SEQ ID NO: 66. The nucleic acid sequence of this embodiment is listed in SEQ ID NO: 65.

Embodiment 9 is similar to embodiment 5, except that the Fc domain is linked to the VEGF binding unit by a linker having a different length. The amino acid sequence of this embodiment is listed in SEQ ID NO: 74. The nucleic acid sequence of this embodiment is listed in SEQ ID NO: 75.

In embodiment 10, an antibody fusion protein of the present invention comprises a pair or dimer of IL-6R scFvs; wherein the amino terminus (N-terminus) of each IL-6R scFv is linked to the carboxy terminus (C-terminus) of a Fc domain of IgG1, the amino terminus of which is linked to a VEGF binding unit that comprises: (a) Ig-like domain 2, or substantially Ig-like domain 2, of human VEGFR-1 (or flt-1) ; and (b) Ig-like domain 3, or substantially Ig-like domain 3, of human VEGFR-2 (or KDR) . The amino acid sequence of this embodiment is listed in SEQ ID NO: 76. The nucleic acid sequence of this embodiment is listed in SEQ ID NO: 77.

In any of the foregoing embodiments, a VEGF binding unit may be linked to a scFv, a Fc domain, or another VEGF binding unit either directly or through a linker. It is preferable that a VEGF binding unit is linked to a scFv, a Fc domain, or another VEGF binding unit through a linker.

In still another embodiment, an antibody fusion protein of the present invention comprises a (Fab’) ₂ fragment of the antibody against IL-6R, wherein each of the light chains and heavy chains is linked to a VEGF binding unit.

In another embodiment, an antibody fusion protein of the present invention comprises a minibody of the antibody against IL-6R, wherein each of the light chains and heavy chains is linked to a VEGF binding unit. Such heavy chains may include the Fc region.

In one embodiment, said antibody against human IL-6R ( “hIL-6R” ) comprises an antibody known as tocilizumab, which is described, for example, in U.S. Patent 10,323,095; 7,479,543; and 5,795,965.

In one aspect, a heavy chain of an antibody fusion protein of the present invention comprises heavy-chain complementarity determining regions CDR1, CDR2, and CDR3 ( “HCCDR1, ” “HCCDR2, ” and “HCCDR3” ) listed in SEQ ID NO: 53, 54, and 55.

In another aspect, a light chain of an antibody fusion protein of the present invention comprises light-chain complementarity determining regions CDR1, CDR2, and CDR3 ( “LCCDR1, ” “LCCDR2, ” and “LCCDR3” ) listed in SEQ ID NO: 56, 57, and 58.

Other antibodies against hIL-6R may be used in an antibody fusion protein of the present invention; such as sarilumab, described in U.S. Patent 7,582,298; or satralizumab, described in U.S. Patent 8,562,991. In some embodiments, an antibody fusion protein of the present invention may also be generated by linking the heavy chains and light chains of sarilumab (SEQ ID NO: 49 and 50) or satralizumab (SEQ ID NO: 51 and 52) to VEGF binding units, as described herein. For example, a heavy chain and a light chain of sarilumab or satralizumab may be linked through flexible linkers to VEGF binding units, each comprising polypeptides of SEQ ID NO: 6 and 8. In other embodiments, a heavy chain of tocilizumab or satralizumab is linked through a flexible linker to a VEGF binding unit comprising a polypeptide of SEQ ID NO: 10; and a light chain of sarilumab or satralizumab is linked through a flexible linker to a VEGF binding unit comprising polypeptides of SEQ ID NO: 6 and 8.

A VEGF binding unit included in an antibody fusion protein of the present invention is capable of binding at least one member of the VEGF family ( “VEGF family member” ) ; thereby, rendering said at least one VEGF family member substantially unavailable for binding to a VEGF receptor on endothelial cells. As a result, said antibody fusion protein substantially inhibits biological activity of said at least one VEGF family member in promoting angiogenesis; thereby, controlling a pathological condition having etiology in aberrant angiogenesis.

In some embodiments, the present invention also provides a binding construct that comprises or consists of a plurality of antibody fusion or chimeric proteins as herein described that are linked to or associated with each other by covalent bonds or other forms of attachment; wherein the antibody portions of such a binding construct may be the same or different. A binding construct of the present invention is capable of binding IL-6R and at least a VEGF family member or a portion thereof and does so with high affinity.

The VEGF binding units may be linked to the C-terminus (carboxy terminus) or the N-terminus (amino terminus) of the light chains and heavy chains of the antibody portion. Preferably, the VEGF binding units are linked to the C-terminus of the light chains and heavy chains of the antibody portion. More preferably, the VEGF binding units are linked to the C-terminus of the light chains and heavy chains of the antibody portion through flexible linkers.

A light chain or heavy chain of the antibody portion of the antibody fusion protein is preferably linked to one VEGF binding unit. However, a light chain or heavy chain of the antibody portion may be linked to more than one VEGF binding unit. In this case, the VEGF binding units may be joined together directly or through a linker. An antibody fusion protein or a binding construct may further include a heterologous peptide or other chemical moieties. Such additions can modify its properties such as stability, solubility, toxicity, serum half-life, immunogenicity, detectability, or other properties.

The term “high affinity” is used in a physiological context pertaining to the relative affinity of the antibody fusion protein for IL-6R and VEGF family members in vivo in a mammal, such as a laboratory test animal, a domesticated farm or pet animal, or a human. Antibody fusion proteins binding IL-6R and various VEGF members in the present invention have characteristic affinities for their ligands in vivo, typically measured in terms of sub-nanomolar dissociation constants (K _d) . For the purposes of this invention, an antibody fusion protein of the present invention can bind to IL-6R or its targeted VEGF family member (s) with a K _d less than or equal to about 1, or about 5, or about 10, or about 50, or about 100, or about 500, or about 1000 times the K _d of the natural IL-6/IL-6R or growth factor/receptor pair.

In one aspect, the Ig-like domains of a VEGF binding unit may be linked together in any order, directly or through an intervening linker.

In another aspect, a targeted VEGF family member may bind to one or more VEGF binding units of the antibody fusion or chimeric protein.

In still another aspect, a VEGF binding unit comprises substantially an Ig-like domain of a VEGF receptor.

In another aspect, a VEGF binding unit comprises two Ig-like domains selected from the group consisting of Ig-like domains of human VEGFR-1, VEGFR-2, and VEGFR-3.

In still another aspect, each of the light chains and heavy chains of an antibody fusion or chimeric protein of the present invention is linked to a VEGF binding unit that comprises: (a) Ig-like domain 2, or substantially Ig-like domain 2, of human VEGFR-1 (or flt-1) ; and (b) Ig-like domain 3, or substantially Ig-like domain 3, of human VEGFR-2 (or KDR) . In one embodiment, such VEGF binding units are linked to the C-termini of the light chains and the heavy chains of the antibody portion.

In still another aspect, each of the light chains of an antibody fusion or chimeric protein of the present invention is linked to a first VEGF binding unit that comprises: (a) Ig-like domain 2, or substantially Ig-like domain 2, of human VEGFR-1 (or flt-1) ; and (b) Ig-like domain 3, or substantially Ig-like domain 3, of human VEGFR-2 (or KDR) ; and each of the heavy chains of the antibody fusion protein is linked to a second VEGF binding unit that comprises Ig-

like domains

1 and 2, or substantially Ig-

like domains

1 and 2, of VEGFR-3 (or flt-4) . In another embodiment, the second VEGF binding unit comprises Ig-

like domains

2 and 3, or substantially Ig-

like domains

2 and 3, of VEGFR-3. In still another embodiment, the second VEGF binding unit comprises at least an Ig-like domain selected from the group consisting of Ig-

like domains

1, 2, and 3, or substantially Ig-

like domains

1, 2, and 3, of VEGFR-3. In yet another embodiment, such VEGF binding units are linked to the C-termini of the light chains and the heavy chains of the antibody portion.

In some embodiments, two or more VEGF binding units act together to bind a single ligand molecule of the VEGF family (wherein the ligand may comprise a monomer or dimer) . In some other embodiments, the binding units act independently, i.e., each binding unit binds a separate ligand molecule.

In one embodiment, a polypeptide linker is inserted between the C-terminus of the light chain or heavy chain of the antibody portion and a VEGF binding unit. The polypeptide linker may be the same or different for the light chain and heavy chain.

In one aspect, the amino acid sequences of the various portions or embodiments of an antibody fusion protein of the present invention are listed in Table 1.

Table 1

Amino Acid Sequences

In another aspect, the nucleic acid sequences encoding the amino acid sequences of the various portions or embodiments of an antibody fusion protein of the present invention are listed in Table 2.

Table 2

Nucleic Acid Sequences

In yet another aspect, the antibody fusion or chimeric protein of the present invention comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 74, or SEQ ID NO: 76.

In still another aspect, the antibody fusion or chimeric protein of the present invention comprises an amino acid sequence that is at least 95%identical to SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, or SEQ ID NO: 32, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 74, or SEQ ID NO: 76.

In still another aspect, one or more amino acid substitutions can be made in anyone of the above-described amino acid sequences. Preferably, such substitution is a conserved substitution, wherein an amino acid in one of the following groups is substituted with another in the same group: (1) A, S, T; (2) D, E; (3) N, Q; (4) R, K; (5) I, L, M, V; and (6) F, Y, W; and such substitution is selected so as to preserve substantially the binding activity of the fusion polypeptide. In one embodiment, an antibody fusion protein of the present invention having a conserved substitution has a K _d value for IL-6R or VEGF ligand less than about 120%of that before such substitution. Preferably, the K _d value is less than about 110%of that before such substitution. More preferably, the K _d value is less than about 105%of that before such substitution.

Most conserved substitutions are not expected to produce radical changes in the characteristics of the Ig-like domain or domains of the fusion polypeptide. However, when it is difficult to predict the exact effect of the substitution in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. For example, an Ig-like domain variant typically is made by site-specific mutagenesis of the nucleic acid encoding the intact fusion polypeptide, expression of the variant nucleic acid in recombinant cell culture, purification of the variant fusion polypeptide from the cell culture and detecting the ability of the variant fusion polypeptide to specifically bind to IL-6R or a VEGF ligand. An exemplary binding assay which can be employed to determine if a particular substitution or substitutions in an Ig-like domain or domains affect the capability of the fusion polypeptide to bind to and inhibit the activity of IL-6R or a VEGF family member is described in the article by Park et al., J. Biol. Chem., 269: 25646-25654 (1994) .

The VEGFR-1-D2 binding unit of the fusion protein is capable of binding free VEGF-A, VEGF-B, and placental growth factor ( “PlGF” ) with high affinity (Davis-Smyth et al., EMBO J., 15 (18) : 4919 (1996) ) . The VEGFR-2-D3 binding unit of the fusion protein is capable of binding free VEGF-A, VEGF-C, and VEGF-D with high affinity (Stuttfeld et al., Life, 61 (9) : 915 (2009) ) . The VEGFR-3-D1D2 binding unit of the fusion protein is capable of binding free VEGF-C and VEGF-D with high affinity. Thus, a fusion protein of the present invention is capable of substantially inhibiting the angiogenic activity of these VEGF family members on endothelial cells at the site of the disease.

In still another aspect, the present invention provides isolated nucleic acid molecules encoding heavy chains and light chains of said antibody fusion protein.

In yet another aspect, the present invention provides isolated nucleic acid molecules encoding said antibody fusion protein; wherein said isolated nucleic acid molecules comprise: (a) a nucleic acid sequence encoding a heavy chain of an antibody against human IL-6R ( “hIL-6R antibody” ) or an antigen-binding fragment or domain thereof; (b) a nucleic acid sequence encoding a light chain of said hIL-6R antibody or an antigen-binding fragment or domain thereof; and (c) a nucleic acid sequence encoding an Ig-like domain of a VEGF receptor operatively linked to each of said nucleic acid sequence encoding said heavy chain of hIL-6R antibody or an antigen-binding fragment or domain thereof and said nucleic acid sequence encoding said light chain of hIL-6R antibody or an antigen-binding fragment or domain thereof.

In still another aspect, the present invention provides isolated nucleic acid molecules encoding said antibody fusion protein; wherein said isolated nucleic acid molecules comprise: (a) a nucleic acid sequence encoding a heavy chain of an hIL-6R antibody or an antigen-binding fragment or domain thereof; (b) a nucleic acid sequence encoding a light chain of said hIL-6R antibody or an antigen-binding fragment or domain thereof; and (c) a nucleic acid sequence encoding VEGFR-1-D2 and VEGFR-2-D3 operatively linked to each of said nucleic acid sequence encoding said heavy chain of hIL-6R antibody or an antigen-binding fragment or domain thereof and said nucleic acid sequence encoding said light chain of hIL-6R antibody or an antigen-binding fragment or domain thereof.

In still another aspect, the present invention provides isolated nucleic acid molecules encoding said antibody fusion protein; wherein said isolated nucleic acid molecules comprise: (a) a nucleic acid sequence encoding a heavy chain of an hIL-6R antibody or an antigen-binding fragment or domain thereof, having a sequence listed in SEQ ID NO: 1; (b) a nucleic acid sequence encoding a light chain of said hIL-6R antibody or an antigen-binding fragment or domain thereof, having a sequence listed in SEQ ID NO: 3; and (c) a nucleic acid sequence encoding VEGFR-1-D2 and VEGFR-2-D3, having sequences listed in SEQ ID NO: 5 and 7, which is operatively linked to each of said nucleic acid sequence encoding said heavy chain of hIL-6R antibody or an antigen-binding fragment or domain thereof and said nucleic acid sequence encoding said light chain of hIL-6R antibody or an antigen-binding fragment or domain thereof.

In still another aspect, the present invention provides isolated nucleic acid molecules encoding said antibody fusion protein; wherein said isolated nucleic acid molecules comprise: (a) a nucleic acid sequence encoding a heavy chain of an hIL-6R antibody or an antigen-binding fragment or domain thereof; (b) a nucleic acid sequence encoding a light chain of said hIL-6R antibody or an antigen-binding fragment or domain thereof; (c) a nucleic acid sequence encoding VEGFR-3-D1D2 operatively linked to said nucleic acid sequence encoding said heavy chain of hIL-6R antibody or an antigen-binding fragment or domain thereof; and (d) a nucleic acid sequence encoding VEGFR-1-D2 and VEGFR-2-D3 operatively linked to said nucleic acid sequence encoding said light chain of hIL-6R antibody or an antigen-binding fragment or domain thereof. In some embodiments, the nucleic acid sequence encoding VEGFR-3-D1D2 is replaced by a nucleic acid sequence that encodes at least a VEGFR-3 Ig-like domain selected from the group consisting of VEGFR-3-D1, VEGFR-3-D2, and VEGFR3-D3.

In still another aspect, the present invention provides isolated nucleic acid molecules encoding said antibody fusion protein; wherein said isolated nucleic acid molecules comprise: (a) a nucleic acid sequence encoding a heavy chain of an hIL-6R antibody or an antigen-binding fragment or domain thereof, having a sequence listed in SEQ ID NO: 1; (b) a nucleic acid sequence encoding a light chain of said hIL-6R antibody or an antigen-binding fragment or domain thereof, having a sequence listed in SEQ ID NO: 3; (c) a nucleic acid sequence encoding VEGFR-3-D1D2, having a sequence listed in SEQ ID NO: 9, which is operatively linked to said nucleic acid sequence encoding said heavy chain of hIL-6R antibody or an antigen-binding fragment or domain thereof; and (d) a nucleic acid sequence encoding VEGFR-1-D2 and VEGFR-2-D3, having sequences listed in SEQ ID NO: 5 and 7, which is operatively linked to said nucleic acid sequence encoding said light chain of hIL-6R antibody or an antigen-binding fragment or domain thereof. In some embodiments, the nucleic acid sequence encoding VEGFR-3-D1D2 is replaced by a nucleic acid sequence that encodes at least a VEGFR-3 Ig-like domain selected from the group consisting of VEGFR-3-D1, VEGFR-3-D2, and VEGFR3-D3.

In still another aspect, the present invention provides an isolated nucleic acid molecule encoding a heavy chain of said antibody fusion protein that has a sequence listed in SEQ ID NO: 17, 21, 25, 29, 79, 83, 87, 91, 95, or 99; and an isolated nucleic acid molecule encoding a light chain of said fusion protein that has a sequence listed in SEQ ID NO: 19, 23, 27, 31, 81, 85, 89, 93, 97, or 101.

In yet another aspect, said isolated nucleic acid molecules further can comprise a leading nucleic acid sequence encoding a signal polypeptide. In certain embodiments wherein a VEGF binding unit is linked to the C-terminus of a heavy chain or light chain of the antibody portion, said leading nucleic acid sequence precedes SEQ ID NO: 1 and 3.

In another aspect, the present invention provides isolated nucleic acid molecules encoding said antibody fusion or chimeric protein; wherein said isolated nucleic acid molecules comprise a nucleic acid sequence that, as a result of the degeneracy of the genetic code, differ in one or more codons from a nucleic acid sequence listed in this disclosure. Such different nucleic acid sequence is within the scope of the present invention.

In still another aspect, the present invention provides a vector that comprises any of said nucleic acid molecules, including an expression vector comprising any of said nucleic molecules operatively linked to an expression control sequence. Embodiments of the vector comprise a nucleic acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, and SEQ ID NO: 101.

In one aspect, the present invention provides for the construction of a nucleic acid molecules encoding heavy chains and light chains of an antibody fusion protein disclosed herein, which nucleic acid molecules are inserted into vectors that are able to express the antibody fusion protein when introduced into an appropriate host cell. Appropriate host cells include, but are not limited to, bacterial cells, yeast cells, insect cells, and mammalian cells. Any of the methods known to one skilled in the art for the insertion of DNA fragments into a vector may be used to construct expression vectors encoding chimeric polypeptide molecules under control of transcriptional/translational control signals. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinations (genetic recombination) (See Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory; Current Protocols in Molecular Biology, Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY) .

Expression of nucleic acid molecules encoding an antibody fusion protein of the present invention may be regulated by a second nucleic acid sequence (a promoter) so that the antibody fusion protein is expressed in a host transformed with the nucleic acid molecules. For example, expression of the antibody fusion protein described herein may be controlled by any promoter/enhancer element known in the art.

In general, plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences that are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (see; e.g., Bolivar et al., Gene, 2: 95 (1977) ) . The plasmid pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR322 plasmid, or other microbial plasmid or phage, must also contain, or be modified to contain, promoters that can be used by the microbial organism for expression of proteins.

Those promoters most commonly used in recombinant DNA construction include the β-lactamase (penicillinase) and lactose promoter systems or a tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res., 8: 4057 (1980) ) . While these are the most commonly used, other microbial promoters have been discovered and utilized. For example, the tac promoter is a synthetically produced DNA promoter produced from the combination of promoters from the trp and lac operons (de Boer et al., PNAS, (1983-01-80 (1) : 21–25 (1983) ) . It is commonly used for protein production in Escherichia coli. (Amann et al., Gene, 25: 167 (1983) ) . Any of these promoters may be used in connection with a method of producing an antibody fusion protein of the present invention.

In addition to prokaryotes, eukaryotic microbes, such as yeast cultures, may also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example (Stinchcomb et al., Nature, 282: 39 (1979) ) is commonly used. Other exemplary plasmids are disclosed in US Patent 4,615,974; Struhl et al., PNAS, 76 (3) : 1035 (1979) . The plasmid YRp7 contains the trp1 gene that provides a selection marker for a mutant strain of yeast lacking the ability to grow without tryptophan, for example, ATCC No. 44,076 or RH218 (Jones, Genetics, 85: 23 (1977) ) . The presence of the trp1 lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255: 2073 (1980) ) or other glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylas, and glucokinase (Romanos et al., Yeast, 8: 423 (1992) ; Weinhandl et al., Microb. Cell factories, 13: 5 (2014) ) . In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination. Other promoters, which have the additional advantage of transcription controlled by growth conditions, such as the promoter region for alcohol dehydrogenase 2, and enzymes responsible for maltose and galactose utilization (Romanos et al., Weinhandl et al., supra) . Any plasmid vector containing yeast-compatible promoter, origin of replication and termination sequences is suitable.

In addition to microorganisms, cultures of cells derived from multicellular organisms may also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. However, much interest has been in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years (Tissue Culture, Academic Press, Kruse and Patterson, editors (1973) ) . Examples of such useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7, 293, and MDCK cell lines. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expression vectors are often provided by viral material. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40) . The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment that also contains the SV40 viral origin of replication (Fiers et al., Nature, 273: 113 (1978) ) . Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250-bp sequence extending from the HindIII site toward the BglI site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.

Thus, according to the invention, expression vectors capable of being replicated in a bacterial, a yeast cell, an insect cell, or a mammalian cell host, comprising an antibody fusion protein-encoding nucleic acid as described herein, are used to transfect the host and thereby direct expression of such nucleic acids to produce the fusion polypeptide, which may then be recovered in a biologically active form. As used herein, a biologically active form includes a form capable of binding to at least a VEGF family member.

In some embodiments, the host cell can be E. coli, a COS cell, a HEK 293 cell (also known simply as 293 cells) , or a Chinese hamster ovary ( “CHO” ) cell. Preferably, the host cell is a HEK 293 or CHO cell.

Vector Construction

Construction of suitable vectors containing the desired coding and control sequences employ standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and ligated in the form desired to form the plasmids required. The methods employed are not dependent on the DNA source or intended host. Cleavage is performed by treating with restriction enzyme (or enzymes) in suitable buffer.

A nucleic acid sequence substantially encoding one or more Ig-like domains of VEGFR-1, VEGFR-2, or VEGR-3 can be produced according to the method disclosed in U.S. Patent 6,897,294.

In one embodiment, nucleic acid sequences substantially encoding the Ig-like domain 2 of VEGFR-1 and Ig-like domain 3 of VEGFR-2 are ligated in tandem in the desired order. This construct is then ligated to the 3’ end of the nucleic acid sequences encoding the heavy chain and light chain of the hIL-6R antibody. In another embodiment, this construct is ligated to the 3’ end of the nucleic acid sequence encoding the light chain of the hIL-6R antibody. Another nucleic acid construct comprising nucleic acid sequences substantially encoding the Ig-

like domains

1 and 2, or 2 and 3, or 1, 2, and 3, of VEGFR-3 is ligated to the 3’ end of the nucleic acid sequence encoding the heavy chain of the hIL-6R antibody. Such an entire nucleic acid sequence is referred to as a chimeric nucleic acid sequence.

The entire chimeric nucleic acid sequence is then positioned in a vector which contains a promoter in the reading frame with the gene and compatible with the proposed host cell. A number of plasmids, such as those described in U.S. Patents 4,456,748; 5,460,811; 5,888,808; and 6,333,147, having may be used in a production of an antibody fusion protein of the present invention.

In one embodiment, a chimeric heavy chain and a chimeric light chain of the antibody fusion protein of the present invention is produced according to the method described in U.S. Patent 7,070,959. The chimeric nucleic acid sequence of SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 59, 61, 63, 65, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, or 101 is inserted into the expression vector pEE14.1 (Lonza Biologics) having the CMV promoter.

In one embodiment, CHO K1 cells are transfected with pEE14.1/SEQ ID NO: 17 and pEE14.1/SEQ ID NO: 19 construct and then grown. The antibody fusion protein obtained from the CHO cells may be purified and characterized by binding assay, as described in U.S. Patent 7,070,959.

Similarly, CHO K1 or HEK293 cells are transfected with a pair of vectors pEE14.1/SEQ ID NO: 21 and pEE14.1/SEQ ID NO: 23; or pEE14.1/SEQ ID NO: 25 and pEE14.1/SEQ ID NO: 27; or pEE14.1/SEQ ID NO: 29 and pEE14.1/SEQ ID NO: 31, or pcDNA3.4/SEQ ID NO: 79 and pcDNA3.4/SEQ ID NO: 81, or pcDNA3.4/SEQ ID NO: 83 and pcDNA3.4/SEQ ID NO: 85, or pcDNA3.4/SEQ ID NO: 87 and pcDNA3.4/SEQ ID NO: 89, or pcDNA3.4/SEQ ID NO: 91 and pcDNA3.4/SEQ ID NO: 93, or pcDNA3.4/SEQ ID NO: 95 and pcDNA3.4/SEQ ID NO: 97, or pcDNA3.4/SEQ ID NO: 99 and pcDNA3.4/SEQ ID NO: 101, construct and grown. Antibody fusion proteins obtained from these CHO or HEK293 cells may be similarly purified and characterized.

In one embodiment, an antibody fusion protein of the present invention can bind to hIL-6R and VEGF family members with K _d ≤ 10 ^-9 M. In another embodiment, an antibody fusion protein of the present invention can bind to VEGF family members with K _d ≤ 5x10 ^-10 M. In still another embodiment, an antibody fusion protein of the present invention can bind to VEGF family members with K _d ≤ 10 ^-10 M.

In one aspect, the present invention provides compounds, compositions, and methods for treating or controlling a disease, condition, or disorder having etiology in aberrant angiogenesis.

In another aspect, the present invention provides a method for treating or controlling at least an ocular disease, condition, or disorder, in a subject, which has etiology in aberrant angiogenesis. The method comprises administering to a subject in need of such treating or controlling a composition comprising an antibody fusion protein herein disclosed. Such an antibody fusion protein comprises a pair of heavy chain and light chain having SEQ ID NO: 18 and 20; or SEQ ID NO: 22 and 24; or SEQ ID NO: 26 and 28; or SEQ ID NO: 30 and 32, or SEQ ID NO: 78 and 80; or SEQ ID NO: 82 and 84; or SEQ ID NO: 86 and 88; or SEQ ID NO: 90 and 100. In one embodiment, an antibody fusion protein used in such a method can comprise a pair or dimer of proteins having a sequence as listed in SEQ ID NO: 60, 62, 64, 66, 74, or 76. In another embodiment, an antibody fusion protein used in such a method can comprise a pair or dimer of proteins having a sequence that begins at amino acid 17 of SEQ ID NO: 60, 62, 64, 66, 74, or 76.

In still another aspect, said ocular disease, condition, or disorder is selected from the group consisting of: choroidal neovascularization, neovascular age-related macular degeneration (wet age-related macular degeneration) , polypoidal choroidal vasculopathy ( “PCV” ) , myopic choroidal neovascularization, vascular leak, macular edema resulting from diabetes, uveitis, central and branch retinal vein occlusion, non-proliferative and proliferative diabetic retinopathy, retinopathy of prematurity, corneal neovascularization, corneal inflammation, and neovascular glaucoma.

In one embodiment, the subject is administered with a dose of about 25-4000 micrograms of the fusion polypeptide. In another embodiment, the subject is administered with a dose of about 50-4000, about 100-4000, about 500-4000, about 1000-4000, about 2000-4000, about 50-3000, about 50-2000, about 50-1000, or about 50-500 micrograms of the fusion polypeptide.

In still another aspect, the composition comprising the fusion polypeptide is in the form of an eye drop or an ocular injection (such as intravitreal, intracameral, peri-orbital, subtenon, subretinal, or suprachoroidal injection) . Such a composition comprises an ophthalmic composition. An antibody fusion protein of the present invention may also be incorporated in a medical device that is implantable into or near a diseased tissue.

In one embodiment, the present invention provides a method or composition for treating or controlling an anterior-segment disease, condition, or disorder; such as corneal neovascularization, corneal inflammation, or neovascular glaucoma. The composition comprising the fusion polypeptide may be in the form of an eye drop or intracameral or subconjunctival injection. In another embodiment, the present invention provides a method or composition for treating or controlling a posterior-segment disease, condition, or disorder; such as choroidal neovascularization, neovascular age-related macular degeneration (wet age-related macular degeneration) , polypoidal choroidal vasculopathy ( “PCV” ) , myopic choroidal neovascularization, vascular leak, macular edema resulting from diabetes, uveitis, central and branch retinal vein occlusion, non-proliferative and proliferative diabetic retinopathy, retinopathy of prematurity. In this case, the composition comprising the fusion polypeptide may be administered in the form of an intravitreal injection.

In yet another aspect, an eye drop is administered to the subject at least once per day, at least once per week, or at least once per month until the disease, condition, or disorder is substantially treated or controlled.

In yet another aspect, the composition is administered via sustained drug release to the subject for a period of at least one month, at least two months, at least three months, or at least six months.

In still another aspect, an intravitreal injection or an injection into, or near, a diseased tissue is administered to the subject according to a regimen recommended by a medical practitioner for a particular patient. For example, an injection may be administered at least once per month, at least once every two months, at least once every three months, or at least once every six months until the disease, condition, or disorder is substantially treated or controlled. In one embodiment, treatment may be administered more frequently at the beginning, and then less frequently after a period of time. Such period of time may be determined by a medical practitioner.

The concentration of an antibody fusion protein of the present invention in such an ophthalmic composition can be in the range from about 0.1 to about 200 mg/ml (or, alternatively, from about 0.25 to about 200 mg/ml, or from about 0.25 to about 160 mg/ml, or from about 0.5 to about 100 mg/ml, or from about 0.25 to about 50 mg/ml, or from about 0.5 to about 200 mg/ml, or from about 0.5 to about 160 mg/ml, or from about 0.5 to about 100 mg/ml, or from about 0.5 to about 50 mg/ml, or from about 1 to about 200 mg/ml, or from 1 to about 160 mg/ml, or from about 0.5 to about 100 mg/ml, or from about 1 to about 50 mg/ml) .

In still another aspect, a method for preparing a composition of the present invention comprises combining: (a) an amount of an antibody fusion protein of the present invention; and (b) a physiologically acceptable carrier.

In one embodiment, such a physiologically acceptable carrier can be a sterile saline solution or a physiologically acceptable buffer. In another embodiment, such a carrier comprises a hydrophobic medium, such as a pharmaceutically acceptable oil. In still another embodiment, such as carrier comprises an emulsion of a hydrophobic material and water. In yet another embodiment, an antibody fusion protein of the present invention may be associated or linked with a high-molecular weight material to provide a long circulation time.

Physiologically acceptable buffers include, but are not limited to, a phosphate buffer or a Tris-HCl buffer (comprising tris (hydroxymethyl) aminomethane and HCl) . For example, a Tris-HCl buffer having pH of 7.4 comprises 3 g/l of tris (hydroxymethyl) aminomethane and 0.76 g/l of HCl. In yet another aspect, the buffer is 10X phosphate buffer saline ( “PBS” ) or 5X PBS solution. Non-limiting examples of buffers used for injectable compositions comprising biologics include phosphate, citric acid, acetic acid, tromethamine, histidine, arginine, gluconic acid, lactic acid, tartaric acid, aspartic acid, and glutamic acid.

Other buffers also may be found suitable or desirable in some circumstances, such as buffers based on HEPES (N- {2-hydroxyethyl} peperazine-N’- {2-ethanesulfonic acid} ) having pK _a of 7.5 at 25 ℃ and pH in the range of about 6.8-8.2; BES (N, N-bis {2-hydroxyethyl} 2-aminoethanesulfonic acid) having pK _a of 7.1 at 25℃ and pH in the range of about 6.4-7.8; MOPS (3- {N-morpholino} propanesulfonic acid) having pK _a of 7.2 at 25℃and pH in the range of about 6.5-7.9; TES (N-tris {hydroxymethyl} -methyl-2-aminoethanesulfonic acid) having pK _a of 7.4 at 25℃ and pH in the range of about 6.8-8.2; MOBS (4- {N-morpholino} butanesulfonic acid) having pK _a of 7.6 at 25℃ and pH in the range of about 6.9-8.3; DIPSO (3- (N, N-bis {2-hydroxyethyl} amino) -2-hydroxypropane) ) having pK _a of 7.52 at 25℃ and pH in the range of about 7-8.2; TAPSO (2-hydroxy-3 {tris (hydroxymethyl) methylamino} -1-propanesulfonic acid) ) having pK _a of 7.61 at 25℃and pH in the range of about 7-8.2.

In certain embodiments, a composition of the present invention is formulated in a buffer having an acidic pH, such as from about 4 to about 6.8, or alternatively, from about 5 to about 6.8. In such embodiments, the buffer capacity of the composition desirably allows the composition to come rapidly to a physiological pH after being administered into the patient.

In addition to a buffer, a composition of the present invention can comprise a material selected from the group consisting of surfactants, stabilizers, preservatives, co-solvent, humectants, emollients, chelating agents, tonicity-adjusting agents, and antioxidants.

In one aspect, any of these materials that may be used in a composition of the present invention is a physiologically acceptable material. In certain embodiments, any of these materials that may be used in a composition of the present invention is an ophthalmically acceptable material.

Water-soluble preservatives that may be employed include quaternary ammonium compounds such as benzalkonium chloride and various polyquaternium compounds. These agents may be present in individual amounts of from about 0.001 to about 2%by weight (preferably, about 0.01%to about 0.05%by weight) .

Non-limiting examples of surfactants include, but are not limited to, non-ionic surfactants, for example, polysorbates (such as polysorbate 20, polysorbate 80) , 4- (1, 1, 3, 3-tetramethylbutyl) phenol/poly (oxyethylene) polymers (such as the polymer sold under the trademark Tyloxapol) , poly (oxyethylene) -poly (oxypropylene) block copolymers, glycolic esters of fatty acids and the like, and mixtures thereof.

In one aspect, the pH of the composition is in the range from about 4 to about 8. Alternatively, the pH of the composition is in the range from about 6 to about 8, or from about 6.5 to about 8, or from about 6.5 to about 7.5.

In another aspect, the composition has a pH of about 7. Alternatively, the composition has a pH in a range from about 7 to about 7.5.

In still another aspect, the composition has a pH of about 7.4.

In yet another aspect, a composition also can comprise a viscosity-modifying compound designed to facilitate the administration of the composition into the subject or to promote the bioavailability in the subject. In still another aspect, the viscosity-modifying compound may be chosen so that the composition is not readily dispersed after being administered into an environment of an eye. Such compounds may enhance the viscosity of the composition, and include, but are not limited to: monomeric polyols, such as, glycerol, propylene glycol, ethylene glycol; polymeric polyols, such as, polyethylene glycol; various polymers of the cellulose family, such as hydroxypropylmethyl cellulose ( “HPMC” ) , carboxymethyl cellulose ( “CMC” ) sodium, hydroxypropyl cellulose ( “HPC” ) ; polysaccharides, such as hyaluronic acid and its salts, chondroitin sulfate and its salts, dextrans, such as, dextran 70; water soluble proteins, such as gelatin; vinyl polymers, such as, polyvinyl alcohol, polyvinylpyrrolidone, povidone; carbomers, such as carbomer 934P, carbomer 941, carbomer 940, or carbomer 974P; and acrylic acid polymers. In general, a desired viscosity can be in the range from about 1 to about 400 centipoises ( “cps” ) or mPa. s.

Non-limiting examples of chelating agents include ethylenediaminetetraacetic acid ( “EDTA” ) , diethylenetriaminepentakis (methylphosphonic acid) , etidronic acid, tetrasodium salt of etidronic acid (also known as “HAP” ) .

While the buffer itself is a “tonicity-adjusting agent” and a “pH-adjusting agent” that broadly maintains the ophthalmic solution at a particular ion concentration and pH, additional “tonicity-adjusting agents” can be added to adjust the final tonicity of the solution. Such tonicity-adjusting agents are well known to those of skill in the art and include, but are not limited to, mannitol, sorbitol, dextrose, sucrose, urea, propylene glycol, and glycerin. Also, various salts, including halide salts of a monovalent cation (e.g., NaCl or KCl) can be utilized. Typically, the tonicity of a formulation of the present invention is in the range from about 200 to 400 mOsm/kg. Alternatively, the tonicity of a formulation of the present invention is in the range from about 220 to 400 mOsm/kg, or from about 220 to 350 mOsm/kg, or from about 220 to 300 mOsm/kg, or from about 250 to 350 mOsm/kg.

Non-limiting examples of anti-oxidants include ascorbic acid (vitamin C) and its salts and esters; tocopherols (such as α-tocopherol) and tocotrienols (vitamin E) , and their salts and esters (such as vitamin E TGPS (D-α-tocopheryl polyethylene glycol 1000 succinate) ) ; glutathione; lipoic acid; uric acid; butylated hydroxyanisole ( “BHA” ) ; butylated hydroxytoluene ( “BHT” ) ; tertiary butylhydroquinone ( “TBHQ” ) ; and polyphenolic anti-oxidants (such as gallic acid, cinnanmic acid, flavonoids, and their salts, esters, and derivatives) .

Non-limiting examples of stabilizers includes sucrose, mannitol, sorbitol, and trehalose.

It should be understood that the proportions of the various components or mixtures in the following examples may be adjusted for the appropriate circumstances.

In another aspect, an antibody fusion protein of the present invention and appropriate amounts of one or more desired excipients are incorporated into a formulation for topical administration or injection to a portion of the eye, such as the anterior or posterior segment, or the vitreous humor. An injectable formulation can desirably comprise a carrier that provides a sustained-release of the active ingredients, such as for a period longer than about 1 week (or longer than about 1, 2, 3, 4, 5, or 6 months) . In certain embodiments, an antibody fusion protein of the present invention is included in a delivery device for sustained release of the active ingredients over a long period of time, such as 4, 5, 6 months or longer. An example of such delivery device is described in U.S. Patents 8,399,006 and 9,417,238.

In still another aspect, a composition comprising an antibody fusion protein of the present invention and desired excipients is lyophilized and is reconstituted with a physiologically acceptable liquid carrier substantially immediately before administration to a subject.

In one embodiment, a compound or composition of the present invention can be injected with a fine-gauge needle, such as 25-35 gauge. Typically, an amount from about 25 μl to about 100 μl of a composition comprising about 25-4000 μg of an antibody fusion protein of the present invention is administered into a patient. In one embodiment, the antibody fusion protein has heavy chain and light chain amino acid sequences substantially as listed in SEQ ID NO: 18 and 20; or SEQ ID NO: 22 and 24; or SEQ ID NO: 26 and 28; or SEQ ID NO: 30 and 32, or SEQ ID NO: 78 and 80; or SEQ ID NO: 82 and 84; or SEQ ID NO: 86 and 88; or SEQ ID NO: 90 and 100. In another embodiment, an antibody fusion protein used in such a procedure can comprise a pair or dimer of proteins having a sequence substantially as listed in SEQ ID NO: 60, 62, 64, 66, 74, or 76. In still another embodiment, an antibody fusion protein used in such a procedure can comprise a pair or dimer of proteins having a sequence that begins at amino acid 17 of the sequences substantially as listed in SEQ ID NO: 60, 62, 64, 66, 74, or 76. In yet another embodiment, the antibody fusion protein has heavy chain and light chain amino acid sequences as listed in SEQ ID NO: 18 and 20; or SEQ ID NO: 22 and 24; or SEQ ID NO: 26 and 28; or SEQ ID NO: 30 and 32, or SEQ ID NO: 78 and 80; or SEQ ID NO: 82 and 84; or SEQ ID NO: 86 and 88; or SEQ ID NO: 90 and 100. In still another embodiment, an antibody fusion protein used in such a procedure can comprise a pair or dimer of proteins having a sequence that begins at amino acid 17 of the sequences as listed in SEQ ID NO: 60, 62, 64, 66, 74, or 76. A concentration of such antibody fusion protein is selected from the ranges disclosed above.

In still another aspect, an antibody fusion protein of the present invention is incorporated into an ophthalmic device that comprises a biodegradable material, and the device is implanted into a posterior-segment tissue of a subject to provide a long-term (e.g., longer than about 1 week, or longer than about 1, 2, 3, 4, 5, or 6 months) treatment or control of an angiogenic disease, condition, or disorder. Such a device may be implanted by a skilled physician in the subject’s ocular or periocular tissue. Non-limiting examples of ophthalmic implant systems or devices for the sustained-release of an active ingredient are disclosed in U.S. Patents 5,378,475; 5,773,019; 5,902,598; 6,001,386; 6,051,576; and 6,726,918.

In still another aspect, a method for treating or controlling an ophthalmic angiogenic disease, condition, or disorder comprises administering a composition comprising an antibody fusion protein of the present invention to a subject in need thereof.

In still another aspect, an antibody fusion protein used in said method comprises an antibody against IL-6R or an antigen-binding fragment or domain thereof; wherein at least one of: (1) a light chain or an antigen-binding fragment or domain thereof and (2) a heavy chain or an antigen-binding fragment or domain thereof is linked to a VEGF binding unit that comprises: (1) (a) Ig-like domain 2, or substantially Ig-like domain 2, of human VEGFR-1; and (b) Ig-like domain 3, or substantially Ig-like domain 3, of human VEGFR-2; or (2) at least an Ig-like domain selected from the group consisting of Ig-

like domains

1, 2, and 3, or substantially Ig-

like domains

1, 2, and 3, of human VEGFR-3. In one embodiment, the VEGF binding unit comprises (a) Ig-

like domains

1 and 2, or substantially Ig-

like domains

1 and 2; or (b) Ig-

like domains

2 and 3, or substantially Ig-

like domains

2 and 3; or (c) Ig-

like domains

1, 2, and 3, or substantially Ig-

like domains

1, 2, and 3, of human VEGFR-3.

In still another aspect, a method for treating or controlling an ophthalmic angiogenic disease, condition, or disorder comprises administering a composition comprising an antibody fusion protein having heavy chain and light chain amino acid sequences as listed in SEQ ID NO: 18 and 20; or SEQ ID NO: 22 and 24; or SEQ ID NO: 26 and 28; SEQ ID NO: 30 and 32; SEQ ID NO: 78 and 80; SEQ ID NO: 82 and 84; SEQ ID NO: 86 and 88; SEQ ID NO: 90 and 92; SEQ ID NO: 94 and 96; or SEQ ID NO: 98 and 100; or an antibody fusion protein having an amino acid sequence as listed in SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 74, or SEQ ID NO: 76 to a subject in need thereof. In still another aspect, an antibody fusion protein used in such a method can comprise a pair or dimer of proteins having a sequence that begins at amino acid 17 of the sequences as listed in SEQ ID NO: 60, 62, 64, 66, 74, or 76.

In still another aspect, a method for treating or controlling an ophthalmic angiogenic disease, condition, or disorder having an etiology in aberrant angiogenesis of in the posterior segment of an eye comprises intravitreally injecting a composition comprising an antibody fusion protein having heavy chain and light chain amino acid sequences as listed in SEQ ID NO: 18 and 20; or SEQ ID NO: 22 and 24; or SEQ ID NO: 26 and 28; or SEQ ID NO: 30 and 32; SEQ ID NO: 78 and 80; SEQ ID NO: 82 and 84; SEQ ID NO: 86 and 88; SEQ ID NO: 90 and 92; SEQ ID NO: 94 and 96; or SEQ ID NO: 98 and 100; or an antibody fusion protein having an amino acid sequence as listed in SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66 , SEQ ID NO: 74, or SEQ ID NO: 76 to a subject in need thereof. In still another aspect, an antibody fusion protein used in such a method can comprise a pair or dimer of proteins having a sequence that begins at amino acid 17 of the sequences as listed in SEQ ID NO: 60, 62, 64, 66, 74, or 76.

In another embodiment, such disease, condition, or disorder is selected from the group consisting of: choroidal neovascularization including neovascular age-related macular degeneration (wet age-related macular degeneration) , polypoidal choroidal vasculopathy (PCV) and myopic choroidal neovacular degeneration, vascular leak, macular edema resulting from diabetes, uveitis, central and branch retinal vein occlusion, non-proliferative and proliferative diabetic retinopathy, retinopathy of prematurity, corneal neovascularization, corneal inflammation, and neovascular glaucoma.

In yet another aspect, a composition of the present invention is administered once a week, once a month, once a year, twice a year, four times a year, or at a suitable frequency that is determined to be appropriate for treating or controlling an anterior-segment inflammatory disease, condition, or disorder.

In still another aspect, an antibody fusion protein of the present invention can also be used for the treatment or control of tumors, systemic inflammatory diseases or conditions, or autoimmune diseases such as arthritis. Such treatment or control may be effected by, for example, systemic administration. Dosages and regimens for treating such diseases or conditions may be determined or recommended for the particular disease or condition by medical practitioners.

EXAMPLE 1: Antibody Fusion Proteins Comprising VEGFR-1-D2, VEGFR-2-D3, and a Monoclonal Antibody (mAb) against IL-6R.

Six antibody fusion proteins of the present invention were produced according to a method disclosed herein, denoted as B781401, B781402, B781403, B781404, B781405, and B781406.

B781401 consists of a VEGF-binding unit that consists of VEGFR-1-D2-VEGFR-2-D3 linked directly to the amino terminus of the heavy chain of tocilizumab (an mAb against IL-6R) . The heavy chain of B781401 has the amino acid sequence SEQ ID NO: 78, and the nucleic acid sequence SEQ ID NO: 79. The light chain of B781401 has the amino acid sequence SEQ ID NO: 80, and the nucleic acid sequence SEQ ID NO: 81.

Each of B781402, B781403, and B781404 consists of a VEGF-binding unit that consists of VEGFR-1-D2-VEGFR-2-D3 linked through a flexible linker to the amino terminus of the heavy chain of tocilizumab. The heavy chains of B781402, B781403, and B781404 have the amino acid sequences SEQ ID NO: 82, SEQ ID NO: 86, and SEQ ID NO: 90, respectively; and the nucleic acid sequences SEQ ID NO: 83, SEQ ID NO: 87, and SEQ ID NO: 91, respectively. The light chains of B781402, B781403, and B781404 have the amino acid sequence SEQ ID NO: 84, SEQ ID NO: 88, and SEQ ID NO: 92, respectively, and the nucleic acid sequences SEQ ID NO: 85, SEQ ID NO: 89, and SEQ ID NO: 93, respectively.

B781405 consists of a VEGF-binding unit that consists of VEGFR-1-D2-VEGFR-2-D3 linked directly to the amino terminus of the light chain of tocilizumab. The heavy chain of B781405 has the amino acid sequence SEQ ID NO: 94, and the nucleic acid sequence SEQ ID NO: 95. The light chain of B781405 has the amino acid sequence SEQ ID NO: 96, and the nucleic acid sequence SEQ ID NO: 97.

B781406 consists of a VEGF-binding unit that consists of VEGFR-1-D2-VEGFR-2-D3 linked through a flexible linker to the amino terminus of the light chain of tocilizumab. The heavy chain of B781406 has the amino acid sequence SEQ ID NO: 98, and the nucleic acid sequence SEQ ID NO: 99. The light chain of B781406 has the amino acid sequence SEQ ID NO: 100, and the nucleic acid sequence SEQ ID NO: 101.

The carboxy terminus of the heavy chain of B781401, B781402, B781405, and B781406 has no glycine and lysine amino acid residues. The carboxy terminus of the heavy chain of B781404 has no lysine amino acid residue. The carboxy terminus of the heavy chain of B781403 includes glycine and lysine amino acid residues.

Figures 7A-D show schematic diagrams of antibody fusion proteins B781401-781406.

EXAMPLE 2: Antibody Fusion Proteins Comprising IL-6R Binding scFv Linked to VEGFR-1-D2-VEGFR-2-D3

Four antibody fusion proteins of the present invention were produced according to a method disclosed herein, denoted as EB-DJ1, EB-vvA, EB-vvB, EB-vvC. Each of these fusion proteins comprises a pair of or a dimer of constructs, each of which comprises an IL-6R scFv, a Fc domain of IgG1, and a VEGF-binding unit that comprises human VEGFR-1-D2 and human VEGFR-2-D3. Specifically, each of EB-DJ1 and EB-vvA consists of a dimer of constructs, each of which consists of an IL-6R scFv linked to a Fc domain of IgG1. The carboxy terminus of the IgG1-Fc domain is linked through a flexible linker to a VEGF-binding unit that consists of VEGFR-1-D2-VEGFR-2-D3. A construct of EB-DJ1 has the amino acid sequence SEQ ID NO: 60 and the nucleic acid sequence SEQ ID NO: 59. A construct of EB-vvA has the amino acid sequence SEQ ID NO: 74 and the nucleic acid sequence SEQ ID NO: 75.

EB-vvB consists of a dimer of constructs, each of which consists of an IL-6R scFv linked through a flexible linker to a VEGF-binding unit that consists of VEGFR-1-D2-VEGFR-2-D3. The carboxy terminus of the VEGF-binding unit is linked directly to the IgG1-Fc domain. A construct of EB-vvB has the amino acid sequence SEQ ID NO: 62 and the nucleic acid sequence SEQ ID NO: 61.

EB-vvC consists of a dimer of constructs, each of which consists of a VEGF-binding unit that consists of VEGFR-1-D2-VEGFR-2-D3 that is linked directly to the IgG1-Fc domain. The carboxy terminus of the IgG1-Fc domain is linked through a flexible linker to an IL-6R scFv. A construct of EB-vvC has the amino acid sequence SEQ ID NO: 76 and the nucleic acid sequence SEQ ID NO: 77.

EXAMPLE 2: Comparison of the ELISA Binding Affinities of EB-DJ1, EB-vvA, EB-vvB, and EB-vvC with Aflibercept, B781403 and B781405 for recombinant human VEGF-A ₁₆₅.

Figure 8 shows schematic diagrams of antibody fusion proteins EB-DJ1, EB-vvA, EB-vvB, and EB-vvC.

EXAMPLE 3: Transient Expression and Purification of B781401-781406.

Genes encoding the amino acid sequences of B781401-781406 as indicated by the SEQ ID NO: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, and 101 were synthetized and expression vectors based on the vector pcDNA3.4 for expressing the proteins were constructed. The expression vectors were used to transiently transfect HEK293 cells with chemically defined culture media. The produced proteins were purified by Protein-A affinity column, ultrafiltration and then subjected to 0.2μm sterile filtration to get the bulk of high purity proteins. Expression of B781401-781406 in HEK293 cells produced proteins with a MW ～200KDa (non-reduced form at SDS-PAGE) with ～100%purity when analyzed by SEC-HPLC. SEC-HPLC chromatogram for B781401 is shown in Fig. 9. The results for the expression and purification of B781401-781406 are shown in Table 3.

Table 3

Expression and Purification of B781401-78140, Produced in HEK293 Cells

Genes encoding the amino acid sequences of B781401-781406 as indicated by the SEQ ID NO: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, and 101 were synthetized and expression vectors based on the vector pcDNA3.4 for expressing the proteins were constructed. The expression vectors were used to transiently transfect CHO cells with chemically defined culture media. The produced proteins were purified by Protein-A affinity column, ultrafiltration and then subjected to 0.2μm sterile filtration to get the bulk of high purity proteins. Expression of B781401-781406 in CHO cells produced proteins with a MW ～200KDa (non-reduced form at SDS-PAGE) with ～93%purity when analyzed by SEC-HPLC. SEC-HPLC chromatogram for B781401 is shown in Fig. 10. The results for the expression and purification of B781401-781406 are shown in Table 4.

Table 4

Expression and Purification of B781401-78140, Produced in CHO Cells

EXAMPLE 4: Comparison of the Binding Affinities of B781401-781406 with Aflibercept

for Recombinant Human VEGF-A ₁₆₅ Using Enzyme Linked Immunosorbent Assay (ELISA) .

ELISA assays were performed using 96-well plates coated with recombinant human VEGF-A ₁₆₅ (2μg/ml, 100μl/well) at +4℃ for 16 hours. After non-specific blocking using 1%BSA at 25℃ for 1 hour, a series of dilutions of test antibodies were added into the coated wells and incubated at 25℃ for 1 hour. The bound Abs were detected using a secondary Ab (goat anti-human IgG1-Fc) conjugated with HRP followed by OD ₄₅₀ reading. The binding affinities of B781401-781406 for recombinant human VEGF-A ₁₆₅ are at sub-nanomolar scales and comparable with aflibercept. The results are shown in Fig. 11.

EXAMPLE 5: Comparison of the ELISA Binding Affinities of B781401-781406 with Aflibercept for Recombinant Human VEGF-B ₁₆₇.

ELISA assays were performed using 96-well plates coated with recombinant human VEGF-B ₁₆₇ (2μg/ml, 100μl/well) at +4℃ for 16 hours. After non-specific blocking using 1%BSA at 25℃ for 1 hour, a series of dilutions of test antibodies were added into the coated wells and incubated at 25℃ for 1 hour. The bound Abs were detected using a secondary Ab (goat anti-human IgG1-Fc) conjugated with HRP followed by OD ₄₅₀ reading. The binding affinities of B781401-781406 for recombinant human VEGF-A ₁₆₅ are at sub-nanomolar scales and comparable with aflibercept. The results are shown in Fig. 12.

EXAMPLE 6: Comparison of the ELISA binding affinities of B781401-781406 with aflibercept for recombinant human PlGF.

ELISA assays were performed using 96-well plates coated with recombinant human PlGF (2μg/ml, 100μl/well) at +4℃ for 16 hours. After non-specific blocking using 1%BSA at 25℃ for 1 hour, a series of dilutions of test antibodies were added into the coated wells and incubated at 25℃ for 1 hour. The bound Abs were detected using a secondary Ab (goat anti-human IgG1-Fc) conjugated with HRP followed by OD ₄₅₀ reading. The binding affinities of B781401-781406 for recombinant human PlGF are at sub-nanomolar scales and comparable with aflibercept. The results are shown in Fig. 13.

EXAMPLE 7: Comparison of the ELISA Binding Affinities of B781401-781406 with Tocilizumab for Recombinant Human IL-6R.

ELISA assays were performed using 96-well plates coated with recombinant human IL-6R (2μg/ml, 100μl/well) at +4℃ for 16 hours. After non-specific blocking using 1%BSA at 25℃ for 1 hour, a series of dilutions of test antibodies were added into IL-6R-coated wells and incubated at 25℃ for 1 hour. The bound Abs were detected using a secondary Ab (goat anti-human IgG1-Fc) conjugated with HRP followed by OD ₄₅₀ reading. The binding affinities of B781401-781406 for recombinant human IL-6R are at sub-nanomolar scales and comparable with tocilizumab. The results are shown in Fig. 17.

EXAMPLE 8: Comparison of the Effect of B781401-781406 with Aflibercept on Inhibiting VEGF-A ₁₆₅-Mediated VEGFR2 Signaling in VEGFR2-NFAT-RE Luciferase Reporter Cells.

Recombinant human VEGF-A ₁₆₅ were mixed with serial dilutions of test Abs, incubated at room temperature for 30 minutes, and then added into wells containing 4 x10 ⁴ VEGFR2 (KDR) -NFAT-RE luciferase reporter cells/well followed by an incubation at 37℃for 6 hours. Luciferase signal was detected by a plate reader after addition of 50μl of Bright-Lite. Validation of VEGFR2 signaling luciferase assay using Bevacizumab (Avastin) as a positive control (see Fig. 15A) . The effects of B781401-781406 on inhibiting VEGF-A ₁₆₅ mediated VEGFR2 signaling are comparable to aflibercept (see Fig. 15B) .

EXAMPLE 9: Comparison of the Effect of B781401-781406 with Tocilizumab on Inhibiting IL-6R Signaling.

Measurement of phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) was conducted in TF-1 cells in response to recombinant IL-6 stimuation using a phospho-STAT3 (Tyr705) cellular kit from Cisbio according to the manufacturer’s instructions. Detection of phosphorylated STAT3 involves a sandwich immunoassay which includes an anti-phospho STAT3 antibody coupled with an anti-total antibody labeled with eitherdonor or acceptor fluors. Activation of the IL-6/IL-6R signaling in TF-1 cells causes an increase in Homogeneous Time Resolved Fluorescence (HTRF) signal, whereas inhibition will demonstrate the opposite effect. B781401, B781402, B781404, and B781405 showed comparable effects with tocilizumab on inhibiting phosphorylation of STAT3 in TF-1 cells stimulated by recombinant human IL-6 ligand. The results are shown in Fig. 16.

EXAMPLE 10: Comparison of the Effect of B781401-781406 with Siltuximab and Tocilizumab on Inhibiting IL-6R Signaling.

Measurement of IL-6 mediated TF-1 cell proliferation was conducted using

Luminescent Cell Viability Assay with a kit obtained from Promega according to the manufacturer’s instructions. Activation of IL-6R signaling in TF-1 cells results in TF-1 cell proliferation which is reflected by increased luminescent signal, whereas inhibition of IL-6R signaling will demonstrate the opposite effect. B781404, B781405, and B781406 showed comparable effects on inhibiting TF-1 cell proliferation stimulated by recombinant human IL-6 ligand. Siltuximab is a mAb against IL-6. The results are shown in Fig. 17.

A summary of the profiles of B781401-781406 is shown in Table 5 for the transient expression in HEK293 and CHO cells, ELISA binding affinities for human VEGF-A165, VEGF-B167, PlGF, and IL-6R, as well as inhibition in three cell-based functional assays

Table 5

Summary of the Profiles of B781401-781406

EXAMPLE 11: Comparison of the ELISA Binding Affinities of EB-DJ1, EB-vvA, EB-vvB, and EB-vvC with Aflibercept, B781403, and B781405 for Recombinant Human VEGF-A ₁₆₅.

ELISA assays were performed using 96-well plates coated with recombinant human VEGF-A ₁₆₅ (2μg/ml, 100μl/well) at +4℃ for 16 hours. After non-specific blocking using 1%BSA at 25℃ for 1 hour, a series of dilutions of test antibodies were added into the coated wells and incubated at 25℃ for 1 hour. The bound Abs were detected using a secondary Ab (goat anti-human IgG1-Fc) conjugated with HRP followed by OD ₄₅₀ reading. The binding affinities of EB-DJ1, EB-vvA, EB-vvB, and EB-vvC for recombinant human VEGF-A ₁₆₅ are at sub-nanomolar scales and comparable with aflibercept, B781403 and B781405. The results are shown in Fig. 18.

EXAMPLE 12: Comparison of the ELISA Binding Affinities of EB-DJ1, EB-vvA, EB-vvB, and EB-vvC with Aflibercept, B781402, B781403, and B781405 for Recombinant Human VEGF-B ₁₆₇.

ELISA assays were performed using 96-well plates coated with recombinant human VEGF-B ₁₆₇ (2μg/ml, 100μl/well) at +4℃ for 16 hours. After non-specific blocking using 1%BSA at 25℃ for 1 hour, a series of dilutions of test antibodies were added into VEGF-B ₁₆₇-coated wells and incubated at 25℃ for 1 hour. The bound Abs were detected using a secondary Ab (goat anti-human IgG1-Fc) conjugated with HRP followed by OD ₄₅₀ reading. The binding affinities of EB-DJ1, EB-vvA, EB-vvB, and EB-vvC for recombinant human VEGF-B ₁₆₇ are at sub-nanomolar scales and comparable with aflibercept, B781402, B781403, and B781405. The results are shown in Fig. 19.

EXAMPLE 13: Comparison of the ELISA Binding Affinities of EB-DJ1, EB-vvA, EB-vvB, and EB-vvC with Aflibercept, B781403, and B781405 for Recombinant Human PlGF.

ELISA assays were performed using 96-well plates coated with recombinant human PlGF (2μg/ml, 100μl/well) at +4℃ for 16 hours. After non-specific blocking using 1%BSA at 25℃ for 1 hour, a series of diluted test antibodies were added into the coated wells and incubated at 25℃ for 1 hour. The bound Abs were detected using a secondary Ab (goat anti-human IgG1-Fc) conjugated with HRP followed by OD ₄₅₀ reading. The binding affinities of EB-DJ1, EB-vvA, EB-vvB and EB-vvC for recombinant human PlGF are at sub- nanomolar scales and comparable with aflibercept, B781403 and B781405. The results are shown in Fig. 20.

EXAMPLE 14: Comparison of the ELISA Binding Affinities of EB-DJ1, EB-vvA, EB-vvB, and EB-vvC with Tocilizumab, B781402, B781403, and B781405 for Recombinant Human IL-6R.

ELISA assays were performed using 96-well plates coated with recombinant human IL-6R (2μg/ml, 100μl/well) at +4℃ for 16 hours. After non-specific blocking using 1%BSA at 25℃ for 1 hour, a series of dilutions of test antibodies were added into Il-6R-coated wells and incubated at 25℃ for 1 hour. The bound Abs were detected using a secondary Ab (goat anti-human IgG1-Fc) conjugated with HRP followed by OD ₄₅₀ reading. The binding affinities of EB-DJ1, EB-vvA, EB-vvB, and EB-vvC for recombinant human IL-6R are at sub-nanomolar scales and comparable with tocilizumab, B781402, B781403 and B781405. The results are shown in Fig. 21.

A summary of the profiles of EB-DJ1, EB-vvA, EB-vvB, and EB-vvC in their binding affinities for human VEGF-A165, VEGF-B167, PlGF and IL-6R is shown in Table 6

Table 6

Summary of the Profiles of EB-DJ1, EB-vvA, EB-vvB, and EB-vvC

NUCLEIC ACID AND AMINO ACID SEQUENCE LISTING

ATG CCC CTG CTT CTC CTC TTG CCG TTG CTC TGG GCT GGT GCT

While specific embodiments of the present invention have been described in the foregoing, it will be appreciated by those skilled in the art that many equivalents, modifications, substitutions, and variations may be made thereto without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

An antibody fusion protein or antigen-binding fragment or domain thereof that are capable of binding substantially to IL-6R and one or more VEGF family members.
The antibody fusion protein of claim 1, comprising an antibody against IL-6R or an antigen-biding fragment or domain thereof linked to a VEGF binding unit that comprises an Ig-like domain selected from the group consisting of Ig-like domains of a first VEGF receptor, Ig-like domains of a second VEGF receptor, Ig-like domains of a third VEGF receptor, and combinations thereof.
The antibody fusion protein of claim 1, comprising an antibody against IL-6R or an antigen-biding fragment or domain thereof; wherein at least one of (1) a light chain or antigen-binding fragment or domain thereof, and (2) a heavy chain or antigen-binding fragment or domain thereof is linked to a VEGF binding unit that comprises a plurality of Ig-like domains selected from the group consisting of Ig-like domains of a first VEGF receptor, Ig-like domains of a second VEGF receptor, Ig-like domains of a third VEGF receptor, and combinations thereof.
The antibody fusion protein of claim 3, wherein said IL-6R is human IL-6R.
The antibody fusion protein of claim 4; wherein said VEGF binding unit comprises: (1) (a) Ig-like domain 2, or substantially Ig-like domain 2, of human VEGFR-1; and (b) Ig-like domain 3, or substantially Ig-like domain 3, of human VEGFR-2; or (2) (a) Ig-like domains 1 and 2, or substantially Ig-like domains 1 and 2, of human VEGFR-3; or (b) Ig-like domains 2 and 3, or substantially Ig-like domains 2 and 3, of human VEGFR-3; or (c) Ig-like domains 1, 2 and 3, or substantially Ig-like domains 1, 2, and 3, of human VEGFR-3.
The antibody fusion protein of claim 4; wherein a light chain or an antigen-binding fragment or domain thereof and a heavy chain or an antigen-binding fragment or domain thereof of the IL-6R antibody portion is linked to a VEGF binding unit comprising (a) Ig-like domain 2, or substantially Ig-like domain 2, of human VEGFR-1; and (b) Ig-like domain 3, or substantially Ig-like domain 3, of human VEGFR-2.
The antibody fusion protein of claim 4; wherein (1) a light chain or an antigen-binding fragment or domain thereof of the IL-6R antibody portion is linked to a first VEGF binding unit comprising (a) Ig-like domain 2, or substantially Ig-like domain 2, of human VEGFR-1; and (b) Ig-like domain 3, or substantially Ig-like domain 3, of human VEGFR-2; and (2) a heavy chain or an antigen-binding fragment or domain thereof of the antibody fusion protein is linked to a second VEGF binding unit comprising (a) Ig-like domains 1 and 2, or substantially Ig-like domains 1 and 2, of human VEGFR-3; or (b) Ig-like domains 2 and 3, or substantially Ig-like domains 2 and 3, of human VEGFR-3; or (c) Ig-like domains 1, 2 and 3, or substantially Ig-like domains 1, 2, and 3, of human VEGFR-3.
The antibody fusion protein of claim 6; wherein the heavy chain thereof has complementarity determining region HCCDR1, HCCDR2, and HCCDR3 amino acid sequences of SEQ ID NO: 53, 54, and 55.
The antibody fusion protein of claim 6; wherein the light chain thereof has complementarity determining region LCCDR1, LCCDR2, and LCCDR3 amino acid sequences of SEQ ID NO: 56, 57, and 58.
The antibody fusion protein of claim 6; wherein a pair of heavy chain and light chain thereof has amino acid sequences selected from the group consisting of SEQ ID NO: 34 and 36, SEQ ID NO: 38 and 40, SEQ ID NO: 42 and 44, SEQ ID NO: 46 and 48, SEQ ID NO: 78 and 80, SEQ ID NO: 82 and 84, SEQ ID NO: 86 and 88 SEQ ID NO: 90 and 92, SEQ ID NO: 94 and 96, and SEQ ID NO: 98 and 100.
The antibody fusion protein of claim 2, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 74, and SEQ ID NO: 76.
The antibody fusion protein of claim 2, comprising an amino acid sequence that begins at amino acid 17 of an amino acid sequence selected from the group consisting of SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 74, and SEQ ID NO: 76.
The antibody fusion protein of any one of claims 1-12; wherein said antibody fusion protein binds to IL-6R and a VEGF family member with dissociation constant K _d ≤ 10 ^-9 M.
The antibody fusion protein of any one of claims 1-12; wherein said antibody fusion protein binds to IL-6R and a VEGF family member with dissociation constant K _d ≤ 10 ^-10 M
An isolated nucleic acid molecule encoding an antibody fusion protein of any of claims 1-12.
An isolated nucleic acid molecule having a sequence substantially as listed in SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 31, 59, 61, 63, 65, 75, or 77.
A vector comprising the nucleic acid molecule of claim 15 or 16.
The vector of claim 17, comprising said nucleic molecule operatively linked to an expression control sequence.
A host-vector system comprising the expression vector of claim 18 in a host cell.
A host-vector system comprising an expression vector pair comprising a pair of nucleic acid sequences selected from the group consisting of: SEQ ID NO: 17 and 19; SEQ ID NO: 21 and 23; SEQ ID NO: 25 and 27; SEQ ID NO: 29 and 31; SEQ ID NO: 79 and 81; SEQ ID NO: 83 and 85; SEQ ID NO: 87 and 89; SEQ ID NO: 91 and 93; SEQ ID NO: 95 and 97; and SEQ ID NO: 99 and 101.
A host-vector system comprising an expression vector comprising a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 75, and SEQ ID NO: 77.
A method of producing a substantially purified antibody fusion protein, which method comprises: (a) growing cells of the host-vector system of claim 20 or 21 under conditions permitting production of the antibody fusion protein; and (b) recovering the antibody fusion protein to produce a recovered antibody fusion protein; and (c) purifying said recovered antibody fusion protein to produce the substantially purified antibody fusion protein.
A method for treating or controlling at least a disease, condition, or disorder, in a subject in need thereof, which has etiology in aberrant angiogenesis or inflammation; wherein said method comprises administering to said subject an amount of a composition of an antibody fusion protein comprising amino acid sequences substantially as listed in SEQ ID NO: 34 and 36; SEQ ID NO: 38 and 40; SEQ ID NO: 42 and 44; SEQ ID NO: 46 and 48; SEQ ID NO: 78 and 80; SEQ ID NO: 82 and 84; SEQ ID NO: 86 and 88; SEQ ID NO: 90 and 92; SEQ ID NO: 94 and 96; SEQ ID NO: 98 and 100; SEQ ID NO: 60; SEQ ID NO: 62; SEQ ID NO: 64; SEQ ID NO: 66; SEQ ID NO: 74; or SEQ ID NO: 76.
The method of claim 23; wherein said disease, condition, or disorder is selected from the group consisting of: choroidal neovascularization, neovascular age-related macular degeneration, polypoidal choroidal vasculopathy, myopic choroidal neovascularization, vascular leak, macular edema, non-proliferative and proliferative diabetic retinopathy, corneal neovascularization, corneal inflammation, myopic neovascularization, and neovascular glaucoma.
The method of claim 24; wherein the subject is administered with a dose of about 25-4000 micrograms of the antibody fusion protein.
The method of claim 25; wherein the composition is administered to the subject as an eye drop, a punctal plug, intracameral, retrobulbar, subconjunctival, peribulbar, subtenon, juxta-scleral, trans-scleral, intravitreal, subretinal, or suprachoroidal injection.
The method of claim 25; wherein the composition is administered to the subject for a period of at least one month if intraocularly administered.
The method of claim 25; wherein the composition is administered to the subject at a frequency of at least once per month if intraocularly administered.
A method for treating or controlling at least a systemic disease, condition, or disorder, in a subject in need thereof, which has etiology in aberrant angiogenesis or inflammation; wherein said method comprises administering to said subject an amount of a composition of an antibody fusion protein or antigen-binding thereof that is capable of binding substantially to IL-6R and one or more VEGF family members.
A method for treating or controlling at least a systemic disease, condition, or disorder, in a subject in need thereof, which has etiology in aberrant angiogenesis or inflammation; wherein said method comprises administering to said subject an amount of a composition of an antibody fusion protein comprising amino acid sequences substantially as listed in SEQ ID NO: 34 and 36; SEQ ID NO: 38 and 40; SEQ ID NO: 42 and 44; SEQ ID NO: 46 and 48; SEQ ID NO: 78 and 80; SEQ ID NO: 82 and 84; SEQ ID NO: 86 and 88; SEQ ID NO: 90 and 92; SEQ ID NO: 94 and 96; SEQ ID NO: 98 and 100; SEQ ID NO: 60; SEQ ID NO: 62; SEQ ID NO: 64; SEQ ID NO: 66; SEQ ID NO: 74; and SEQ ID NO 76.
The method of claim 30; wherein said systemic disease, condition, or disorder involves tumor growth, tumor metastasis, or both.
The method of claim 30; wherein said systemic disease, condition, or disorder is atherosclerosis.
The method of claim 30; wherein said systemic disease, condition, or disorder is psoriasis.