WO2024114641A1

WO2024114641A1 - C5/vegf bispecific binding molecules

Info

Publication number: WO2024114641A1
Application number: PCT/CN2023/134770
Authority: WO
Inventors: Chiungkuang CHEN
Original assignee: Shenzhen Oculgen Biomedical Technology Co., Ltd
Priority date: 2022-11-28
Filing date: 2023-11-28
Publication date: 2024-06-06

Abstract

The present disclosure provides bispecific binding molecules against human vascular endothelial growth factor (VEGF/VEGF-A) and against human complement 5 (C5), including protein sequences, methods for their production, pharmaceutical compositions containing the bispecific binding molecules, and uses thereof.

Description

C5/VEGF BISPECIFIC BINDING MOLECULES

FIELD OF THE INVENTION

The present invention relates to bispecific binding molecules against human vascular endothelial growth factor (VEGF/VEGF-A) and against complement component 5 (C5) , methods for preparation and therapeutic uses thereof.

BACKGROUND

Age-related macular degeneration (AMD) is the leading cause of blindness in elderly (Rein et al., Arch Ophthalmology, 127: 533-540, 2009) . AMD is typically a disease of the elderly and is the leading cause of blindness in individuals >50 years of age in developed countries. In the absence of adequate prevention or treatment measures, the number of cases of AMD with visual loss is expected to grow in parallel with the aging population.

AMD is characterized by progressive degeneration of the photoreceptors, outer retina, and retinal pigment epithelium at the macula. Advanced AMD occurs in two forms, dry (atrophic) and wet AMD. Anti-VEGF agents (e.g. anti-VEGF antibodies, VEGF Traps, etc. ) are now widely used in the treatment of wet AMD, by inhibition of neovascularization and angiogenesis. In most patients this can reduce the progression of choroidal neovascularization and vision loss resulting from the downstream effects of neovascularization, and has been observed to reduce some aspects of inflammation in animal models.

Late stages of AMD is either characterized by choroidal neovascularization, or Geographic Atrophy (GA) . GA tends to affect more than 20%of AMD patients. More than 8 million patients worldwide are effected by GA, or at least one eye from AMD patients is diagnosed with GA. Currently there is no effective treatment to treat or even slow down the progression of the GA symptom such as missing some letters while reading or missing portion of eye vision, require additional light for reading or normal activities in the dark and lost visual resolution requiring detailed vision (faces recognition and visual color fading due to death center of vision retinal pigment epithelial (RPE) cells) .

Anti-VEGF therapies are not effective in treating GA (Park, D.H . et al, Front. Immunol., 15 May 2019) . It was even reported that anti-VEGF treatment can potentially increase development of GA (Gemenetzi, M., et al, Eye (Lond) . 2017 Jan; 31 (1) : 1–9) . Complement inhibitors targeting modulation of complement proteins C3, C5, factor B, factor D, and properdin were under study for their potential to treat GA, but there has been no success yet. In a phase II clinical trial (COMPLETE, NCT00935883) , an anti-C5 antibody eculizumab failed to decrease the growth rate of GA significantly.

Therefore, there exists significant clinical therapeutic needs for the treatment of AMD and GA, and in particular for prevention of progression from AMD to geographic atrophy (GA) .

SUMMARY OF THE INVENTION

The present disclosure provides multi-specific molecules capable of inhibiting VEGF and/or C5. In some embodiment, the multi-specific molecules are designed to inhibit C5 related and/or a VEGF related disease. Methods for producing the multi-specific molecules, including the processes involving nucleic acids, vectors, expression vectors, and host vector system, are also disclosed.

In one aspect, the present disclosure provides a multi-specific molecule comprising a fusion protein comprising: (a) a complement component 5 (C5) binding domain, (b) a vascular endothelial growth factor (VEGF) binding domain, and (c) a multimerizing component; wherein: the C5 binding domain comprises an antigen-binding fragment of an anti-C5 antibody, the VEGF binding domain comprises one or more extracellular immunoglobulin-like (Ig) domain of one or more VEGF receptor (VEGFR) , and the multimerizing component comprises a polypeptide having a length between 1 and 200 amino acids and having at least one cysteine residue.

In another aspect, the present disclosure provides a pharmaceutical composition comprising the multi-specific molecule provided herein, and one or more pharmaceutically acceptable carriers.

In another aspect, the present disclosure provides an isolated polynucleotide encoding the multi-specific molecule provided herein.

In another aspect, the present disclosure provides a vector comprising the isolated polynucleotide provided herein.

In another aspect, the present disclosure provides a host expression system comprising the vector provided herein.

In another aspect, the present disclosure provides a method of expressing the multi-specific molecule provided herein, comprising culturing the host expression system provided herein under the condition at which the vector provided herein is expressed.

In another aspect, the present disclosure provides a method of treating, preventing or alleviating a C5 related and/or a VEGF related disease, disorder or condition in a subject, comprising administering to the subject a therapeutically effective amount of the multi-specific molecule provided herein and/or the pharmaceutical composition provided herein.

In another aspect, the present disclosure provides a method of treating, preventing or alleviating a disease, disorder or condition associated with an increased level and/or activity of C5 and/or VEGF in a subject, comprising administering to the subject a therapeutically effective amount of the multi-specific molecule provided herein and/or the pharmaceutical composition provided herein.

In another aspect, the disease, disorder or condition is selected from the group consisting of ocular disease, cancer, inflammatory disease, autoimmune disease, angiogenesis, vascular permeability, edema, and inflammation.

In another aspect, the present disclosure provides a method of modulating C5 and/or VEGF activity in a cell, comprising exposing the cell to the multi-specific molecule provided herein.

In another aspect, the present disclosure provides a multi-specific molecule provided herein and/or the pharmaceutical compositions provided herein for use in treating, preventing or alleviating a C5 related and/or a VEGF related disease, disorder or condition in a subject.

In another aspect, the present disclosure provides the use of the multi-specific molecule provided herein and/or the pharmaceutical composition provided herein in the manufacture of a medicament for treating, preventing or alleviating a C5 related and/or a VEGF related disease, disorder or condition in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the first structure of VEGF/C5 bispecific antibodies (BSP1, BSP1a) .

Figure 2 depicts the second structure of VEGF/C5 bispecific antibodies (BSP2) .

Figure 3 depicts the third structure of VEGF/C5 bispecific antibodies (BSP3) .

Figure 4 depicts the fourth structure of VEGF/C5 bispecific antibodies (BSP4) .

Figure 5 depicts the fifth structure of VEGF/C5 bispecific antibodies (BSP5) .

Figure 6 depicts the sixth structure of VEGF/C5 bispecific antibodies (BSP6) .

Figure 7 and 8 shows the Dose-response curves of BSP1 and PC1 (Aflibercept) in duplicate trials of VEGF-mediated cell proliferation assay.

Figure 9A and 9B shows the Dose-response curves of BSP1 and PC2 (eculizumab) in duplicate trials of CH50 assay.

Figure 10 shows the chronic animal/PD model of vascular leakage score vs time with S. D.

Figure 11 shows the chronic animal/PD model vascular leakage progression over time (0, 2, 4 and 8 weeks) .

Figure 12 shows the chronic animal/PD model vascular leakage progression over time (0, 12 and 16 weeks) .

Figure 13 shows the effect of BSP1 on the eye over time (0, 2, 4, 8, 12 and 16 weeks) .

Figure 14 shows the effect of PBS on the eye over time (0, 2, 4, 8, 12 and 16 weeks) .

Figure 15 shows the SEC-HPLC chromatograms of sample (BSP1) at 40℃in the single clone drug stability test.

Figure 16 shows CE-SDS-NR electropherograms of the sample/BSP1 at 40℃ in the single clone drug stability test.

Figure 17 shows icIEF electropherograms of the sample/BSP1 at 40℃ in the single clone drug stability test.

Figure 18 shows SEC-HPLC chromatograms of the sample/BSP1 at 5 ℃ in the single clone drug stability test.

Figure 19 shows CE-SDS-NR electropherograms of the sample/BSP1 at 5 ℃ in the single clone drug stability test.

Figure 20 shows icIEF electropherograms of the sample/BSP1 at 5 ℃ in the single clone drug stability test.

Figure 21 shows certain sequences disclosed in the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

Before the detailed description of the inventions is provided, the following are noted and defined.

All the description provided herein is merely intended to illustrate various embodiments of the inventions provided in the present disclosure. As such, the specific modifications discussed are not to be construed as limitations on the scope of the disclosure. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the disclosure, and it is understood that such equivalent embodiments are to be included herein.

All references cited in the present disclosure, including patent applications, issued patents, published articles or other publications, are incorporated by reference in their entirety, which are for the purpose of providing methodologies that might be used in connection with the description provided herein. With respect to any term that is presented in one or more publications that is similar to, or identical with, a term that has been expressly defined in this disclosure, the definition of the term as expressly provided in this present disclosure will control in all respects.

All technical and scientific terms used, unless expressly defined otherwise, in this present disclosure, are generally deemed to have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.

As used herein, i.e., throughout the whole disclosure, the articles “a, ” “an, ” and “the” are to be construed to mean “one or more” or “at least one” unless specified otherwise. By way of example, “amolecule” means one molecule or more than one molecule.

As used herein, the terms “about, ” “approximately, ” “around” or alike, refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1%to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In particular embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 10%, 5%, or 1%.

As used herein, the terms “comprise, ” “comprises, ” “comprising, ” “include, ” “includes, ” “including, ” “contain, ” “contains, ” “containing” , “have, ” “has, ” “having” and the like, are synonymous and are used inclusively, in an open-ended fashion, and do not exclude additional elements, features, steps, acts, operations, and so forth.

As used herein, the term “or” is used in its inclusive sense (and not in its exclusive sense) so that when used, for example, to connect a list of elements, the term “or” means one, some, or all of the elements in the list.

As used herein, the phrase “at least one” means one or more, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

I. Definitions and Abbreviations

In this section, definitions for some general terms are provided. Definition for other terms may be found in other sections of the disclosure that follow.

The terms “polypeptide” , “peptide” , and “protein” are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds, which includes proteins, polypeptides, oligopeptides, peptides, and fragments thereof. The protein may be made up of naturally occurring amino acids and/or synthetic (e.g., modified or non-naturally occurring) amino acids. Thus “amino acid” , or “peptide residue” , as used herein means both naturally occurring and synthetic amino acids. The terms “polypeptide” , “peptide” , and “protein” includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; fusion proteins with detectable fusion partners, e.g., fusion proteins including as a fusion partner a fluorescent protein, β-galactosidase, luciferase, etc.; and the like.

As used herein, the term “amino acid” refers to a building block of a protein, a peptide, a polypeptide or an amino acid polymer, and the term “amino acid” further refers to a naturally occurring or synthetic amino acid, as well as any amino acid analog and amino acid mimetics that functions in a manner similar to the naturally occurring amino acid. Naturally occurring amino acids are those encoded by the genetic codes, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. As used within this application, naturally occurring amino acids include the group of naturally occurring carboxy alpha-amino acids comprising alanine (three letter code: Ala, one letter code: A) , arginine (Arg, R) , asparagine (Asn, N) , aspartic acid (Asp, D) , cysteine (Cys, C) , glutamine (Gln, Q) , glutamic acid (Glu, E) , glycine (Gly, G) , histidine (His, H) , isoleucine (Ile, I) , leucine (Leu, L) , lysine (Lys, K) , methionine (Met, M) , phenylalanine (Phe, F) , proline (Pro, P) , serine (Ser, S) , threonine (Thr, T) , tryptophan (Trp, W) , tyrosine (Tyr, Y) , and valine (Val, V) .

As used herein, the term “domain” refers to a globular structure formed by one or more regions of one or more polypeptide chains comprising peptide loops (e.g., comprising 3 to 4 peptide loops) that are stabilized, for example, by β-pleated sheet and/or intrachain disulfide bond (s) . Examples may include a Fab domain (see below for more details) . It is noted that in the present disclosure, the two terms “domain” and “region” may be used interchangeably.

As used herein, the term “nucleic acid, ” “nucleic acid molecule, ” “nucleotide, ” “polynucleotide” or alike, is construed to refer to a nucleotide polymer of any length, and can include both DNA and RNA, and can be single-stranded or double-stranded.

As used herein, the term “percent (%) sequence identity” is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids (or nucleic acids) . In other words, percent (%) sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of amino acid residues (or bases) that are identical relative to the reference sequence to which it is being compared by the total number of the amino acid residues (or bases) in the candidate sequence or in the reference sequence, whichever is shorter. Conservative substitution of the amino acid residues is not considered as identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI) , see also, Altschul S.F. et al, J. Mol. Biol., 215: 403–410 (1990) ; Stephen F. et al, Nucleic Acids Res., 25: 3389–3402 (1997) ) , ClustalW2 (available on the website of European Bioinformatics Institute, see also, Higgins D.G. et al, Methods in Enzymology, 266: 383-402 (1996) ; Larkin M.A. et al, Bioinformatics (Oxford, England) , 23 (21) : 2947-8 (2007) ) , and ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by the tool, or may customize the parameters as appropriate for the alignment, such as for example, by selecting a suitable algorithm.

A “conservative substitution” with reference to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties. For example, conservative substitutions can be made among amino acid residues with hydrophobic side chains (e.g. Met, Ala, Val, Leu, and Ile) , among residues with neutral hydrophilic side chains (e.g. Cys, Ser, Thr, Asn and Gln) , among residues with acidic side chains (e.g. Asp, Glu) , among amino acids with basic side chains (e.g. His, Lys, and Arg) , or among residues with aromatic side chains (e.g. Trp, Tyr, and Phe) . As known in the art, conservative substitution usually does not cause significant change in the protein conformational structure, and therefore could retain the biological activity of a protein.

The term “antibody” as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, or bispecific antibody that binds to a specific antigen. The term antibody as used herein is construed broadly to encompass conventional immunoglobulin comprising two heavy (H) chains and two light (L) chains, as well as unconventional antibodies such as heavy chain antibodies comprising heavy chain only. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each heavy chain consists of a variable region (V_H) and a first, second, third, and optionally fourth constant region (C_H1, C_H2, C_H3, C_H4 respectively) ; mammalian light chains are classified as λ or κ, while each light chain consists of a variable region (V_L) and a constant region. The antibody has a “Y” shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together via disulfide bonding. Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2, and LCDR3, heavy chain CDRs including HCDR1, HCDR2, HCDR3) . CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273 (4) , 927 (1997) ; Chothia, C. et al., J Mol Biol. Dec 5; 186 (3) : 651-63 (1985) ; Chothia, C. and Lesk, A.M., J. Mol. Biol., 196, 901 (1987) ; Chothia, C. et al., Nature. Dec 21-28; 342 (6252) : 877-83 (1989) ; Kabat E.A. et al., Sequences of Proteins of immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) ; Marie-Paule Lefranc et al, Developmental and Comparative Immunology, 27: 55-77 (2003) ; Marie-Paule Lefranc et al, Immunome Research, 1 (3) , (2005) ; Marie-Paule Lefranc, Molecular Biology of B cells (second edition) , chapter 26, 481-514, (2015) ) . The three CDRs are interposed between flanking stretches known as framework regions (FRs) , which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen-binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgG1 (gamma1 heavy chain) , IgG2 (gamma2 heavy chain) , IgG3 (gamma3 heavy chain) , IgG4 (gamma4 heavy chain) , IgA1 (alpha1 heavy chain) , or IgA2 (alpha2 heavy chain) .

An “antigen” as used herein refers to a compound, composition, peptide, polypeptide, protein or substance (e.g., polypeptide, carbohydrate, nucleic acid, lipid, or other naturally occurring or synthetic compound) that can be specifically recognized and bound by a component of the immune system, e.g., an antibody. As used herein, the term “antigen” encompasses antigenic epitopes, e.g., fragments of an antigen which are antigenic epitopes.

The term “bivalent” as used herein refers to an antibody or an antigen-binding fragment having two antigen-binding sites; the term “monovalent” refers to an antibody or an antigen-binding fragment having only one single antigen-binding site; and the term “multivalent” refers to an antibody or an antigen-binding fragment having multiple antigen-binding sites. In some embodiments, the antibody or antigen-binding fragment thereof is bivalent.

As used herein, the term “multi-specific molecule” refers to an artificial or engineered molecule that can simultaneously bind to at least two different epitopes. The two epitopes may present on the same antigen, or they may present on two different antigens. A bispecific molecule is substantially a type of a multi-specific molecule.

The term “antigen-binding fragment” as used herein refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure. Examples of antigen-binding fragment include, without limitation, a diabody, a Fab, a Fab', a F (ab') ₂, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) ₂, a bispecific dsFv (dsFv-dsFv') , a disulfide stabilized diabody (ds diabody) , a single-chain antibody molecule (scFv) , an scFv dimer (bivalent diabody) , a bispecific antibody, a multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody binds.

“Fab” with regard to an antibody refers to that portion of the antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by a disulfide bond.

“Fab'” refers to a Fab fragment that includes a portion of the hinge region.

“F (ab') ₂” refers to a dimer of Fab’.

“Fv” with regard to an antibody refers to the smallest fragment of the antibody to bear the complete antigen-binding site. An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

A “dsFv” refers to a disulfide-stabilized Fv fragment that the linkage between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some embodiments, a “ (dsFv) ₂” or “ (dsFv-dsFv') ” comprises three peptide chains: two V_H moieties linked by a peptide linker (e.g. a long flexible linker) and bound to two V_L moieties, respectively, via disulfide bridges. In some embodiments, dsFv-dsFv' is bispecific in which each disulfide paired heavy and light chain has a different antigen specificity.

“Single-chain Fv antibody” or “scFv” refers to a multi-specific molecule consisting of a light chain variable region and a heavy chain variable region connected to one another directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85: 5879 (1988) ) .

“Fc” with regard to an antibody (e.g. of IgG, IgA, or IgD isotype) refers to that portion of the antibody consisting essentially of the second and third constant domains of a first heavy chain bound to the second and third constant domains of a second heavy chain via disulfide bonding. Fc with regard to antibody of IgM and IgE isotype further comprises a fourth constant domain. The Fc portion of the antibody is responsible for various effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) , and complement dependent cytotoxicity (CDC) , but does not function in antigen binding.

“Single-chain Fv-Fc antibody” or “scFv-Fc” refers to a multi-specific molecule consisting of a scFv connected to the Fc region of an antibody.

“Camelized single domain antibody, ” “heavy chain antibody, ” or “HCAb” refers to an antibody that contains two V_H domains and no light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231 (1-2) : 25-38 (1999) ; Muyldermans S., J Biotechnol. Jun; 74 (4) : 277-302 (2001) ; WO94/04678; WO94/25591; U.S. Patent No. 6,005,079) . Heavy chain antibodies were originally derived from Camelidae (camels, dromedaries, and llamas) . Although devoid of light chains, camelized antibodies have an authentic antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3; 363 (6428) : 446-8 (1993) ; Nguyen VK. et al. Immunogenetics. Apr; 54 (1) : 39-47 (2002) ; Nguyen VK. et al. Immunology. May; 109 (1) : 93-101 (2003) ) . The variable domain of a heavy chain antibody (VHH domain) represents the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. Nov; 21 (13) : 3490-8. Epub 2007 Jun 15 (2007) ) .

A “nanobody” refers to an antibody fragment that consists of a VHH domain from a heavy chain antibody and two constant domains, CH2 and CH3.

“Diabodies” or “dAbs” include small antibody fragments with two antigen-binding sites, wherein the fragments comprise a V_H domain connected to a V_L domain in the same polypeptide chain (V_H-V_L or V_L-V_H) (see, e.g. Holliger P. et al., Proc Natl Acad Sci U S A. Jul 15; 90 (14) : 6444-8 (1993) ; EP404097; WO93/11161) . By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain, thereby creating two antigen-binding sites. The antigen–binding sites may target the same or different antigens (or epitopes) . In certain embodiments, a “bispecific ds diabody” is a diabody target two different antigens (or epitopes) . In certain embodiments, an “scFv dimer” is a bivalent diabody or bivalent ScFv (BsFv) comprising V_H-V_L (linked by a peptide linker) dimerized with another V_H-V_L moiety such that V_H's of one moiety coordinate with the V_L's of the other moiety and form two binding sites which can target the same antigens (or epitopes) or different antigens (or epitopes) . In other embodiments, an “scFv dimer” is a bispecific diabody comprising V_H1-V_L2 (linked by a peptide linker) associated with V_L1-V_H2 (also linked by a peptide linker) such that V_H1 and V_L1 coordinate and V_H2 and V_L2 coordinate and each coordinated pair has a different antigen specificity.

A “domain antibody” refers to an antibody fragment containing only the variable region of a heavy chain or the variable region of a light chain. In certain instances, two or more VH domains are covalently joined with a peptide linker to create a bivalent or multivalent domain antibody. The two VH domains of a bivalent domain antibody may target the same or different antigens.

The term “vector” as used herein refers to a vehicle into which a genetic element may be operably inserted so as to bring about the expression of that genetic element, such as to produce the protein, RNA or DNA encoded by the genetic element, or to replicate the genetic element. A vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the genetic element it carries within the host cell. Examples of vectors include plasmids, phagemids, cosmids, and artificial chromosomes such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , or P1-derived artificial chromosome (PAC) , bacteriophages such as lambda phage or M13 phage, and animal viruses. A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. A vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating. A vector can be an expression vector or a cloning vector. The present disclosure provides vectors (e.g., expression vectors) containing the nucleic acid sequence provided herein encoding the antibody or an antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker.

The phrase “host cell” as used herein refers to a cell into which an exogenous polynucleotide and/or a vector can be or has been introduced.

The phrase “operably linked” refers to juxtaposition such that the normal function of the components can be performed.

II. C5/VEGFR Multi-Specific Molecules

In one aspect, the present disclosure provides a multi-specific molecule comprising a fusion protein comprising: (a) a complement component 5 (C5) binding domain, (b) a vascular endothelial growth factor (VEGF) binding domain, and (c) a multimerizing component. In certain embodiments, the multi-specific molecule is bispecific.

The fusion protein as used herein can be a single chain polypeptide or a polypeptide complex comprising two or more polypeptide chains associated together.

A. Antigen-binding fragment

In certain embodiments, the fusion protein comprises a complement component 5 (C5) binding domain, wherein the C5 binding domain comprises an antigen-binding fragment of an anti-C5 antibody.

C5 is a component of the complement system, a part of the innate immune system that plays an important role in inflammation, host homeostasis, and host defense against pathogens. The C5 protein includes C5 alpha and beta chains which are linked by a disulfide bridge. The C5 protein can be proteolytically processed to generate multiple protein products, including C5 alpha chain, C5 beta chain, C5a anaphylatoxin and C5b.

An “anti-C5 antibody” is an antibody capable of specifically binding to C5, for example human C5. The anti-C5 antibody can be a conventional IgG antibody, or alternatively can be a single domain antibody, such as camelized single domain antibody comprising a heavy chain variable (VH) region, or a llama anti-C5 single domain antibody.

In certain embodiments, the anti-C5 antibody comprises eculizumab or is derived from eculizumab.

In certain embodiments, the antigen-binding fragment of the anti-C5 antibody comprises one or more complementarity determining regions (CDRs) contained in the heavy chain variable region and the light chain variable region of eculizumab. In certain embodiments, the antigen-binding fragment of the anti-C5 antibody comprises three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable (VH) region comprising the amino acid sequence as set forth in SEQ ID NO: 1, and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) contained within a light chain variable (VL) region comprising the amino acid sequence as set forth in SEQ ID NO: 2.

In certain embodiments, the antigen-binding fragment of the anti-C5 antibody comprises the six CDRs of eculizumab. In certain embodiments, the antigen-binding fragment of the anti-C5 antibody provided herein comprises a HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 3, a HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 4, a HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 5, a LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 6, a LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 7, and a LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 8.

Table 1A. Sequences of eculizumab

In certain embodiments, the antigen-binding fragment of the anti-C5 antibody is derived from or comprises a variant of eculizumab, for example without limitation, an affinity variant of eculizumab, or a glycosylation variant of eculizumab.

The term “variant” with respect to a parent antibody refers to any antibody which has a structure or sequence derived from the parent antibody and whose structure/sequence is sufficiently similar to those in the parent antibody. Modifications to obtain a variant includes, for example, addition, deletion and/or substitution of one or more of the amino acid residues. The variant may have one or more conservative amino acid substitutions.

In certain embodiments, the antigen-binding fragment of the anti-C5 antibody is derived from a variant of eculizumab, and comprises one or more amino acid residue substitutions or modifications relative to eculizumab, yet retains binding specificity and/or affinity to C5. The substitutions or modifications can be in one or more CDR sequences and/or VH and/or VL sequences of eculizumab.

In certain embodiments, the antigen-binding fragment of the anti-C5 antibody provided herein comprises one or more amino acid residue substitutions in one or more of the CDR sequences, and/or one or more of the FR sequences of eculizumab. In certain embodiments, the antigen-binding fragment of the anti-C5 antibody provided herein comprises no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitution (s) in the CDR sequences and/or FR sequences listed in Table 1A in total.

In certain embodiments, the variant of eculizumab, or the antigen-binding fragment derived from such a variant, has comparable or improved affinity to human C5, relative to the parent antibody eculizumab.

The term “affinity” as used herein refers to the strength of non-covalent interaction between an antibody or fragment thereof and an antigen. Affinity of an antibody to an antigen can be measured by the equilibrium dissociation constant K_D using methods well known in the art (see, generally, Davies et al. Ann. Rev. Biochem. 1990, 59: 439-15 473) . For instance, Biacore is a classic device for detecting molecular interactions based on surface plasmon resonance (SPR) technology and the gold standard for antibody affinity determination. It is well known in the art that enzyme-linked immunosorbent assay (ELISA) is also a common mean to determine the binding affinity.

In certain embodiments, the variant of eculizumab or the antigen-binding fragment derived from such a variant is capable of specifically binding to C5 at a KD value of no more than 10 nM (e.g. no more than 8 nM, no more than 5 nM, no more than 2 nM, no more than 1 nM, no more than 800 pM, no more than 700 pM, no more than 600 pM, no more than 500 pM, no more than 400 pM, or no more than 300 pM) as measured by Biacore.

Methods are known in the art to make and obtain an affinity variant of a parent antibody. For example, variants can be screened for its binding affinity to the intended target (e.g. human C5) , and variant with high affinity for the antigen can be identified.

In some embodiments, the variant of eculizumab or a fusion protein derived from such a variant of the invention have improved druggability properties, for example, when expressed in mammalian cells such as CHO cells, have one or more properties selected from: (i) superior expression than wild-type eculizumab or the fusion protein thereof, (ii) higher feasibility of purification to high purity, and (iii) higher stability.

In some embodiments of the invention, the variant of eculizumab or the fusion protein thereof exhibit increased expression levels compared to wild-type eculizumab or the fusion protein thereof. In some embodiments, the increased expression occurs in a mammalian cell expression system. Expression levels can be determined by any suitable method that allows quantitative or semi-quantitative analysis of the amount of the variant of eculizumab or the fusion protein thereof in cell culture supernatants, preferably after one-step affinity chromatography purification. For example, the amount of the variant of eculizumab or the fusion protein thereof in a sample can be assessed by Western blot or ELISA. In some embodiments, the expression level of the variant of eculizumab or the fusion protein thereof in mammalian cells is increased by at least 1.1 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times times, at least 5 times, at least 10 times, at least 20 times, or at least 30 times more, compared with wild-type eculizumab or the fusion protein thereof.

In some embodiments, the variant of eculizumab or the fusion protein thereof of the present invention exhibits greater purity relative to the wild-type of eculizumab or the fusion protein thereof. In some embodiments, the protein purity is determined by the SEC-HPLC, CE-SDS-NR, or icIEF technique. In some preferred embodiments, after purification, the variant of eculizumab or the fusion protein thereof can reach a purity of more than 65%, 70%, 75%, 80%, or 85%, preferably more than 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, or 99%.

In one embodiment, the variant of eculizumab or the fusion protein thereof have improved thermostability at a temperature ranging from -20 ℃ to 60 ℃. In one embodiment, the variant of eculizumab or the fusion protein thereof have no significant aggregation and degradation at a temperature ranging from -20 ℃ to 60℃. In one embodiment, the variant of eculizumab or the fusion protein thereof have no significant aggregation and degradation at 5 ℃. In one embodiment, the variant of eculizumab or the fusion protein thereof have no significant aggregation and degradation at 40 ℃. In one embodiment, the variant of eculizumab or the fusion protein thereof have a degree of aggregation or degradation less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%, after 14 days storage at 5 ℃.

In certain embodiments, the variant of eculizumab provided herein comprises an HCDR1 having no more than 3, 2, or 1 amino acid substitutions in SEQ ID NO: 3, an HCDR2 having no more than 3, 2, or 1 amino acid substitutions in SEQ ID NO: 4, HCDR3 having no more than 3, 2, or 1 amino acid substitutions in SEQ ID NO: 5, LCDR1 having no more than 3, 2 or 1 amino acid substitution in SEQ ID NO: 6, LCDR2 having no more than 3, 2, or 1 amino acid substitution in SEQ ID NO: 7, and/or LCDR3 having no more than 3, 2, or 1 amino acid substitutions in SEQ ID NO: 8.

In certain embodiments, the antigen-binding fragment of the anti-C5 antibody provided herein comprises a HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 3, a HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 4, a HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 5 or SEQ ID NO: 27, a LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 6 or SEQ ID NO: 29 or SEQ ID NO: 41 or SEQ ID NO: 38, a LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 7, and a LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 8 or SEQ ID NO: 30 or SEQ ID NO: 35.

In certain embodiments, the antigen-binding fragment of the anti-C5 antibody provided herein comprises a HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 3, a HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 4, a HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 27, a LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 7, and a LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 30.

In certain embodiments, the antigen-binding fragment of the anti-C5 antibody provided herein comprises a HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 3, a HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 4, a HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 27, a LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 41, a LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 7, and a LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 35.

. In certain embodiments, the antigen-binding fragment of the anti-C5 antibody provided herein comprises a HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 3, a HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 4, a HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 5, a LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 38, a LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 7, and a LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 30.

Table 1B. Sequences of eculizumab variants

In certain embodiments, the antigen-binding fragment of the anti-C5 antibody comprises a VH region comprising the amino acid sequence as set forth in SEQ ID NO: 1, or a homologous sequence thereof having at least 80%sequence identity to SEQ ID NO: 1 yet retaining binding specificity to C5.

In certain embodiments, the antigen-binding fragment of the anti-C5 antibody comprises a VL region comprising an amino acid sequence as set forth in SEQ ID NO: 2, or a homologous sequence thereof having at least 80%sequence identity to SEQ ID NO: 2 yet retaining binding specificity to C5.

In certain embodiments, the antigen-binding fragment of the anti-C5 antibody comprises a VH region comprising the amino acid sequence as set forth in SEQ ID NO: 28, or a homologous sequence thereof having at least 80%sequence identity to SEQ ID NO: 28 yet retaining binding specificity to C5.

In certain embodiments, the antigen-binding fragment of the anti-C5 antibody comprises a VL region comprising an amino acid sequence as set forth in SEQ ID NO: 31, or a homologous sequence thereof having at least 80%sequence identity to SEQ ID NO: 31 yet retaining binding specificity to C5.

In certain embodiments, the antigen-binding fragment of the anti-C5 antibody comprises a VL region comprising an amino acid sequence as set forth in SEQ ID NO: 36, or a homologous sequence thereof having at least 80%sequence identity to SEQ ID NO: 36 yet retaining binding specificity to C5.

In certain embodiments, the antigen-binding fragment of the anti-C5 antibody comprises a VL region comprising an amino acid sequence as set forth in SEQ ID NO: 39, or a homologous sequence thereof having at least 80%sequence identity to SEQ ID NO: 39 yet retaining binding specificity to C5.

The terms “homologous” , “substantially homologous” , and “substantial homology” as used herein denote a sequence of amino acids having at least 50%, 60%, 70%, 80%or 90%identity wherein one sequence is compared to a reference sequence of amino acids. The percentage of sequence identity or homology is calculated by comparing one to another when aligned to corresponding portions of the reference sequence.

In certain embodiments, the antigen-binding fragment of the anti-C5 antibody comprises a VH region comprising an amino acid sequence as set forth in SEQ ID NO: 1, and a VL region comprising an amino acid sequence as set forth in SEQ ID NO: 2.

In certain embodiments, the antigen-binding fragment of the anti-C5 antibody comprises a VH region comprising an amino acid sequence as set forth in SEQ ID NO: 28, and a VL region comprising an amino acid sequence as set forth in SEQ ID NO: 31.

In certain embodiments, the antigen-binding fragment of the anti-C5 antibody comprises a VH region comprising an amino acid sequence as set forth in SEQ ID NO: 28, and a VL region comprising an amino acid sequence as set forth in SEQ ID NO: 36.

In certain embodiments, the antigen-binding fragment of the anti-C5 antibody comprises a VH region comprising an amino acid sequence as set forth in SEQ ID NO: 1, and a VL region comprising an amino acid sequence as set forth in SEQ ID NO: 39.

Without wishing to be bound by any theory, but it is found that certain variants of eculizumab achieve unexpected effects, for example, in stability and purification, which makes them advantageous for manufacturing, especially scale-up manufacturing, and process development. For example, the eculizumab mutant #1 as provided herein is found to be more stable than eculizumab, with higher purity in the stability test (see details in Example 4) , and much higher protein production yield in recombinant expression (see details in Example 4) .

In certain embodiments, the antigen-binding fragment of the anti-C5 antibody comprises a Fab domain. In certain embodiments, the Fab domain comprises a heavy chain polypeptide comprising the VH domain and a light chain polypeptide comprising the VL domain, in which the VH domain and the VL domain associate to form the C5-binding domain.

In certain embodiments, the Fab domain comprises a heavy chain polypeptide comprising the VH region comprising the amino acid sequence as set forth in SEQ ID NO: 1 and a heavy chain constant region 1 (CH1) . In certain embodiments, the CH1 domain comprises the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the Fab domain comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21.

In certain embodiments, the Fab domain comprises a heavy chain polypeptide comprising the VH region comprising the amino acid sequence as set forth in SEQ ID NO: 28 and a heavy chain constant region 1 (CH1) . In certain embodiments, the CH1 domain comprises the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the Fab domain comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 32.

In certain embodiments, the Fab domain comprises a light chain polypeptide comprising the VL region comprising the amino acid sequence as set forth in SEQ ID NO: 2 and a light chain constant region (CL) . In certain embodiments, the CL domain comprises the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the Fab domain comprises a light chain comprising the amino acid sequence of SEQ ID NO: 17.

In certain embodiments, the Fab domain comprises a light chain polypeptide comprising the VL region comprising the amino acid sequence as set forth in SEQ ID NO: 31 and a light chain constant region (CL) . In certain embodiments, the CL domain comprises the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the Fab domain comprises a light chain comprising the amino acid sequence of SEQ ID NO: 33.

In certain embodiments, the Fab domain comprises a light chain polypeptide comprising the VL region comprising the amino acid sequence as set forth in SEQ ID NO: 36 and a light chain constant region (CL) . In certain embodiments, the CL domain comprises the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the Fab domain comprises a light chain comprising the amino acid sequence of SEQ ID NO: 37.

In certain embodiments, the Fab domain comprises a light chain polypeptide comprising the VL region comprising the amino acid sequence as set forth in SEQ ID NO: 39 and a light chain constant region (CL) . In certain embodiments, the CL domain comprises the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the Fab domain comprises a light chain comprising the amino acid sequence of SEQ ID NO: 40.

In certain embodiments, the Fab domain comprises a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 21 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 17, wherein the heavy chain and the light chain associate to form the C5-binding domain.

In certain embodiments, the Fab domain comprises a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 32 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 33, wherein the heavy chain and the light chain associate to form the C5-binding domain.

In certain embodiments, the Fab domain comprises a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 32 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 37, wherein the heavy chain and the light chain associate to form the C5-binding domain.

In certain embodiments, the Fab domain comprises a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 21 and a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 40, wherein the heavy chain and the light chain associate to form the C5-binding domain.

In certain embodiments, the antigen-binding fragment of the anti-C5 antibody comprises a single chain Fab. In certain embodiments, the single chain Fab comprises the heavy chain polypeptide provided herein and the light chain polypeptide provided herein, which are operably linked via a linker. In certain embodiments, the single chain Fab comprises the heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 21 operably linked to the light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 17, wherein the heavy chain polypeptide and the light chain polypeptide associate to form the C5-binding domain.

In certain embodiments, the single chain Fab comprises the heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 32 operably linked to the light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 33, wherein the heavy chain polypeptide and the light chain polypeptide associate to form the C5-binding domain.

In certain embodiments, the single chain Fab comprises the heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 32 operably linked to the light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 37, wherein the heavy chain polypeptide and the light chain polypeptide associate to form the C5-binding domain.

In certain embodiments, the single chain Fab comprises the heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 21 operably linked to the light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 40, wherein the heavy chain polypeptide and the light chain polypeptide associate to form the C5-binding domain.

In certain embodiments, the antigen-binding fragment of the anti-C5 antibody comprises a single chain Fv (scFv) domain. In certain embodiments, the scFv domain comprises the VH region comprising an amino acid sequence as set forth in SEQ ID NO: 1 and the VL region comprising an amino acid sequence as set forth in SEQ ID NO: 2, which are operably linked via a linker.

In certain embodiments, the scFv domain comprises the VH region comprising an amino acid sequence as set forth in SEQ ID NO: 28 and the VL region comprising an amino acid sequence as set forth in SEQ ID NO: 31, which are operably linked via a linker.

In certain embodiments, the scFv domain comprises the VH region comprising an amino acid sequence as set forth in SEQ ID NO: 28 and the VL region comprising an amino acid sequence as set forth in SEQ ID NO: 36, which are operably linked via a linker.

In certain embodiments, the scFv domain comprises the VH region comprising an amino acid sequence as set forth in SEQ ID NO: 1 and the VL region comprising an amino acid sequence as set forth in SEQ ID NO: 39, which are operably linked via a linker.

In certain embodiments, the VH region is operably linked to the N terminus of the VL region. In certain embodiments, the VL region is operably linked to the N terminus of the VH region.

In certain embodiments, the C5 binding domain comprises a VHH domain.

B. VEGFR

In certain embodiments, the fusion protein in the multi-specific molecule provided herein comprises a vascular endothelial growth factor (VEGF) binding domain.

VEGF is an important pro-angiogenic factor, which regulates endothelial proliferation, permeability, and survival with high efficacy and specificity (Folkman et al., Science 1987, 235: 442; Giampietro et al., Cancer Metastasis Rev. 1994; Ferrara, Endocrine Rev. 2004, 25 (4) : 581-611. ) .

Three VEGF receptors have been identified, including VEGFR-1 (fms-like tyrosine kinase, Flt-1) , VEGFR-2 (fetal liver kinase 1-murine homologue/Kinase insert Domain containing Receptor-human homologue, KDR/Flk-1) , and VEGFR-3 (Flt-4) . VEGFR-1 and VEGFR-2 are expressed primarily on endothelial cells. VEGFR-3 is mainly expressed on lymphatic vessels and neuropilin, and is also expressed on neuronal cells.

VEGFR has seven immunoglobulin-like domains in the extracellular domain. As used herein, an “extracellular” domain is the portion of a cell surface receptor that is outside the surface of the cell and usually includes the ligand binding site (s) .

The immunoglobulin-like domains of a VEGFR, in the native conformation of the VEGFR in the cell membrane, are oriented extracellularly where they can bind to VEGF. Each VEGFR has seven extracellular Ig domains, which from N terminus to C terminus, are numbered as Ig domain 1, Ig domain 2, Ig domain 3, Ig domain 4, Ig domain 5, Ig domain 6 and Ig domain 7.

Upon binding to VEGF, VEGFR undergoes dimerization and ligand-dependent tyrosine phosphorylation in the cell and results in a mitogenic, chemotactic and prosurvival signal. Blockade of VEGF binding to its receptor VEGFR has been shown to be effective to treat angiogenesis in pathological conditions. Blocking antibodies against VEGF or soluble VEGF receptor fragments can inhibit the binding of VEGF to VEGFRs on vascular endothelial cells, thus block the VEGF-initiated signal transduction, and the pathological angiogenesis resulting from high VEGF expression.

A VEGF binding domain as used herein can be any suitable domain that can bind to VEGF. Examples include, a VEGF binding domain derived from an anti-VEGF antibody, or derived from a VEGF receptor.

In certain embodiments, the VEGF binding domain comprises one or more extracellular immunoglobulin-like (Ig) domains of one or more VEGF receptors (VEGFR) .

In certain embodiments, the VEGFR is selected from the group consisting of VEGFR-1, VEGFR-2 and VEGFR-3.

In certain embodiments, the Ig domain of a VEGFR may be selected from the group consisting of Ig domain 1, Ig domain 2, Ig domain 3 and Ig domain 4.

In certain embodiments, the VEGF binding domain comprises two or more different Ig domains of two or more different VEGFRs.

In certain embodiments, the VEGF binding domain comprises a first Ig domain of a first VEGFR operably linked to N terminus of a second Ig domain of a second VEGFR, either directly or via a first linker.

In certain embodiments, the first Ig domain is an Ig domain 2 and the second Ig domain is an Ig domain 2 or an Ig domain 3.

In certain embodiments, the first VEGFR is VEGFR-1, and the second VEGFR is VEGFR-2.

In certain embodiments, the VEGF binding domain comprises an Ig domain 2 of VEGFR-1 and an Ig domain 3 of VEGFR-2. In certain embodiments, the Ig domain 2 of VEGFR-1 comprises the amino acid sequence as set forth in SEQ ID NO: 9, and Ig domain 3 of VEGFR-2 comprises the amino acid sequence as set forth in SEQ ID NO: 10.

In certain embodiments, the Ig domain 2 of VEGFR-1 and Ig domain 3 of VEGFR-2 are joined directly or via the first linker.

In certain embodiments, the Ig domain 2 of VEGFR-1 is operably linked to the N-terminus of the Ig domain 3 of VEGFR-2, either directly or via the first linker.

The first linker joins the two Ig domains of the VEGFR (s) . In certain embodiment, the first linker comprises a peptide linker. The peptide linker can be a synthetic peptide or a peptide derived from a naturally-occurring polypeptide.

In certain embodiments, the first linker comprises the amino acid sequence of SEQ ID NO: 18 (TNTII) .

In certain embodiments, the VEGF binding domain comprises a VEGF trap, which competes with the naturally occurring VEGF cellular receptor to inhibit VEGF. such as aflibercept. Aflibercept is angiogenesis inhibitor that has been developed as therapeutic to treat angiogenesis-related diseases. In certain embodiments, the VEGF binding domain comprises the amino acid sequence of SEQ ID NO: 11.

In certain embodiments, the VEGF binding domain comprises the amino acid sequence as set forth in SEQ ID NO: 11, or an amino acid sequence having at least 80%sequence identity thereof yet retaining binding specificity to VEGF.

Table 2. Sequences of Exemplary VEGF binding domain in the C5/VEGF bispecific antibody

C. Peptide Linker Sequences

In certain embodiments, in the multi-specific molecules provided herein, the C5 binding domain is operably linked to N terminus of the VEGF binding domain directly or via a second linker. An example is shown in Figure 1, 2 or 3.

In certain embodiments, the C5 binding domain comprises a Fab domain comprising a heavy chain polypeptide and a light chain polypeptide, and the heavy chain polypeptide is operably linked to the N terminus of the VEGF binding domain. An example is shown in Figure 1.

In certain embodiments, the C5 binding domain comprises a Fab domain comprising a heavy chain polypeptide and a light chain polypeptide, and the light chain polypeptide is operably linked to the N terminus of the VEGF binding domain.

In certain embodiments, the C5 binding domain comprises a scFv domain that is operably linked to the N terminus of the VEGF binding domain. An example is shown in Figure 2. In certain embodiments, the scFv domain comprises the VL region operably linked to the N terminus of the VH region.

In certain embodiments, the C5 binding domain comprises a VHH domain that is operably linked to the N terminus of the VEGF binding domain. An example is shown in Figure 3.

In certain embodiment, the second linker comprises a peptide linker. The peptide linker can be a synthetic peptide or a peptide derived from a naturally-occurring polypeptide. Linker considerations include the effect on physical or pharmacokinetic properties of the resulting compound, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable as well as planned degradation) , viscosity, rigidity, flexibility, immunogenicity, modulation of antibody binding, the ability to be incorporated into a micelle or liposome, and the like.

In certain embodiments, the peptide linker may be a GS linker. As used herein, “GS linker” is the peptide linker comprises one, two, three, four or more repeats of glycine (G) or serine (S) . In certain embodiments, the GS linker may comprise one, two, three, four, five, six, seven, eight, nine, ten or more repeats of SEQ ID NO: 13 (GGGS) or SEQ ID NO: 14 (GGGGS) or SEQ ID NO: 15 (GGGGSGGGGSGGGGS) .

D. Multimerizing component

In certain embodiments, the fusion protein in the multi-specific molecule provided herein comprises a multimerizing component.

As used herein, a “multimerizing component” refers to a component that has the ability to associate with another multimerizing component to form a homodimer or a heterodimer.

In certain embodiments, the multimerizing component comprises a polypeptide fragment comprising one or more amino acid residues and having at least one cysteine residue. The cysteine residue can dimerize to form a disulfide bond, thus allowing formation of a dimer.

In some embodiment, the multimerizing component comprises a polypeptide having a length between 1 and 200 amino acids and having at least one cysteine residue. In some embodiment, the multimerizing component comprises a polypeptide having a length between 1 and 180, 1 to 150, 1 to 120, 1 to 100, 1 to 80, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, or 1 to 10 amino acid residues.

Any suitable polypeptide can be used as the multimerizing component, including those that contain cysteine residues that can form inter-chain disulphide bond, or those that can associate with each other via electrostatic interactions, hydrogen bonds, hydrophobic interactions, and so on. Examples of multimerizing component include, without limitation Fc domain, and Leucine zipper motif.

In some embodiment, the multimerizing component comprises an antibody Fc domain or a fragment thereof. For example, a multimerizing component may comprise an immunoglobulin CH3 domain. For another example, a multimerizing component may comprise an immunoglobulin CH2 and CH3 domain. In some embodiment, the Fc domain of an IgG is selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

Without wishing to be bound by any theory, it is believed that Fc domain is advantageous in prolonging half-life or increasing stability of the multi-specific molecules.

In some embodiment, the Fc domain comprises a human Fc domain.

In certain embodiments, the Fc domain is derived from human immunoglobulin (Ig) .

In certain embodiments, the Fc domain is derived from human IgG, optionally human IgG1, IgG2, IgG3 or IgG4.

In certain embodiments, the Fc domain is derived from human IgG1.

In certain embodiments, the Fc domain comprises the amino acid sequence as set forth in SEQ ID NO: 12, or an amino acid sequence having at least 80%sequence identity thereof yet retaining the capability to multimerize.

In certain embodiments, the Fc domain is mutated. In some embodiments, the Fc domain comprises a mutation of substitution, deletion, insertion and/or addition to improve aggregation.

In certain embodiments, the Fc domain comprises a mutation at position 235 and/or 309 of the human IgG1 in accordance with EU numbering system.

In certain embodiments, the Fc domain comprises a substitution at position 235 and/or 309 of the human IgG1 in accordance with EU numbering system.

In certain embodiments, the Fc domain comprises a L235K and/or L309K mutation in accordance with EU numbering system.

In certain embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence having at least 80%sequence identity thereof yet retaining the capability to multimerize and the L235K and/or L309K mutation in accordance with EU numbering system.

In certain embodiments, the Fc domain comprises a mutation of substitution, deletion, insertion and/or addition for example to reduce or eliminate one or more effector functions, or to improve pH-dependent binding to neonatal Fc receptor (FcRn) .

In certain embodiments, the Fc domain provided herein has reduced effector functions, and comprise one or more amino acid substitution (s) in IgG1 at a position selected from the group consisting of: 234, 235, 237, and 238, 268, 297, 309, 330, and 331, in accordance with EU numbering system. In certain embodiments, the Fc domain provided herein is of IgG1 isotype and comprises one or more amino acid substitution (s) selected from the group consisting of: N297A, N297Q, N297G, L235E, L234A, L235A, L234F, L235E, P331S, and any combination thereof, in accordance with EU numbering system.

In certain embodiments, the Fc domain provided herein is of IgG2 isotype, and comprises one or more amino acid substitution (s) selected from the group consisting of: H268Q, V309L, A330S, P331S, V234A, G237A, P238S, H268A, and any combination thereof (e.g. H268Q/V309L/A330S/P331S, V234A/G237A/P238S/H268A/V309L/A330S/P331S) , in accordance with EU numbering system.

In certain embodiments, the Fc domain provided herein is of IgG4 isotype, and comprises one or more amino acid substitution (s) selected from the group consisting of: N297A, N297Q, N297G, L235E, L234A, L235A, and any combination thereof, in accordance with EU numbering system.

In certain embodiments, the Fc domain comprise one or more amino acid substitution (s) that improves pH-dependent binding to neonatal Fc receptor (FcRn) . Such a variant can have an extended pharmacokinetic half-life, as it binds to FcRn at acidic pH which allows it to escape from degradation in the lysosome and then be translocated and released out of the cell. Methods of engineering an antibody and antigen-binding fragment thereof to improve binding affinity with FcRn are well-known in the art, see, for example, Vaughn, D. et al, Structure, 6 (1) : 63-73, 1998; Kontermann, R. et al, Antibody Engineering, Volume 1, Chapter 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. et al, Cancer Research, 70: 3269-3277 (2010) ; and Hinton, P. et al, J. Immunology, 176: 346-356 (2006) .

In certain embodiments, the multimerizing component is operably linked to the C terminus of the VEGF binding domain, directly or via a third linker.

In certain embodiment, the third linker comprises a peptide linker. The peptide linker can be a synthetic peptide or a peptide derived from a naturally- occurring polypeptide. In certain embodiment, the third linker comprises a fragment derived from the hinge region of an antibody. In certain embodiment, the third linker comprises the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25.

In certain embodiments, the peptide linker may be a GS linker.

E. Multi-Specific Molecule

In some embodiments, the multi-specific molecule provided herein are capable of specifically binding to both human C5 and human VEGF. The multi-specific molecules provided herein retain the specific binding affinity to both human C5 and human VEGF, in certain embodiments are at least comparable to, or even better than, the parent anti-C5 antibody such as eculizumab and the parent VEGF-binding molecule such as aflibercept.

In certain embodiments, the multi-specific molecule descried herein comprises the fusion protein comprising the C5-binding domain which is a Fab domain. In certain embodiments, the Fab domain comprises a heavy chain polypeptide and a light chain polypeptide. In certain embodiments, the heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 21, and/or the light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 17. In certain embodiments, the heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 32, and/or the light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 33.

In certain embodiments, the heavy chain polypeptide is operably linked to the N terminus of the VEGF binding domain, either directly or via a linker (e.g. the second linker provided herein) . In certain embodiments, the VEGF binding domain is operably linked to the N terminus of the multimerizing component, either directly or via a linker (e.g. the third linker provided herein) . In certain embodiments, the fusion protein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 16 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 17, wherein the first polypeptide and the second polypeptide associates to form the C5-binding domain. In certain embodiments, the fusion protein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 34 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 33, wherein the first polypeptide and the second polypeptide associates to form the C5-binding domain.

Table A. Sequences of the full length C5/VEGF bispecific antibody

In certain embodiments, the heavy chain polypeptide is operably linked to the C terminus of the multimerizing component, either directly or via a linker (e.g. the second linker provided herein) . In certain embodiments, the multimerizing component is operably linked to the C terminus of the VEGF binding domain, either directly or via a linker (e.g. the third linker provided herein) . An example is shown in Figure 4.

In certain embodiments, the fusion protein comprises the C5 binding domain comprising an scFv comprising the VH region and the VL region operably linked via a linker.

In certain embodiments, the fusion protein comprises the scFv operably linked to the N terminus of the VEGF binding domain, either directly or via a linker. In certain embodiments, the fusion protein comprises the scFv operably linked to the C terminus of the multimerizing component, either directly or via a linker, and the multimerizing component is operably linked to the C terminus of the VEGF binding domain, either directly or via a linker. An example is shown in Figure 5.

In certain embodiments, the fusion protein comprises the C5 binding domain comprising a VHH domain.

In certain embodiments, the fusion protein comprises the VHH domain operably linked to the N terminus of the VEGF binding domain, either directly or via a linker. In certain embodiments, the fusion protein comprises the VHH domain operably linked to the C terminus of the multimerizing component, either directly or via a linker, and the multimerizing component is operably linked to the C terminus of the VEGF binding domain, either directly or via a linker. An example is shown in Figure 6.

Without wishing to be bound by any theory, but it is found that the efficacy of the fusion protein is influenced by the positioning of the C5-binding domain. Specifically, when the C5-binding domain is operably linked to the C terminus of the multimerizing component (see, e.g. Figures 4-6) , the fusion protein demonstrates reduced efficacy. In contrast, linking the C5-binding domain to the N terminus of the VEGF binding domain (see, e.g. Figures 1-3) appears to enhance its efficacy. In certain embodiments, the multi-specific molecule comprises a dimer of the fusion protein, in which the multimerizing component in the fusion protein associates to form a dimer.

In certain embodiments, the multi-specific molecules provided herein is capable of specifically binding to C5 at a K_D value of 2000 pM or less, 1800 pM or less, 1500 pM or less, 1200 pM or less, 1000 pM or less, 500 pM or less, 400 pM or less, 300 pM or less, 250 pM or less, 220 pM or less as determined by BIACORE as described in Example 2 of the present disclosure.

In certain embodiments, the multi-specific molecules provided herein binds to VEGF with a K_D of 1000 pM or less, 500 pM or less, 400 pM or less, 300 pM or less, 250 pM or less, 220 pM or less, 200 pM or less, 190 pM or less, 160 pM or less, 130 pM or less, 100 pM or less, 90 pM or less, 80 pM or less, 70 pM or less, 60 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 20 pM or less, or 10 pM or less, as determined by BIACORE as described in Example 2 of the present disclosure.

In certain embodiments, the multi-specific molecules provided herein inhibits proliferation of HUVEC cells at 50 nM or less, 20 nM or less, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, as determined by anti-VEGF cell proliferation functional assay described in Example 3.1 of the present disclosure.

In certain embodiments, the multi-specific molecules provided herein blocks effects of C5 activity at an IC50 value of 500 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, as determined by hemolytic assay described in Example 3.2 of the present disclosure.

In certain embodiments, the multi-specific molecules provided herein have superior effect (such as vascular leaking inhibition capability, leaky vessels regression and longer duration) in comparison with aflibercept.

F. Polynucleotides and Recombinant Methods

The present disclosure provides isolated polynucleotides that encode the multi-specific molecules provided herein. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) , alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19: 5081 (1991) ; Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985) ; and Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994) ) .

The polynucleotides encoding the multi-specific molecules disclosed herein may be generated using methods known in the art. In certain embodiments, the sequence of the polynucleotides may be obtained based on the amino acid sequences of the multi-specific molecules, and nucleic acids can be generated using synthetic methods. Alternatively, the polynucleotides provided herein can also be obtained from another available nucleic acid that encodes a polypeptide with a sequence homologous to the polypeptides in the multi-specific molecules disclosed herein. Then a DNA manipulation process can be applied to manipulate the sequence of the parent multi-specific-molecule-encoding nucleic acid, such as introducing mutations, insertion, deletion, etc., so as to obtain the nucleic acid encoding the multi-specific molecules disclosed herein.

The isolated polynucleotide that encodes the multi-specific molecules can be inserted into one or more vector (s) for further cloning (amplification of the DNA) or for expression, using recombinant techniques known in the art. Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α) , a transcription termination sequence, and one or more other regulatory elements.

The present disclosure provides vectors comprising the isolated polynucleotides provided herein. In certain embodiments, the polynucleotides provided herein encodes the multi-specific molecules, with at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, papovavirus (e.g., SV40) , lambda phage, and M13 phage, plasmid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT. RTM., pCDM8, pCDNA1.1/amp, pcDNA3.1, pRc/RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos etc.

Vectors comprising the polynucleotide sequence encoding the multi-specific molecules can be introduced to a host cell for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.

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

Suitable host cells for the expression of glycosylated multi-specific molecules provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar) , Aedes aegypti (mosquito) , Aedes albopictus (mosquito) , Drosophila melanogaster (fruiffly) , and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.

However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651) ; human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36: 59 (1977) ) ; baby hamster kidney cells (BHK, ATCC CCL 10) ; Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980) ) ; mouse sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980) ) ; monkey kidney cells (CV1 ATCC CCL 70) ; African green monkey kidney cells (VERO-76, ATCC CRL-1587) ; human cervical carcinoma cells (HELA, ATCC CCL 2) ; canine kidney cells (MDCK, ATCC CCL 34) ; buffalo rat liver cells (BRL 3A, ATCC CRL 1442) ; human lung cells (W138, ATCC CCL 75) ; human liver cells (Hep G2, HB 8065) ; mouse mammary tumor (MMT 060562, ATCC CCL51) ; TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383: 44-68 (1982) ) ; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2) . In some embodiments, the host cell is a mammalian cultured cell line, such as CHO, BHK, NS0, 293 and their derivatives.

Host cells are transformed with the above-described expression or cloning vectors for multi-specific molecule production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. In another embodiment, the multi-specific molecules may be produced by homologous recombination known in the art. In certain embodiments, the host cell is capable of producing the multi-specific molecules provided herein.

The present disclosure also provides a method of expressing the multi-specific molecules provided herein, comprising culturing the host cell provided herein under the condition at which the vector of the present disclosure is expressed. The host cells used to produce the multi-specific molecules provided herein may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma) , Minimal Essential Medium (MEM) , (Sigma) , RPMI-1640 (Sigma) , and Dulbecco's Modified Eagle's Medium (DMEM) , Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58: 44 (1979) , Barnes et al., Anal. Biochem. 102: 255 (1980) , U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30, 985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor) , salts (such as sodium chloride, calcium, magnesium, and phosphate) , buffers (such as HEPES) , nucleotides (such as adenosine and thymidine) , antibiotics (such as GENTAMYCIN^TM drug) , trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) , and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to a person skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to a person skilled in the art.

When using recombinant techniques, the multi-specific molecule can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the multi-specific molecule is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992) describe a procedure for isolating multi-specific molecules which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5) , EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the multi-specific molecule is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

The multi-specific molecules prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.

In certain embodiments, Protein A immobilized on a solid phase is used for immunoaffinity purification of the multi-specific molecules comprising Fc domain. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the multi-specific molecule. Protein A can be used to purify multi-specific molecules that are based on human gamma1, gamma2, or gamma4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983) ) . Protein G is recommended for all mouse isotypes and for human gamma3 (Guss et al., EMBO J. 5: 1567 1575 (1986) ) . The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the multi-specific molecules comprises a CH3 domain, the Bakerbond ABX^TM resin (J.T. Baker, Phillipsburg, N.J. ) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE^TM chromatography on an anion or cation exchange resin (such as a polyaspartic acid column) , chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the multi-specific molecules to be recovered.

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

III. Pharmaceutical Formulations and Administration

The present disclosure also provides pharmaceutical compositions comprising the multi-specific molecule provided herein, and one or more pharmaceutically acceptable carriers.

The present disclosure further provides pharmaceutical compositions comprising the polynucleotides encoding the multi-specific molecule provided herein, and one or more pharmaceutically acceptable carriers.

The present disclosure further provides pharmaceutical compositions comprising the expression vector comprising the polynucleotides encoding the multi-specific molecule provided herein, and one or more pharmaceutically acceptable carriers. In certain embodiments, the expression vector comprises a viral vector or a non-viral vector. Examples of viral vectors include, without limitation, adeno-associated virus (AAV) vector, lentivirus vector, retrovirus vector, and adenovirus vector. Examples of non-viral vectors include, without limitation, naked DNA, plasmid, exosome, mRNA, and so on. In certain embodiments, the expression vector is suitable for gene therapy in human. Suitable vectors for gene therapy include, for example, adeno-associated virus (AAV) , or adenovirus vector. In certain embodiments, the expression vector comprises a DNA vector or an RNA vector. In certain embodiments, the pharmaceutically acceptable carriers are polymeric excipients, such as without limitation, microspheres, microcapsules, polymeric micelles and dendrimers. The polynucleotides, or polynucleotide vectors of the present disclosure may be encapsulated, adhered to, or coated on the polymer-based components by methods known in the art (see for example, W. Heiser, Nonviral gene transfer techniques, published by Humana Press, 2004; U.S. patent 6025337; Advanced Drug Delivery Reviews, 57 (15) : 2177-2202 (2005) ) .

As used herein, the term “pharmaceutical composition” refers to a formulation containing an active ingredient in a form suitable for administration to a subject.

As used herein, the term “pharmaceutically acceptable” indicates that the designated carrier, vehicle, diluent, excipient (s) , salt and/or medium is generally chemically and/or physiologically compatible with other ingredients, such as the active ingredient (i.e. the multi-specific molecules disclosed herein) comprising the formulation, and is physiologically compatible with a subject receiving the pharmaceutical composition.

A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is bioactivity acceptable and nontoxic to a subject. In the context of the present disclosure, a pharmaceutical acceptable carrier for use in the pharmaceutical composition disclosed herein may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

The carrier can be solvents, dispersion media, isotonic agents and the like. The carrier can be liquid, semi-solid or solid carriers. In some embodiments, carriers may be water, saline solutions or other buffers (such as serum albumin and gelatin) , carbohydrates (such as monosaccharides, disaccharides, and other carbohydrates including glucose, sucrose, trehalose, mannose, mannitol, sorbitol, or dextrins) , gel, lipids, liposomes, resins, porous matrices, binders, fillers, coatings, stabilizers, preservatives, antioxidants (including ascorbic acid and methionine) , chelating agents (such as EDTA) , salt forming counter-ions (such as sodium) , non-ionic surfactants [such as TWEEN^TM, PLURONICS^TM or polyethylene glycol (PEG) ] , or combinations thereof. Pharmaceutical acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art.

The compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

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

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

The composition can comprise an ophthalmic depot formulation comprising an active agent for subconjunctival administration. The ophthalmic depot formulation comprises microparticles of essentially pure active agent, e.g., the bispecific antibody according to the invention. The microparticles comprising the bispecific antibody according to the invention can be embedded in a biocompatible pharmaceutically acceptable polymer or a lipid encapsulating agent. The depot formulations may be adapted to release all of substantially all the active material over an extended period of time. The polymer or lipid matrix, if present, may be adapted to degrade sufficiently to be transported from the site of administration after release of all or substantially all the active agents. The depot formulation can be liquid formulation, comprising a pharmaceutical acceptable polymer and a dissolved or dispersed active agent. Upon injection, the polymer forms a depot at the injections site, e.g. by gelifying or precipitating.

In embodiments, the pharmaceutical compositions are formulated into an injectable composition. The injectable pharmaceutical compositions may be prepared in any conventional form, such as for example liquid solution, suspension, emulsion, or solid forms suitable for generating liquid solution, suspension, or emulsion. Preparations for injection may include sterile and/or non-pyretic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use, and sterile and/or non-pyretic emulsions. The solutions may be either aqueous or nonaqueous.

In certain embodiments, unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile and not pyretic, as is known and practiced in the art.

In certain embodiments, a sterile, lyophilized powder is prepared by dissolving the multi-specific molecule as disclosed herein in a suitable solvent. The solvent may contain an excipient which improves the stability or other pharmacological components of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agents. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides a desirable formulation. In one embodiment, the resulting solution will be apportioned into vials for lyophilization. Each vial can contain a single dosage or multiple dosages of the polypeptide complex, the polypeptide complex. Overfilling vials with a small amount above that needed for a dose or set of doses (e.g., about 10%) is acceptable so as to facilitate accurate sample withdrawal and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4 ℃ to room temperature.

Reconstitution of a lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, for reconstitution the sterile and/or non-pyretic water or other liquid suitable carrier is added to lyophilized powder. The precise amount depends upon the selected therapy being given, and can be empirically determined.

In certain embodiments, a composition is further provided, comprising a pharmaceutically acceptable carrier, diluent or adjuvant, and an active ingredient. The active ingredient can be the multi-specific molecules thereof disclosed herein.

IV. Kits

In another aspect, the present invention provides kits containing the multi-specific molecule provided herein and directions for using the multi-specific molecule. The kit may also include a container and optionally one or more vial, test tube, flask, bottle, or syringe. Other formats for kits will be apparent to those of skill in the art and are within the scope of the present invention.

Such kits can further include, if desired, one or more of various conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers etc., as will be readily apparent to a person skilled in the art. Instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.

In some embodiments, therapeutic kits of the invention can contain one or more doses of a multi-specific molecule present in a pharmaceutical composition described herein, a suitable device for intravitreal injection of the pharmaceutical composition, and an instruction detailing suitable subjects and protocols for carrying out the injection. In these embodiments, the compositions are typically administered to the subject in need of treatment via intravitreal injection.

V. Medical Use

In another aspect, the present invention provides a method for treating, preventing or alleviating a disease condition in a subject that is in need of such treatment, comprising: administering to the subject a therapeutically effective amount of the multi-specific molecule of the present disclosed herein, or the polynucleotide encoding the multi-specific molecule provided herein, or the pharmaceutical composition provided herein.

As used herein, the term “subject” or “individual” or “animal” or “patient” refers to human or non-human animal, including a mammal or a primate, in need of diagnosis, prognosis, amelioration, prevention and/or treatment of a disease or disorder. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows, bears, and so on.

As used herein, “treatment” of a condition may include, alleviating a condition, slowing the onset or rate of development of a condition, delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combinations thereof.

As used herein, the term “disorder, ” “disease, ” “condition” or alike, refers to a condition that affects a subject who would nonetheless benefits from treatment with the multi-specific molecule.

As used herein, the term “therapeutically effective amount” of a therapeutic agent refers to an amount of the therapeutic agent that, when taken by a subject in an appropriate manner, can generate sufficient therapeutic effects to the subject. It is to be understood that just like other therapeutic drugs, the therapeutically effective amount of the multi-specific molecules as provided herein will be influenced by various factors known in the art, such as for example body weight, age, past medical history, present medications, state of health of the subject and potential for cross-reaction, allergies, sensitivities and adverse side-effects, as well as the administration route and extent of disease development. Dosages may be proportionally reduced or increased by one of ordinary skill in the art (e.g., physician or veterinarian) as indicated by these and other circumstances or requirements.

In certain embodiments, the disease or disorder is a C5-related and/or a VEGF related disease or disorder. In some embodiments, the disease, disorder or condition is selected from the group consisting of ocular diseases, cancer, inflammatory disease, autoimmune disease, angiogenesis, vascular permeability, edema, and inflammation.

In certain embodiments, the C5-related and/or a VEGF related disease or disorder is an ocular disease.

In some embodiments, the ocular disease is selected from the group consisting of age-related macular degeneration (AMD) , geographic atrophy (GA) , macular edema, macular edema following retinal vein occlusion (RVO) , diabetic macular edema (DME) , diabetic retinopathy (DR) , retinal central vein occlusion, corneal neovascularization (CNV) , retinal pigmentosa (RP) , ocular angiogenesis (ocular neovascularization affecting choroidal, corneal or retinal tissue) , retinopathy of prematurity (ROP) , pathological myopia, vascular glaucoma, retinoblastoma, retinal vein occlusion, uveitis and neuromyelitis optica.

In some embodiments, the ocular disease is age-related macular degeneration (AMD) . AMD is a disease characterized by progressive degenerative abnormalities in the macula, a region in the central portion of the retina. Age-related macular degeneration is a complex, gradually progressing disorder of the eye that leads to distortions and/or blind spots (scotoma) , changes in dark adaptation (diagnostic of rod cell health) , changes in color interpretation (diagnostic of cone cell health) , a decrease in visual acuity, or irreversible blindness.

In some embodiments, the AMD is dry AMD. Non-exudative AMD is the non-neovascular ( “dry” ) form of the disease ( “dry AMD” ) . Dry AMD accounts for approximately 90%of all AMD cases. Dry AMD can be characterized by degeneration of the macula and, with continued progression over multiple years, may ultimately result in atrophy of the central retina associated with central vision loss, also known as geographic atrophy (GA) . Dry AMD is a significant cause of moderate and severe loss of central vision and is bilateral in most patients. In dry AMD, thinning of the retinal pigment epithelial cells (RPE) in the macula develops, along with other age-related changes to the adjacent retinal tissue layers.

In some embodiments, the AMD is wet AMD. Choroidal neovascularization can be an early sign of wet AMD. Once neovascularization arises in non-exudative AMD and begins to leak, the disease is referred to as exudative AMD, the neovascular ( “wet” ) form of the disease ( “wet AMD” ) , with non-exudative AMD still present and potentially progressing in the patient. Wet AMD may cause sudden, often substantial, loss of central vision, particularly if untreated.

In some embodiments, the ocular disease is geographic atrophy. Geographic atrophy (GA) is a chronic progressive degeneration of the macula, as part of late-stage AMD. The macula is the central part of the retina, which is the “film” lining the inside of the eye. In GA, areas of the retina experience cell death (atrophy) . These areas can grow and may result in a dim or blind spot in the vision. GA often first develops near the fovea, the center of the macula, which is the central and clearest part of vision. GA can lead to progressive and permanent vision loss. If one eye develops GA, it is more likely to develop GA in the other eye. GA is characterized by localized sharply demarcated atrophy of outer retinal tissue, retinal pigment epithelium and choriocapillaris.

It is estimated that more than 8 millions people worldwide have GA. Currently there is no treatment for GA.

It is unexpectedly found by the present inventor that, by combination of inhibition on VEGF and C5, synergistic effects can be achieved in chronic diseases animal model for GA, and this is otherwise not achieved by inhibition of either of the single target, for example, not achieved by VEGF-Trap (e.g. Aflibercept) , and also not achieved by an anti-C5 antibody such as elulizumab. The multi-specific molecules provided herein show much improved therapeutic efficacy as well as duration of such therapeutic efficacy relative to Aflibercept.

In certain embodiments, the subject has been treated with a VEGF antagonist or a VEGF receptor antagonist. In certain embodiments, the subject has developed resistance to a VEGF antagonist or a VEGF receptor antagonist.

The present invention provides a method for treating or preventing an ocular disease or for inducing the regression or elimination or inhibiting the progression of at least one sign or symptom of an ocular disease in a subject in need thereof by administering a therapeutically effective amount of the combination to the subject. In certain embodiments, the subject suffers from an ocular disease and suffering from one or more of signs or symptoms of the ocular disease.

Exemplary sign or symptom of an ocular disease include, for example, increased rate of loss of vision; drusen in the eye (e.g., of a subject with dry AMD) ; loss of vision; gradual loss of central vision (e.g., in subjects with non-exudative macular degeneration) ; visual distortion; difficulty adapting to low light levels; crooked central vision; haziness of central and/or overall vision; eye pigmentary changes; distorted vision (e.g., metamorphopsia in which a grid of straight lines appears wavy and parts of the grid may appear blank) ; exudative changes (e.g., hemorrhages in the eye, hard exudates, subretinal/sub-RPE/intraretinal fluid) ; slow recovery of visual function after exposure to bright light (e.g., as determined in a photostress test) ; incipient and/or geographic atrophy; drastically decreasing visual acuity (e.g., two levels or more, e.g., 20/20 to 20/80) ; preferential hyperacuity perimetry changes (e.g., in a subject with wet AMD) ; blurred vision; rapid onset of vision loss (e.g., caused by leakage and bleeding of abnormal blood vessels in subjects with exudative macular degeneration) ; central scotomas (shadows or missing areas of vision) ; trouble discerning colors (e.g., specifically dark colors from other dark colors and/or light colors from other light colors) ; loss in contrast sensitivity; and/orstraight lines appear curved in an Amsler grid.

In certain embodiments, the subject is human.

In certain embodiments, the multi-specific molecules as provided herein may be administered at a therapeutically effective amount of about 1 mg to about 20 mg (or 2mg to 20mg, or 4mg to 20 mg, or 4 mg to 12 mg) per intravitreal (IVT) injection. Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response) . For example, a single dose may be administered, or several divided doses may be administered over time.

The multi-specific molecules disclosed herein may be administered by any route known in the art, such as for example parenteral (e.g., intraocular, intravitreal injection, subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, intravitreal injection, sublingual, rectal, or topical) routes.

Many possible modes of delivery can be used, including, but not limited to intraocular application or topical application. In one embodiment the application is intraocular and includes, but is not limited to, subconjunctival injection, intracanieral injection, injection into the anterior chamber via the termporal limbus, intrastromal injection, intracorneal injection, subretinal injection, aqueous humor injection, subtenon injection or sustained delivery device, intravitreal injection (e.g., front, mid or back vitreal injection) . In one embodiment the application is topical and includes, but is not limited to eye drops to the cornea.

In one embodiment the multi-specific molecule or pharmaceutical composition according to the invention is administered via intravitreal application, e.g. via intravitreal injection. This can be performed in accordance with standard procedures known in the art. See, e.g., Ritter et al., J. Clin. Invest. 116 (2006) 3266-76; Russelakis-Carneiro et al., Neuropathol. Appl. Neurobiol. 25 (1999) 196-206; and Wray et al., Arch. Neurol. 33 (1976) 183-5.

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

In some embodiments, the multi-specific molecules disclosed herein may be administered alone or in combination with one or more additional therapeutic means or agents. For example, the multi-specific molecules disclosed herein may be administered in combination with one or more additional therapeutic agents or methods for the treatment of one or more ocular diseases described herein.

In certain of these embodiments, the multi-specific molecules as disclosed herein that is administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments the multi-specific molecules and the additional therapeutic agent (s) may be administered as part of the same pharmaceutical composition. However, the multi-specific molecules administered “in combination” with another therapeutic agent does not have to be administered simultaneously with or in the same composition as the agent. The multi-specific molecules administered prior to or after another agent is considered to be administered “in combination” with that agent as the phrase is used herein, even if the multi-specific molecules and second agent are administered via different routes. Where possible, additional therapeutic agents administered in combination with the multi-specific molecules disclosed herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002) ) or protocols well known in the art.

In other embodiments the multi-specific molecules or pharmaceutical composition according to the invention is administered in combination with one or more additional therapeutic agents or methods for the treatment of one or more ocular diseases described herein.

In other embodiments, the multi-specific molecules or pharmaceutical composition according to the invention is formulated in combination with one or more additional therapeutic agents and administered for the treatment of one or more ocular diseases described herein.

In certain embodiments, the combination treatments provided herein include administration the multi-specific molecule or pharmaceutical composition according to the invention is administered sequentially with one or more additional therapeutic agents for the treatment of one or more ocular diseases described herein.

The additional therapeutic agents include, but are not limited to, an anti-angiogenic agent (such as a VEGF antagonist, a VEGF receptor antagonist, an anti-inflammatory drug, neuroprotective agent, therapeutic agent for AMD, or a C5 inhibitor) .

In certain embodiments, the anti-angiogenic agent is a VEGF antagonist or a VEGF receptor antagonist. Examples include, without limitation, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, small interfering RNAs decreasing expression of VEGFR or VEGF ligand, low molecule weight inhibitors of VEGFR tyrosine kinases and any combinations thereof and these include anti-VEGF aptamers (e.g. Pegaptanib) , soluble recombinant decoy receptors (e.g. VEGF Trap) .

In certain embodiments, the anti-angiogenic agent include anti-inflammatory drugs, m-Tor inhibitors, rapamycin, everolimus, temsirolimus, cyclosporine, anti-TNF agents, anti-complement agents, and nonsteroidal anti-inflammatory agents.

In certain embodiments, the anti-angiogenic agent include corticosteroids, angiostatic steroids, anecortave acetate, angiostatin, endostatin, MMP inhibitors, IGFBP3, SDF-1 blockers, PEDF, gamma-secretase, Delta-like ligand 4, integrin antagonists (e.g. inhibitors of integrin β3 function) , HIF-1 alpha blockade, protein kinase CK2 blockade, and inhibition of stem cell (i.e. endothelial progenitor cell) homing to the site of neovascularization using vascular endothelial cadherin (CD-144) and stromal derived factor (SDF) -I antibodies.

In certain embodiments, the additional therapeutic agent is a complement related agent, such as C1q, C3, C5, factor B, factor D, or factor H.

In certain embodiments, the additional therapeutic agent is a C3 inhibitor, including without limitation, compstatin and/or its analog, H17 (monoclonal antibody, EluSys Therapeutics, Pine Brook, NJ) ; mirococept (CR1-based protein) ; sCR1 (CR1-based protein, Celldex, Hampton, NJ) ; TT32 (CR-1 based protein, Alexion Pharmaceuticals, Inc., Boston, MA) ; HC-1496 (recombinant peptide) ; CB 2782 (enzyme, Catalyst Biosciences, South San Francisco, CA) ; APL-2 (pegylated synthetic cyclic peptide, Apellis Pharmaceuticals, Crestwood, KY) ; or combinations thereof.

In certain embodiments, the additional therapeutic agent is a complement factor B inhibitor that include, but are not limited to: anti-FB SiRNA (Alnylam Pharmaceuticals, Cambridge, MA) ; TA106 (monoclonal antibody, Alexion Pharmaceuticals, Inc., Boston, MA) ; LNP023 (small molecule, Novartis, Basel, Switzerland) ; SOMAmers (aptamers, SomaLogic, Boulder, CO) ; bikaciomab (Novelmed Therapeutics, Cleveland, OH) ; complin (see, Kadam et al., J. Immunol. 2010, DOI: 10.409/jimmunol. 10000200) ; Ionis-FB-LRx (ligand conjugated antisense drug, Ionis Pharmaceuticals, Carlsbad, CA) ; or a combination thereof.

In certain embodiments, the additional therapeutic agent is a complement factor D antagonist, e.g., an anti-complement factor D antibody, e.g., lampalizumab (Roche) .

In certain embodiments, the additional therapeutic agent is an Ang-2 antagonist.

In certain embodiments, the additional therapeutic agent is a neuroprotective agent that can potentially reduce the progression of dry macular degeneration. This class of drug is also known as neurosteroids, including, for example, dehydroepiandrosterone (DHEA) , dehydroepiandrosterone sulfate, and pregnenolone sulfate.

In certain embodiments, the additional therapeutic agent is therapeutic agent for AMD, including but not limited to verteporfin in combination with PDT, pegaptanib sodium, zinc, or an antioxidant (s) , alone or in any combination.

In certain embodiments, the additional therapeutic agent is a C5 inhibitor, including without limitation, eculizumab, ravulizumab, 305LO5, SKY59, pozelimab, tesidolumab, crovalimab, or ABP 959 or a biosimilar thereof.

In another aspect, the present disclosure provides a method of modulating C5 and/or VEGF activity in a cell, comprising exposing the cell to the multi-specific molecule provided herein and/or the pharmaceutical composition provided herein.

In certain embodiments, the cell is selected from the group consisting of a retinal ganglion cell, a rod cell, a cone cell, a glial cell (e.g., Müller glia cell) , a bipolar cell, an amacrine cell, and a horizontal cell.

EXAMPLES:

The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. A person skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the present invention. It is the intention of the inventors that such variations are included within the scope of the invention.

Example 1 Gene synthesis, expression and purification of bispecific molecules

1.1 Experimental methods:

a) VEGF/C5 bispecific antibodies BSP1, BSP2, BSP3, BSP4, BSP5, and BSP6

Nucleic acid sequences encoding VEGF/C5 bispecific molecules BSP1, BSP2, BSP3, BSP4, BSP5, and BSP6 were designed, optimized and synthesized. The complete sequences were sub-cloned into pcDNA3.4 vector. The recombinant plasmids encoding target antibodies respectively were transiently co-transfected into suspension HD 293F cell cultures (Thermofisher Scientific) . The cells transfected with the plasmids were cultured in Medium at 37℃, 8%CO2. After 6 days of culture, supernatants were collected for protein purification, centrifuged, and followed by filtration. Filtered cell culture supernatant was loaded onto an affinity purification column at an appropriate flowrate. After washing and elution with appropriate buffers, the eluted fractions were pooled, concentrated and loaded onto gel filtration chromatography column at an appropriate flow rate to improve the purity. Concentrate the protein to required concentration. The purified protein was analyzed by SDS-PAGE, Western blot, HPLC analysis to determine the molecular weight and purity. The concentration was determined by A280 method.

On the other hand, Aflibercept (ProBio) and mAb eculizumab (ProBio) were used as positive controls to evaluate the viability and functions of the VEGF/C5 bispecific antibodies prepared in the present application.

Table 3. Structures of different VEGF/C5 bispecific antibodies from N terminal to C terminal

In BSP1 to BSP6, the amino acid sequences for VH (eculizumab) is SEQ ID NO: 1, VL (eculizumab) is SEQ ID NO: 2, VEGFR1 (Domain 2) is SEQ ID NO: 9, VEGFR2 (Domain 3) is SEQ ID NO: 10, human IgG1 CH1 is SEQ ID NO: 19, human CL (κ) is SEQ ID NO: 20, and human IgG1 Fc is SEQ ID NO: 12. Each of the BSP1 to the BSP6 is a dimer of the fusion protein as set forth in Table 3. Linkers in the molecules are present but not expressly indicated in Table 3.

b) Affinity Kd maturation/optimization

Experimental Methods:

i) Selection of amino acids for Kd maturation

FASEBA (Fast Screening for Expression, Biophysical-properties and Affinity) was chosen for affinity Kd maturation (Probio and Genscript) . Based on the parental antibody sequence/BSP1, FASEBA screening was conducted to increase the antibody affinity to target antigen.

Parent mAb Eculizumab was expressed in CHO cell, after antibody purification the binding of the mAb to the human Complement C5 Protein with Biacore 8K/T200. In order to determine the amino acids selected for Kd maturation, parental Fab was constructed in FASEBA format and validated binding using SPR. PML (Precise Mutagenesis Library) library construction in FASEBA format in six CDR residues and conduct NGS (Next Generation Sequencing) to check the distribution of the PML library.

ii) Production and characterization of affinity-matured antibody

The DNA encoding from the best affinity-matured antibody obtained from PML was synthesized and subcloned into expression vector for antibody expression in CHO cells. The affinity-matured antibody was purified using protein A column. Biacore 8K was used to study the kinetics of the interaction between the antigen and affinity-matured antibody (ies) and wild type antibody.

Mutations were introduced to HCDR1, HCDR3, HFR2, HFR3, and HFR4, respectively, in the heavy chain variable region. Additional mutations were also introduced to LCDR2, LFR1, and LFR2 respectively, in the light chain variable region. The kinetics of the interaction between the Human Complement C5 and wild-type/mutated eculizumab were studied.

Affinity-matured mutants have been identified having significant improvement in binding affinity to human C5, ranging from 3 to 10 folds improvement.

Eculizumab -mutant #1 is an eculizumab mutant including one mutation in HC and three mutations in LC. Eculizumab -mutant #1 comprises a VH region comprising the amino acid sequence as set forth in SEQ ID NO: 28, and a VL region comprising the amino acid sequence as set forth in SEQ ID NO: 31. Eculizumab -WT is the original eculizumab sequence.

The results are shown in Table 3A. Eculizumab -mutant #1 to #3 have been identified having significant improvement in binding affinity to human C5.

Table 3A. Affinity of wild-type eculizumab and mutated eculizumab

Eculizumab -mutant #1 was selected as the preferred affinity-matured mutant based on superior protein expression yield from 100mL shake flask production. It was used to make the bispecific molecules BSP1a to conduct further studies.

Table 3B Structures of different VEGF/C5 bispecific antibodies from N terminal to C terminal

In BSP1a, the amino acid sequences for VH (eculizumab mutant) is SEQ ID NO: 28, VL (eculizumab mutant) is SEQ ID NO: 31, VEGFR1 (Domain 2) is SEQ ID NO: 9, VEGFR2 (Domain 3) is SEQ ID NO: 10, human IgG1 CH1 is SEQ ID NO: 19, human CL (κ) is SEQ ID NO: 20, and human IgG1 Fc is SEQ ID NO: 12. The BSP1a is a dimer of the fusion protein as set forth in Table 3B. Linkers in the molecules are not expressly indicated in Table 3 and Table 3B.

1.2 Experimental Results of bispecific antibody purity analysis:

The purified protein was analyzed by SDS-PAGE, Western blot, HPLC analysis to determine the molecular weight and purity.

Table 4. Purity analysis of VEGF/C5 bispecific molecules by SDS-Page and SEC-HPLC

The purity of BSP1a purified by SDS-Page is within the range of 94%-96%, and SEC-HPLC is within the range of 95%-99%.

Example 2 Binding Affinity Assay

2.1 Experimental methods:

Binding affinity of the bispecific molecules to the antigen were determined by SPR assay using Biacore 8K (GE Healthcare, Chicago, IL) . Binding assays were carried out by first attaching BSP1 to the Series S Sensor coated with protein A, then various concentration of human recombinant VEGF-165 protein (Acro: VE5-H4210, 1.5625-100 nM) or human recombinant Complement C5 protein (Acro: CO5-H52Ha, 0.15625-5 nM) were injected over the BSP bound surface at flow rate of 30 μL/min for 120 seconds. 10 mM Glycine-HCl pH 1.5, (GenSript, Lot. No. 20211113) was used to dissociate bound analyte from the sensor chip after sample injection. Binding affinity assay of the BSP1a bispecific molecules is conducted in a same manner.

Antibody binding kinetics including ka (association rate constant) , kd (dissociation rate constant) and K_D (dissociation equilibrium constant) were determined by using Biacore 8K evaluation software version 3.0 (Chicago, IL. ) . Plots of the binding response (RU) versus time were recorded, which allow different stages of a binding event to be visualized and evaluated.

2.2 Experimental results:

As shown in Table 5, the binding affinity of BSP1 and BSP1a against C5 was measured, and the K_D average of BSP1 and BSP1a against human C5 and VEGF are provided. PC2 (anti-C5 mAb) had K_D within the range of 400-500pM against C5. PC1 (Aflibercept) had K_D average of 21.7 pM against VEGF.

Table 5. Binding affinity of VEGF/C5 bispecific molecules and controls against VEGF and C5

Example 3 In vitro cellular functional assays (anti-VEGF and anti-C5)

3.1 VEGF-mediated cell proliferation assay (Anti-VEGF)

3.1.1 Experimental methods:

VEGF-mediated HUVEC (Human Umbilical Vein Endothelial Cells, C2519A, Lonza, Basel, Switzerland) proliferation was chosen to measure the cellular function of VEGF molecules. HUVEC cells were harvested by centrifugation and the cells were re-suspended with cell culture medium. The working solution of test items and VEGF protein with assay buffer were prepared and transferred to the corresponding wells of the 96 well Plate. The cells suspension were transferred to 384 well assay plate, followed by transferring the working solution of test items and VEGF protein mixture to said 384 well plate. Assay plates were incubated in incubator (37℃ and 5%CO₂) for 72 hours, before adding CellTiter-Glo to the corresponding wells of assay plate.

The luminescence signal was recorded with PHERA Star. Raw data were exported from PHERA Star FSX system and analyzed using Microsoft Office Excel 2016 and GraphPad Prism 6.

The dose-response curves were fitted and relative IC50 values were obtained using four-parameter function as follows, characterizing sigmoid curve where percentage growth inhibition was against the concentration of the test samples:

Y = Bottom + (Top-Bottom) / (1+10^ ( (LogIC50-X) *HillSlope) )

In which X = logarithmic of concentration and Y = luminescence signal

Target cells were treated with serial dilutions of test items. Luminescence ± SEM for triplicates repeat in each group was plotted.

3.1.2 Experimental results:

The experimental results showed that both positive control 1 (Aflibercept) and BSP1 could inhibit the proliferation of HUVEC cell and the averaged IC50 value of dose response curves were calculated as 1.55 and 1.10 nM. Negative control were not observed to inhibit HUVEC cells proliferation. Both PC1 (Aflibercept) and BSP1 have similar growth inhibition effect in VEGF-mediated HUVEC proliferation assay. The results of growth inhibition effect of test samples (PC1 and BSP1) against target cell line are shown in Figure 7, Figure 8 and Tables 6 and 7.

BSP1a is also tested using similar methods and shows an IC₅₀ (μg/mL) within the range of 0.1 to 0.3 for growth inhibition effect in VEGF-mediated HUVEC proliferation assay.

Table 6. Best-fit values summary for the first experiment of VEGF-mediated cell proliferation assay

Table 7. Best-fit values summary for the second experiment of VEGF-mediated cell proliferation assay

BSP1a is also tested using the VEGF-mediated cell proliferation assay, and BSP1a shows an IC₅₀ (μg/mL) within the range of 0.1 to 0.2 for inhibition of VEGF-mediated cell proliferation.

3.2 Blockade effect of hemolytic assay/CH50 assay (Anti-C5)

3.2.1 Experimental Methods

Hemolytic assay was chosen to measure the cellular function of C5 molecules. Harvest Sheep Red Blood cells (SRBCs) by centrifugation and re-suspend the cells with GVB++ assay buffer. The working solution of Anti-Red Blood Cell Stroma polyclonal antibody with GVB++ assay buffer was prepared and transferred to SRBCs suspension. Then the mixture of the working solution and SRBCs suspension was well mixed and the plate was incubated at 37℃ and 5%CO₂ for approximately 30 minutes. Similarly, the working solution of PNHS and test items with GVB++assay buffer was prepared and transferred to the corresponding wells of the 96 well assay Plate. Then the mixture was well mix and the plate was incubated at RT for approximately 30 minutes. The Sheep Red Blood cells (SRBCs) suspension primed by incubating anti-RBC stroma antibody was taken out and the cell suspension was transferred to the 96 well assay plate mentioned above. The assay plate was incubated at 37℃ and 5%CO₂ for approximately 1 hour. Then the assay plate was taken from incubator and supernatant collected by centrifuging was transferred to new 96 well test plate. Hemoglobin shedding was performed with Hemoglobin Assay kit. Detection reagent was added to above 96 well test plate, followed by incubating at room temperature for 5 minutes.

The absorbance (OD 400nM) signal was read with PHERA star. The dose-response curves were fitted and relative EC50 values were obtained using four-parameter function as follows, characterizing sigmoid curve where percentage growth inhibition was against the concentration of the test samples:

Y = Bottom + (Top-Bottom) / (1+10^ ( (LogEC50-X) *Hillslope) )

In which X = logarithmic of concentration and Y = Absorbance signal

SRBCs were treated with serial dilutions of test items. Absorbance signal SEM for triplicates repeat in each group was plotted (Fig. 9A) . Hemoglobin amount ±SEM for triplicates repeat in each group was plotted (Fig. 9B) .

3.2.2 Experimental results

For CH50 assay (C5 target test) , both eculizumab (Positive control 2 (PC2) ) and BSP1 had significant blocked effect on Complement C5 and the dose-response curve could be observed. The EC₅₀ values of PC2 (eculizumab) and Test item BSP1 were 21.01 nM and 18.08 nM respectively (Table 8) . BSP1a was tested using similar methods and showed an EC50 (nM) within a range of 18 to 22 nM for CH50 assay.

Table 8. Best-fit values summary for the CH50 assay in terms of Absorbance signal

Example 4 Single clone drug stability assay for BSP1

4.1 Experimental Methods

Stability assays were performed to assess the developability of the multi-specific molecule (s) .

4.2 Experimental Results:

Samples of BSP1 and BSP1a was stored at a concentration of 40 mg/mL in PBS at 40℃ or 5℃ for 14 days. Samples were taken at Day 0, Day 7 and Day 14, respectively, and subject to SEC-HPLC test, CE-SDS-NR test and icIEF (imaged capillary isoelectric focusing) test. A summary is provided at the below Tables 9 and 10.

Table 9. Experimental conditions of sample (BSP1) preparation used in the single clone drug stability test

Table 10. Experimental settings used in the single clone drug stability test

Results:

The 40℃ stability test results are shown in Table 11, where the sample of D0 serves as starting point. In the SEC-HPLC tests, the sample showed that high molecular weight (HMW) percentage increased, main peak percentage decreased, and low molecular weight (LMW) percentage increased. In the CE-SDS-NR tests, the sample showed that the main peak percentage slightly decreased. In the icIEF tests, the sample showed the shift towards the lower pI (acidic) region with the increase of incubation time.

The 5 ℃ stability test results are shown in Table 12, where the sample of D0 serves as starting point. Compared with D0, no obvious changes were observed in SEC-HPLC, CE-SDS-NR, and icIEF tests after incubation of 14 days at 5 ℃.

Stability assay results are shown in Figure 15 to 20 and Table 11 and 12, which supports that BSP1 is the most stable compound over other orientations (BSP2 to BSP6, data not provided) with no change at 5℃ and no significant aggregation and degradation at 40℃ during 14 days and suitable for further drug development.

Table 11. Results of the stable cell-line single clone drug stability test of BSP1 under the condition of 40℃

Table 12. Results of the single clone drug stability test of BSP1 under the condition of 5℃

Example 5 Chronic animal model for persistent retinal neovascularization (up to 12 months)

The objective of the animal study was to evaluate the efficacy and safety of biologics agents BSP1, BSP1a and Aflibercept (Positive Control (PC) , which was a commercially available product) by intravitreal injection (IVT) in the persistent retinal neovascularization (PRNV or dl-AAA) rabbit model. The PRNV model simulates angiogenic retinal diseases to identify indications that may benefit from the drug or novel therapies of the angiogenic retinal diseases in human. The efficacy of BSP1 IVT injection is compared with that of aflibercept IVT injection by their inhibitory effects on animal pharmacodynamics (PD) model which known as DLAAA/PRVN model (see, for details, C. Patel et al, Exp. Eye Res., 2020, 195: 108031) .

5.1 Experimental Methods:

Dutch Belted rabbits were selected in this study for the best results feasibility. For each animal, only one eye will be used to establish the retinal injury model (dl-AAA, less than 70%rate of the PRNV) for characterization and the fellow eye will be used as a control. For further drug testing, retinal injury will be done on one eye. However, the compound will be administered to both eyes to investigate the drug toxicity/effect with and without disease. A unique component of this protocol is the evaluation of ophthalmologic endpoints on conscious rather than on anesthetized animals. This removes potential interferences from anesthetic agents during the examinations, and moreover, increases the likelihood that animals will survive for what could amount to years of follow-up.

Table 13. Animal study timepoints and procedures

5.1.1 Standards of PRNV model for pharmacology study:

Persistent retinal neovascularization (PRNV) with the FA Leakage over 3 months prior to dosing. Neovascular angiographic leak area and intensity are defined by NaF angiography (0.05 mls, 10%, OCT/FA in3 fields, 55°) stable angiographic leak area > 2 disc diameters and leak severity of ≥ score 2 (may difference with study purpose) .

5.1.2 Efficacy of the BSP1, BSP1a and PC on FA leak reduction and the change of retina profile in PRNV model (dl-AAA eyes) :

Fundus photography (FP) : Fundus pictures taken serially are useful to monitor treatment response, inflammatory changes, and the drug distribution.

Fluorescein Angiography (FA) : FA imaging location is same as color images. FA images are taken according to the to the following schedule: Later-phase FA images will be recorded in both the dl-AAA and the control eyes at baseline, week 1, 2, 4, 8, 12 and 16 (may prolong to 24) weeks after the treatment. Angiographic leak will be observed and evaluated by clinical observation in FA for the treatment of BSP1, BSP1a and PC.

Optical coherence tomography (OCT) : OCT is an established medical imaging technique that uses light to capture with micrometer-resolution three-dimensional images of the retinal and choroid structure, map and measure their thickness. The analysis and measurements of the retinal and choroid thickness in OCT will be performed in correlation with the color fundus or FA findings.

Slit lamp examination is performed to evaluate safety and tolerability, revealing an important inflammatory reaction in anterior chamber of the rabbit’s eyes received treatment of the test articles.

The observations will be performed by an ophthalmologist for all eyes immediately after the injections and follow up at the day 1 and day 2. If there are signs of inflammation, the follow up will be continued based on the drug effects and durations.

The standardization of Uveitis Nomenclature (SUN) is applied in all our studies. Cells and flare in the anterior chamber are observed using a slit lamp beam of 1 X 1 mm in height and width, when thrown at an angle of 45-60°. Based on the finding, the inflammation can be categorized from 0 to 3+/ (100%) grade.

5.2 Experimental results:

5.2.1 Effect of the test articles on angiographic leakage

The effect of the BSP1, BSP1a and PC1 on angiographic leakage was evaluated by late-phase fluorescein angiography (FA) at 2, 4, 8, 12 and 16 weeks after the dosing of intravitreal injection (Table 13) . A total of 10 rabbits were used in the study. Seven rabbits received BSP1 in both eyes of the dl-AAA model eye (OD) and theeye (OS) ; 3 rabbits received PC in dl-AAA model eye (OD) and PBS for the eye (OS) as NC. A representative image of the leak profile in each timepoints between the two groups were shown in Fig. 11 and 12. A representative image of the eyes profile in each timepoints between the two groups were shown in Fig. 13 and 14.

All PRNV model eyes (n = 10) of the two treatment groups showed an absence of angiographic leakage from the PRNV sites observed at 2-week post treatment. The different effects on duration and intensity of the in inhibition of the PRNV leakage in FA were observed between the two agents. The recurrence of leakage mainly started around 6～8 weeks with PC1 (Aflibercept) group and 12～16 weeks with BSP1 or BSP1a group.

In order to evaluate and compare the effect of BSP1, BSP1a and PC1 on chronic animal/PD model vascular leakage, vascular leakage scores of BSP1, BSP1a and PC1, which were measured by calculating their corresponding vascular leakage percentage area (%) based on FA images, were plotted versus time (0, 2, 4, 8, 12 and 16 weeks) in Figure 10. Furthermore, FA images (Figure 11) were taken for each group at week 0, 2, 4, 8, 12 and 16 for compound treated vascular leakage evaluation (OD) and compound treated non-DLAAA induced eye as control (OS) .

According to Figure 11 and Table 14, BSP1 has indicated superior vascular leaking inhibition capability, regression on induced leaky vessels, vessel proliferation and longer duration in comparison with the animal group treated by known VEGF inhibitor, PC1 (Aflibercept) .

Table 14. Chronic animal/PD model vascular leakage score vs time (wks) w SD (standard deviation)

Efficacy of BSP1a is investigated as well using the same model. BSP1a also indicated superior vascular leaking inhibition capability, regression on induced leaky vessels, vessel proliferation and longer duration in comparison with the animal group treated by known VEGF inhibitor, PC1 (Aflibercept) . Results of BSP1a is at least comparable to BSP1 as shown in Figure 10, Figure 11, Figure 12, Figure 13, and Table 14.

BSP1 and BSP1a showed much better therapeutic effects over aflibercept in animal model for vascular leakage controlled and anti-inflammation. In particular, BSP1 and BSP1a successfully delayed onset of leakage until week 12, and the significantly slowed down the progression of the leakage to the extent the only an average of less than 30%leakage was observed at week 16. In contrast, animals treated with aflibercept showed obvious leakage at week 8, and the leakage progression was fast because the average leakage reached more than 66%at week 12. This clearly shows superior results of BSP1 and BSP1a over Aflibercept in therapeutic effects and long-lasting duration.

Example 6. BSP1a was expected to have advantageous in purification and recovery rate over BSP1

Mutations in BSP1a are capable of improving protein long term stability and scale up production yield/recovery rate at high protein concentration for further drug process and development.

6.1 Long Term Stability

Stability assays of BSP1 and BSP1a were performed to assess the developability, using the experimental methods as described in Example 4.

Stability assay results are shown in Table 15, BSP1a has superior stability profile and protein expression yield over BSP1.

Table 15. Results of the single clone drug stability test of BSP1 and BSP1a under the condition of 5℃

6.2 Protein Production Yield

Both BSP1 and BSP1a constructs were further developed into stable cell line using CHOK1 cells (thermos fisher) . BSP1a has superior protein production yield of 6.6g/L over BSP1 of 3.6g/L.

Claims

A multi-specific molecule comprising a fusion protein comprising

(a) a complement component 5 (C5) binding domain,

(b) a vascular endothelial growth factor (VEGF) binding domain, and

(c) a multimerizing component;

wherein:

the C5 binding domain comprises an antigen-binding fragment of an anti-C5 antibody,

the VEGF binding domain comprises one or more extracellular immunoglobulin-like (Ig) domain of one or more VEGF receptor (VEGFR) , and

the multimerizing component comprises a polypeptide having a length between 1 and 200 amino acids and having at least one cysteine residue.
The multi-specific molecule of claim 1, wherein the C5 binding domain is operably linked to N terminus of the VEGF binding domain.
The multi-specific molecule of claim 1 or 2, wherein the VEGF binding domain is operably linked to N terminus of the multimerizing component.
The multi-specific molecule of any one of the preceding claims, wherein the multimerizing component comprises an antibody Fc domain.
The multi-specific molecule of any one of the preceding claims, wherein the antigen-binding fragment is a Fab, a Fab’, a F (ab) ₂, a F (ab’) ₂, a single-chain Fab, a VHH, a Fd, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) ₂, a bispecific dsFv (dsFv-dsFv’) , a diabody, a disulfide stabilized diabody (ds diabody) , a single chain Fv (scFv) , an scFv dimer (bivalent diabody) , a camelized single domain antibody, a nanobody, a Tetrabody, a domain antibody, or a bivalent domain antibody.
The multi-specific molecule of any one of the preceding claims, wherein the antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable (VH) region comprising the amino acid sequence as set forth in SEQ ID NO: 1, and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) contained within a light chain variable (VL) region comprising the amino acid sequence as set forth in SEQ ID NO: 2.
The multi-specific molecule of any one of the preceding claims, wherein the antigen-binding fragment comprises a HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 3, a HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 4, a HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 5, a LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 6, a LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 7, and a LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 8.
The multi-specific molecule of any one of the preceding claims, wherein the antigen-binding fragment comprises a VH region comprising the amino acid sequence as set forth in SEQ ID NO: 1, or a homologous sequence thereof having at least 80%sequence identity to SEQ ID NO: 1 yet retaining binding specificity to C5.
The multi-specific molecule of any one of the preceding claims, wherein the antigen-binding fragment comprises a VL region comprising an amino acid sequence as set forth in SEQ ID NO: 2, or a homologous sequence thereof having at least 80%sequence identity to SEQ ID NO: 2 yet retaining binding specificity to C5.
The multi-specific molecule of any one of the preceding claims, wherein the antigen-binding fragment comprises a VH region comprising the amino acid sequence as set forth in SEQ ID NO: 1, and a VL region comprising the amino acid sequence as set forth in SEQ ID NO: 2.
The multi-specific molecule of any one of claims 1-5, wherein the antigen-binding fragment comprises

(a) three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable (VH) region comprising the amino acid sequence as set forth in SEQ ID NO: 28, and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) contained within a light chain variable (VL) region comprising the amino acid sequence as set forth in SEQ ID NO: 31; or

(b) three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable (VH) region comprising the amino acid sequence as set forth in SEQ ID NO: 28, and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) contained within a light chain variable (VL) region comprising the amino acid sequence as set forth in SEQ ID NO: 36; or

(c) three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable (VH) region comprising the amino acid sequence as set forth in SEQ ID NO: 1, and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) contained within a light chain variable (VL) region comprising the amino acid sequence as set forth in SEQ ID NO: 39.
The multi-specific molecule of any one of claims 1-5 and 11, wherein the antigen-binding fragment comprises

(a) a HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 3, a HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 4, a HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 5 or SEQ ID NO: 27, a LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 6 or SEQ ID NO: 29 or SEQ ID NO: 41 or SEQ ID NO: 38, a LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 7, and a LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 8 or SEQ ID NO: 30 or SEQ ID NO: 35; or

(b) a HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 3, a HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 4, a HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 27, a LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 7, and a LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 30; or

(c) a HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 3, a HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 4, a HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 27, a LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 41, a LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 7, and a LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 35; or

(d) a HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 3, a HCDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 4, a HCDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 5, a LCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 38, a LCDR2 comprising the amino acid sequence as set forth in SEQ ID NO: 7, and a LCDR3 comprising the amino acid sequence as set forth in SEQ ID NO: 30.
The multi-specific molecule of any one of claims 1-5, 11 and 12, wherein the antigen-binding fragment comprises a VH region comprising the amino acid sequence as set forth in SEQ ID NO: 28, or a homologous sequence thereof having at least 80%sequence identity to SEQ ID NO: 28 yet retaining binding specificity to C5.
The multi-specific molecule of any one of the preceding claims 1-5, 11-13, wherein the antigen-binding fragment comprises a VL region comprising an amino acid sequence as set forth in SEQ ID NO: 31, 36, or 39, or a homologous sequence thereof having at least 80%sequence identity to SEQ ID NO: 31, 36, or 39 yet retaining binding specificity to C5.
The multi-specific molecule of any one of claims 1-5, 11-14, wherein the antigen-binding fragment comprises

(a) a VH region comprising the amino acid sequence as set forth in SEQ ID NO: 28, and a VL region comprising the amino acid sequence as set forth in SEQ ID NO: 31; or

(b) a VH region comprising the amino acid sequence as set forth in SEQ ID NO: 28, and a VL region comprising the amino acid sequence as set forth in SEQ ID NO: 36; or

(c) a VH region comprising the amino acid sequence as set forth in SEQ ID NO: 1, and a VL region comprising the amino acid sequence as set forth in SEQ ID NO: 39.
The multi-specific molecule of any one of claims 6-15, wherein the antigen-binding fragment further comprises one or more amino acid residue substitutions or modifications yet retains binding specificity to C5.
The multi-specific molecule of any one of the preceding claims, wherein the VEGFR is selected from the group consisting of VEGFR-1, VEGFR-2 and VEGFR-3.
The multi-specific molecule of any one of the preceding claims, wherein the Ig domain is selected from the group consisting of Ig domain 1, Ig domain 2, Ig domain 3 and Ig domain 4.
The multi-specific molecule of any one of the preceding claims, wherein the VEGF binding domain comprises two or more different Ig domains of two or more different VEGFRs.
The multi-specific molecule of any one of the preceding claims, wherein the VEGF binding domain comprises a first Ig domain of a first VEGFR operably linked to N terminus of a second Ig domain of a second VEGFR, either directly or via a first linker.
The multi-specific molecule of claim 20, wherein the first Ig domain is an Ig domain 2 and the second Ig domain is an Ig domain 2 or an Ig domain 3.
The multi-specific molecule of claim 20, wherein the first VEGFR is VEGFR-1, and the second VEGFR is VEGFR-2.
The multi-specific molecule of any one of the preceding claims, wherein the VEGF binding domain comprises an Ig domain 2 of VEGFR-1 and an Ig domain 3 of VEGFR-2.
The multi-specific molecule of claim 23, wherein the Ig domain 2 of VEGFR-1 is operably linked to the N-terminus of the Ig domain 3 of VEGFR-2, either directly or via the first linker.
The multi-specific molecule of claim 23, wherein the Ig domain 2 of VEGFR-1 comprises the amino acid sequence as set forth in SEQ ID NO: 9, and Ig domain 3 of VEGFR-2 comprises the amino acid sequence as set forth in SEQ ID NO: 10.
The multi-specific molecule of any one of claims 20-25, wherein the first linker comprises a peptide linker.
The multi-specific molecule of claim 26, wherein the first linker comprises the amino acid sequence of SEQ ID NO: 18 (TNTII) .
The multi-specific molecule of any one of the preceding claims, wherein the VEGF binding domain comprises the amino acid sequence as set forth in SEQ ID NO: 11, or an amino acid sequence having at least 80%sequence identity thereof yet retaining binding specificity to VEGF.
The multi-specific molecule of any one of the preceding claims, wherein the C5 binding domain is operably linked to the VEGF binding domain directly or via a second linker.
The multi-specific molecule of any one of the preceding claims, wherein the second linker comprises a peptide linker, optionally a GS linker.
The multi-specific molecule of claim 30, wherein the second linker comprises a GS linker.
The multi-specific molecule of claim 31, wherein the GS linker comprises one, two, three, four or more repeats of SEQ ID NO: 13 (GGGS) or SEQ ID NO: 14 (GGGGS) .
The multi-specific molecule of any one of claims 4-32, wherein the Fc domain is derived from human immunoglobulin (Ig) .
The multi-specific molecule of claim 33, wherein the Fc domain is derived from human IgG, optionally human IgG1, IgG2, IgG3 or IgG4.
The multi-specific molecule of claim 34, wherein the Fc domain is derived from human IgG1.
The multi-specific molecule of any one of claims 4-32, wherein the Fc domain is mutated.
The multi-specific molecule of claim 36, wherein the Fc domain comprises a mutation at position 235 and/or 309 of the human IgG1 in accordance with EU numbering system.
The multi-specific molecule of claim 36, wherein the Fc domain comprises a substitution at position 235 and/or 309 of the human IgG1 in accordance with EU numbering system.
The multi-specific molecule of claim 36, wherein the Fc domain comprises a L235K and/or L309K mutation in accordance with EU numbering system.
The multi-specific molecule of any one of the preceding claims, wherein the Fc domain comprises the amino acid sequence as set forth in SEQ ID NO: 12, SEQ ID NO: 26, or an amino acid sequence having at least 80%sequence identity thereof yet retaining the capability to multimerize.
The multi-specific molecule of any one of the preceding claims, wherein the multimerizing component is operably linked to the C terminus of the VEGF binding domain, directly or via a third linker, optionally, the third linker is a peptide liner, further optionally the third linker comprises the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25.
The multi-specific molecule of any one of the preceding claims, wherein the fusion protein comprises the C5 binding domain comprising a Fab domain comprising a heavy chain polypeptide and a light chain polypeptide.
The multi-specific molecule of claim 42, wherein the heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 21, and/or the light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 17.
The multi-specific molecule of claim 42, wherein

(a) the heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 32, and/or the light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 33; or

(b) the heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 32, and/or the light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 37; or

(c) the heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 21, and/or the light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 40.
The multi-specific molecule of claim 42, wherein the heavy chain polypeptide is operably linked to the N terminus of the VEGF binding domain, either directly or via the second linker.
The multi-specific molecule of claim 42, wherein the fusion protein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 16 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 17.
The multi-specific molecule of claim 42, wherein the fusion protein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 34 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 33.
The multi-specific molecule of claim 42, wherein the heavy chain polypeptide is operably linked to the C terminus of the multimerizing component, either directly or via a linker, and the multimerizing component is operably linked to the C terminus of the VEGF binding domain, either directly or via a linker.
The multi-specific molecule of any one of the preceding claims, wherein the fusion protein comprises the C5 binding domain comprising an scFv comprising the VH region and the VL region operably linked via a linker.
The multi-specific molecule of claim 49, wherein the fusion protein comprises the scFv operably linked to the N terminus of the VEGF binding domain, either directly or via a linker.
The multi-specific molecule of claim 50, wherein the fusion protein comprises the scFv operably linked to the C terminus of the multimerizing component, either directly or via a linker, and the multimerizing component is operably linked to the C terminus of the VEGF binding domain, either directly or via a linker.
The multi-specific molecule of any one of the preceding claims, wherein the fusion protein comprises the C5 binding domain comprising a VHH domain.
The multi-specific molecule of claim 52, wherein the fusion protein comprises the VHH domain operably linked to the N terminus of the VEGF binding domain, either directly or via a linker.
The multi-specific molecule of claim 53, wherein the fusion protein comprises the VHH domain operably linked to the C terminus of the multimerizing component, either directly or via a linker, and the multimerizing component is operably linked to the C terminus of the VEGF binding domain, either directly or via a linker.
The multi-specific molecule of any one of the preceding claims, wherein the multi-specific molecule comprises a dimer of the fusion protein.
A pharmaceutical composition comprising the multi-specific molecule of any one of the preceding claims, and one or more pharmaceutically acceptable carriers.
An isolated polynucleotide encoding the multi-specific molecule of any one of the preceding claims.
A vector comprising the isolated polynucleotide of claim 57.
A host expression system comprising the vector of claim 58.
The host expression system of claim 59, which is a microorganism, a yeast, or a mammalian cell.
A method of expressing the multi-specific molecule of any one of claims 1-55, comprising culturing the host expression system of any one of the preceding claims under the condition at which the vector of any one of the preceding claims is expressed.
A method of treating, preventing or alleviating a C5 related and/or a VEGF related disease, disorder or condition in a subject, comprising administering to the subject a therapeutically effective amount of the multi-specific molecule of any one of claims 1-55 and/or the pharmaceutical composition of claim 56.
A method of treating, preventing or alleviating a disease, disorder or condition associated with an increased level and/or activity of C5 and/or VEGF in a subject, comprising administering to the subject a therapeutically effective amount of the multi-specific molecule of any one of claims 1-55 and/or the pharmaceutical composition of claim 56.
The method of claim 62 or 63, wherein the disease, disorder or condition is selected from the group consisting of ocular disease, cancer, inflammatory disease, autoimmune disease, angiogenesis, vascular permeability, edema, and inflammation.
The method of claim 64, wherein the ocular disease is selected from the group consisting of age-related macular degeneration (AMD) , geographic atrophy (GA) , macular edema, macular edema following retinal vein occlusion (RVO) , diabetic macular edema (DME) , diabetic retinopathy (DR) , retinal central vein occlusion, corneal neovascularization (CNV) , retinal pigmentosa (RP) , ocular angiogenesis (ocular neovascularization affecting choroidal, corneal or retinal tissue) , retinopathy of prematurity (ROP) , pathological myopia, vascular glaucoma, retinoblastoma, retinal vein occlusion, uveitis and neuromyelitis optica.
The method of claim 64, wherein the ocular disease is AMD or GA.
The method of claim 66, wherein the AMD is wet AMD.
The method of claim 66, wherein the AMD is dry AMD.
The method of any one of claims 62-68, wherein the subject is human.
The method of any one of claims 62-69, wherein the administration is via intraocular, intravitreal injection, topical, subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, intradermal injection, oral, intranasal, intraocular, intravitreal injection, sublingual, or rectal administration.
The method of any one of claims 62-70, further comprising administering a therapeutically effective amount of a second therapeutic agent.
The method of claim 71, wherein the second therapeutic agent is selected from the group consisting of anti-angiogenic agent, inflammatory drugs, m-Tor inhibitors, rapamycin, everolimus, temsirolimus, cyclosporine, anti-TNF agents, anti-complement agents, and nonsteroidal anti-inflammatory agents.
A method of modulating C5 and/or VEGF activity in a cell, comprising exposing the cell to the multi-specific molecule of any one of claims 1-55.
The method of claim 73, wherein the cell is selected from the group consisting of a retinal ganglion cell, a rod cell, a cone cell, a glial cell (e.g., Müller glia cell) , a bipolar cell, an amacrine cell, and a horizontal cell.
The multi-specific molecule of any one of claims 1-55 and/or the pharmaceutical compositions of claim 56 for use in treating, preventing or alleviating a C5 related and/or a VEGF related disease, disorder or condition in a subject.
Use of the multi-specific molecule of any one of claims 1-55 and/or the pharmaceutical composition of claim 56 in the manufacture of a medicament for treating, preventing or alleviating a C5 related and/or a VEGF related disease, disorder or condition in a subject.