CN115677854A

CN115677854A - Antibodies against activin receptor-like kinase 1 and uses thereof

Info

Publication number: CN115677854A
Application number: CN202110836999.3A
Authority: CN
Inventors: 缪小牛; 胡化静; 罗羿; 王平
Original assignee: Biotheus Inc
Current assignee: Biotheus Inc
Priority date: 2021-07-23
Filing date: 2021-07-23
Publication date: 2023-02-03

Abstract

The invention provides an antibody or an antigen binding fragment thereof aiming at an activin receptor-like kinase 1 (ALK-1), wherein the antibody or the antigen binding fragment thereof has improved affinity to the ALK-1, can more effectively inhibit an ALK-1/TGF-beta-1/Smaddl signal pathway and block Smaddl phosphorylation and downstream transcription reaction, thereby having the effect of inhibiting tumor blood vessel growth. The invention also relates to the therapeutic use of these antibodies or antigen-binding fragments thereof for inhibiting tumor angiogenesis-related diseases.

Description

Antibodies against activin receptor-like kinase 1 and uses thereof

Technical Field

The invention provides an antibody or an antigen binding fragment thereof for activin receptor-like kinase 1 (ALK-1), wherein the antibody or the antigen binding fragment thereof has improved affinity for ALK-1, can more effectively inhibit an ALK-1/TGF-beta-1/Smaddl signal pathway, and can block Smaddl phosphorylation and downstream transcription reaction, thereby having the effect of inhibiting tumor blood vessel growth. The invention also relates to therapeutic uses of these antibodies or antigen binding fragments thereof for inhibiting tumor vascular growth.

Background

ALK-1 (activin receptor-like kinase-1), an activin receptor-like kinase-1, is a member of the TGF- β receptor family, is mainly expressed in vascular endothelial cells, is involved in the growth and migration of endothelial cells, and has an important role in regulating angiogenesis or repair. Angiogenesis is a physiological process involving the formation of new blood vessels from pre-existing blood vessels and/or circulating endothelial stem cells. This is a normal process in growth, development and wound healing. However, angiogenesis is also an important step in the transition of tumors from a dormant state to a malignant state. In the development of diseases such as tumors, the body loses the ability to maintain a balance of angiogenesis. The neovasculature harbors abnormal tissue, destroys normal tissue, and for some tumors, allows tumor cells to escape to the circulatory system and reside in other organs (tumor metastases). Therefore, angiogenesis inhibitors are a very promising class of antitumor drugs that target this abnormal process and thereby block or slow tumor growth.

By targeting targets with highly selective endothelial function, such as ALK-1, dimerization of type II receptors can be inhibited, and in turn Smadl phosphorylation and downstream transcriptional responses blocked, thereby inhibiting angiogenesis. The potential of anti-ALK-1 monoclonal antibodies as angiogenesis inhibitors has been reported, for example, in PCT International application WO2007040912.GT90001 is a fully humanized IgG2 monoclonal antibody which binds to the extracellular domain (ECD) of ALK-1, and can generate the effects of inhibiting the growth of tumor blood vessels and the like by preventing the blocking of TGF-beta receptor, thereby influencing the tumor microenvironment and slowing the tumor progression. However, GT90001 has a low affinity, which may affect the drug efficacy and increase the difficulty of subsequent industrialization. There is therefore a need to develop more effective antibodies against ALK-1, thereby providing an effective therapeutic or adjuvant means for the treatment of tumors.

Disclosure of Invention

Antibodies of the invention

In one aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to activin receptor-like kinase 1 (ALK-1), the antibody or antigen-binding fragment thereof comprising:

(i) A heavy chain variable region (VH) comprising the sequence set forth in any one of SEQ ID NOs:1, 3-11; and, a light chain variable region (VL) comprising the sequence set forth in any one of SEQ ID NOs: 12-18; or the like, or a combination thereof,

(ii) A heavy chain variable region (VH) comprising the sequence set forth in any one of SEQ ID NOs: 3-11; and a light chain variable region (VL) comprising the sequence set forth in any one of SEQ ID NOs:2, 12-18.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO. 1; and a VL comprising a sequence set forth in any one of SEQ ID NOs: 12-18.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

(a) A VH comprising the sequence shown in SEQ ID NO. 1; and, a VL comprising the sequence set forth in SEQ ID NO. 15; or the like, or a combination thereof,

(b) VH comprising the sequence shown as SEQ ID NO 1; and, a VL comprising the sequence shown in SEQ ID NO 16; or the like, or, alternatively,

(c) VH comprising the sequence shown as SEQ ID NO 1; and VL comprising the sequence shown in SEQ ID NO. 18.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in any one of SEQ ID NOs: 3-11; and VL comprising the sequence shown in SEQ ID NO. 2.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in any one of SEQ ID NOs: 3-9; and a VL comprising a sequence set forth in any one of SEQ ID NOs:12, 13, 16-18.

(a) A VH comprising the sequence shown in any one of SEQ ID NOs:3, 4, 6, 7, 9; and, a VL comprising the sequence shown in SEQ ID NO 18; or the like, or, alternatively,

(b) A VH comprising the sequence shown in any one of SEQ ID NOs:3, 5, 6, 9; and, a VL comprising the sequence shown in SEQ ID NO 17; or the like, or, alternatively,

(c) A VH comprising a sequence set forth in any one of SEQ ID NOs: 3-9; and, a VL comprising the sequence shown in SEQ ID NO 16; or the like, or, alternatively,

(d) A VH comprising the sequence shown as

SEQ ID NO

7 or 8; and VL comprising the sequence shown in SEQ ID NO. 13; or the like, or a combination thereof,

(e) A VH comprising the sequence shown in SEQ ID NO. 6; and VL comprising the sequence shown in SEQ ID NO. 12.

In certain embodiments, an antibody or antigen-binding fragment thereof of the invention may further comprise a constant region sequence derived from a mammalian (e.g., human) immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions, or additions compared to the sequence from which it is derived.

In certain embodiments, the heavy chain of an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) as compared to the sequence from which it is derived; and/or the presence of a gas in the gas,

the light chain of the antibody or antigen-binding fragment thereof of the invention comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof having conservative substitutions of up to 20 amino acids (e.g., conservative substitutions of up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it is derived.

In some embodiments, the variants of the heavy chain constant region (CH) may have conservative substitutions of one or more amino acids compared to the sequence from which they are derived. In such embodiments, the variants of the heavy chain constant region (CH) may have the same or substantially the same effector function as compared to the wild-type sequence from which they are derived.

In other embodiments, the variant of the heavy chain constant region (CH) may comprise one or more amino acid mutations to alter one or more of the following properties of the antibody of the invention: fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function or complement function, etc. A functional change, e.g., an alteration in the affinity of an antibody for an effector ligand (e.g., fcR or complement C1 q), and thus an alteration (e.g., a decrease) in effector function, can be produced by substituting at least one amino acid residue in the constant region of the antibody with a different residue. The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, and the like.

In certain embodiments, the heavy chain constant region is an IgG heavy chain constant region, e.g., an IgG1, igG2, igG3, or IgG4 heavy chain constant region. In certain embodiments, the heavy chain constant region is a human IgG1, igG2, igG3, or IgG4 heavy chain constant region.

In certain embodiments, the light chain constant region is a kappa light chain constant region. In certain embodiments, the light chain constant region is a human kappa light chain constant region.

In certain exemplary embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise the heavy chain constant region (CH) shown in SEQ ID NO 19; and/or, the light chain constant region (CL) shown in SEQ ID NO: 20.

In certain embodiments, the antibody or antigen-binding fragment thereof is selected from IgG, igM, igE, igD, or IgA, e.g., igG1, igG2, igG3, or IgG4.

In certain embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of scFv, di-scFv, (scFv) ₂ 、Fab、Fab’、(Fab’) ₂ Fv, disulfide-linked Fv.

In certain embodiments, an antibody or antigen-binding fragment thereof of the invention possesses one or more of the following characteristics:

(1) Specifically binds to ALK-1 (e.g., human ALK-1) (e.g., its extracellular domain (ECD));

(2) At less than about 5nM, e.g., less than about 4.5nM, 4nM, 3.5nM, 3nM, 2.5nM, 2nM, 1.5nM, 1K of nM, 0.5nM, 0.4nM or less _D Binds to ALK-1 (e.g., human ALK-1); preferably, said K _D As determined by surface plasmon resonance techniques (e.g., fortebio);

(3) Blocking or inhibiting Smadl phosphorylation;

(4) Blocking or inhibiting ALK-1/TGF-beta-1/Smaddl signaling pathway

(5) Inhibiting tumor angiogenesis.

Herein, an antibody or antigen-binding fragment thereof of the invention may include variants that differ from the antibody or antigen-binding fragment thereof from which it is derived only by conservative substitutions of one or more (e.g., conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids) amino acid residues, or that have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the antibody or antigen-binding fragment thereof from which it is derived, and that substantially retain the above-described biological functions of the antibody or antigen-binding fragment thereof from which it is derived.

Preparation of antibodies

The antibody of the present invention can be prepared by various methods known in the art, for example, by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into a host cell. The transfected host cells are then cultured under specific conditions and the antibodies of the invention are expressed.

The antigen-binding fragments of the invention can be obtained by hydrolysis of the intact antibody molecule (see Morimoto et al, j. Biochem. Biophysis. Methods 24, 107-117 (1992) and Brennan et al, science 229 (1985). Alternatively, these antigen-binding fragments can be produced directly by recombinant host cells (reviewed in Hudson, curr. Opin. Immunol.11:548-557 (1999); little et al, immunol.Today,21 (364-370) (2000)). For example, fab' fragments can be obtained directly from the host cell; fab 'fragments can be chemically coupled to form F (ab') ₂ Fragments (Carter et al, bio/Techno)log, 10. In addition, fv, fab or F (ab') ₂ The fragments may also be isolated directly from the culture medium of the recombinant host cell. Other techniques for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.

Thus, in another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof of the invention, or a heavy chain variable region and/or a light chain variable region thereof. In certain embodiments, the isolated nucleic acid molecule encodes an antibody or antigen-binding fragment thereof of the present invention, or a heavy chain variable region and/or a light chain variable region thereof.

In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the present invention and a second nucleotide sequence encoding a light chain or light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different isolated nucleic acid molecules. When the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules, the isolated nucleic acid molecules of the invention comprise a first nucleic acid molecule comprising the first nucleotide sequence and a second nucleic acid molecule comprising the second nucleotide sequence.

In another aspect, the invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule as described above. In certain embodiments, the vectors of the invention are, for example, plasmids, cosmids, phages and the like.

In certain embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention and a second nucleotide sequence encoding a light chain or light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector of the present invention comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

In certain embodiments, the vector comprises a first nucleotide sequence encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention, and/or a second nucleotide sequence encoding the light chain variable region of the antibody or antigen-binding fragment thereof of the present invention; wherein the first nucleotide sequence and the second nucleotide sequence are provided on the same or different vectors.

In certain embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain of the antibody or antigen-binding fragment thereof of the present invention, and/or a second nucleotide sequence encoding a light chain of the antibody or antigen-binding fragment thereof of the present invention; wherein the first nucleotide sequence and the second nucleotide sequence are provided on the same or different vectors.

In another aspect, the invention provides a host cell comprising an isolated nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., E.coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.).

In another aspect, there is provided a method of making an antibody or antigen-binding fragment thereof of the invention comprising culturing a host cell as described above under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

Pharmaceutical compositions and therapeutic uses

The antibody or the antigen binding fragment thereof can inhibit an ALK-1/TGF-beta-1/Smaddl signal pathway, block Smaddl phosphorylation and downstream transcription reaction, and further has the effect of inhibiting the growth of tumor blood vessels.

Thus, in another aspect, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof, an isolated nucleic acid molecule, vector or host cell of the invention, and a pharmaceutically acceptable carrier and/or excipient.

In certain exemplary embodiments, the pharmaceutically acceptable carrier and/or excipient comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), glucose solutions (e.g., 5% glucose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), ringer's solution, and any combination thereof.

In another aspect, the present invention provides a method for inhibiting tumor angiogenesis, inhibiting tumor growth, and/or treating a tumor in a subject, comprising: administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof, or a pharmaceutical composition of the invention. The invention also relates to the use of an antibody or antigen-binding fragment thereof, an isolated nucleic acid molecule, a vector, a host cell or a pharmaceutical composition of the invention in the manufacture of a medicament for inhibiting tumor angiogenesis, inhibiting tumor growth, inhibiting tumor metastasis and/or treating a tumor in a subject.

In certain embodiments, the antibody or antigen-binding fragment thereof is used alone or in combination with another pharmaceutically active agent (e.g., an anti-tumor drug, such as an immune checkpoint inhibitor). The antibody or antigen-binding fragment thereof of the invention and the additional pharmaceutically active agent may be administered simultaneously, separately or sequentially.

In certain embodiments, the tumor is selected from a solid tumor, such as hepatobiliary cancer, renal cell carcinoma, colorectal cancer, mesothelioma, or urethral cancer.

The antibody or antigen-binding fragment thereof of the present invention, or the pharmaceutical composition of the present invention may be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The antibodies or antigen binding fragments thereof or pharmaceutical compositions of the invention should be sterile and stable under the conditions of manufacture and storage. One preferred dosage form is an injection. Such injectable formulations may be sterile injectable solutions. For example, sterile injectable solutions can be prepared by the following methods: the antibody or antigen-binding fragment thereof of the present invention is incorporated in a suitable solvent at the necessary dosage and, optionally, with other desired ingredients (including, but not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filter sterilization. In addition, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for storage and use. Such sterile lyophilized powders may be dispersed in a suitable carrier, e.g., water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffered solution (e.g., phosphate buffered solution), ringer's solution, and any combination thereof, prior to use.

The antibody or antigen-binding fragment thereof of the invention, or the pharmaceutical composition of the invention, may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powder, ointment, or drops), or nasal route. However, for many therapeutic uses, the preferred route/mode of administration is parenteral (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and/or mode of administration will vary depending on the intended purpose. In certain embodiments, the antibody or antigen-binding fragment thereof or pharmaceutical composition of the invention is administered by intravenous injection or bolus injection.

The pharmaceutical compositions of the invention may include a "therapeutically effective amount" of an antibody or antigen-binding fragment thereof of the invention. A "therapeutically effective amount" refers to an amount of a therapeutic agent administered to alleviate, to some extent, one or more of the symptoms of the disease being treated. In treating tumors, a therapeutically effective amount refers to an amount that has at least one of the following effects: tumor volume is reduced, tumor metastasis is inhibited (i.e., slowed to some extent, better stopped), tumor growth is inhibited (i.e., slowed to some extent, better stopped), and one or more symptoms associated with the tumor are alleviated (or, better eliminated) to some extent.

In this context, the dosage regimen may be adjusted to obtain the optimal desired response (e.g., therapeutic response). For example, the dosage may be given in a single dose, may be given multiple times over a period of time, or may be reduced or increased proportionally with the exigencies of the therapeutic situation.

Herein, the subject may be a mammal, such as a human.

Definition of terms

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the laboratory procedures in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, as used herein, are conventional procedures that are widely used in the relevant fields. Meanwhile, in order to better understand the present invention, the following provides definitions and explanations of related terms.

As used herein, the term "ALK-1" (interchangeably referred to as "activin receptor-like kinase-1") refers to a type I cell surface receptor that transforms growth factor beta receptor type 1 (TGF-beta-1). As used herein, the term "ALK-1" refers to any native or variant (whether native or synthetic) ALK-1 polypeptide. The term "native sequence" specifically encompasses naturally occurring truncated or secreted forms (e.g., extracellular domain sequences), naturally occurring variant forms (e.g., alternatively spliced forms), and naturally occurring allelic variants. Human ALK-1 is a 503 amino acid polypeptide comprising a signal peptide (amino acids: 1-21), an N-terminal extracellular TGF- β -1 ligand binding domain or ECD (amino acids: 22-118), a single-pass transmembrane domain (amino acids: 119-141), a glycine/serine (GS) -rich regulatory domain (amino acids: 142-202), and a C-terminal serine-threonine kinase domain (202-492).

As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one Light Chain (LC) and one Heavy Chain (HC). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the antibody isotypes are defined as IgM, igD, igG, igA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit a variety of effector functions, such as may mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). The VH and VL regions can also be subdivided into regions of high denaturation, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each V _H And V _L By the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino-terminal to the carboxyl-terminal arrangement of 3 CDR and 4 FR composition. The variable regions (VH and VL) of each heavy/light chain pair form the antigen-binding sites, respectively. The allocation of amino acids in each region or domain may follow Kabat, sequences of Proteins of Immunological Interest (National Institutes of Health, bethesda, md. (1987 and 1991)), or Chothia&Lesk (1987) J.mol.biol.196:901-917; chothia et al (1989) Nature 342, 878-883.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs, designated CDR1, CDR2 and CDR3, in the variable regions of the heavy and light chains. The precise boundaries of these CDRs may be defined according to various numbering systems known in the art, for example, according to the Kabat numbering system (Kabat et al, sequences of Proteins of Immunological Interest,5th Ed. Public Health service, national Institutes of Health, bethesda, md., 1991), chothia numbering system (Chothia & Lesk (1987) J.mol.biol.196:901-917, chothia et al (1989) Nature 342. For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between the different numbering systems is well known to the person skilled in the art (see for example Lefranc et al, dev. Company. Immunol.27:55-77, 2003).

As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibodies can be of different isotypes, e.g., igG (e.g., igG1, igG2, igG3, or IgG4 subtypes), igA1, igA2, igD, igE, or IgM antibodies.

As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion. See generally, fundamental Immunology, ch.7 (Paul, W., ed., 2nd edition, raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes ₂ Fv, disulfide-linked Fv, scFv, di-scFv, (scFv) ₂ And polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding capacity to the polypeptideAnd (4) dividing. Engineered antibody variants are reviewed in Holliger et al, 2005; nat Biotechnol, 23.

As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" and two "full-length light chains". Wherein "full-length heavy chain" refers to a polypeptide chain consisting of, in the N-terminal to C-terminal direction, a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a Hinge Region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain; and, when the full length antibody is of IgE isotype, optionally further comprising a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. Two pairs of full length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between HR of the two full length heavy chains. The full length antibodies of the invention may be from a single species, e.g., human; chimeric antibodies or humanized antibodies are also contemplated. The full-length antibody of the present invention comprises two antigen-binding sites formed by VH and VL pairs, respectively, that specifically recognize/bind to the same antigen.

As used herein, the term "Fd" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al, nature 341 544 (1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F (ab') ₂ Fragment "means an antibody fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; the term "Fab 'fragment" means a reductively linked F (ab') ₂ The fragment obtained after disulfide bonding of the two heavy chain fragments of the fragment consists of one complete fragment of Fd of the light and heavy chains (consisting of the VH and CH1 domains).

As used herein, the term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that can form an entire antigen binding site. It is generally believed that the six CDRs confer antigen binding specificity on the antibody. However, even one variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) is able to recognize and bind antigen, although its affinity may be lower than the entire binding site.

As used herein, the term "Fc" means an antibody fragment formed by disulfide bonding of the second and third constant regions of a first heavy chain and the second and third constant regions of a second heavy chain of an antibody. The Fc fragment of an antibody has a number of different functions, but is not involved in antigen binding.

As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein The VL and VH are connected by a linker (linker) (see, e.g., bird et al, science 242 423-426 (1988); huston et al, proc. Natl. Acad. Sci. USA 85. Such scFv molecules can have the general structure: NH (NH) ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of a repetitive GGGGS amino acid sequence or a variant thereof. For example, a peptide having an amino acid sequence (GGGGS) ₄ But variants thereof can also be used (Holliger et al (1993), proc.natl.acad.sci.usa 90. Other linkers useful in the present invention are described by Alfthan et al (1995), protein Eng.8:725-731, choi et al (2001), eur.J. Immunol.31:94-106, hu et al (1996), cancer Res.56:3055-3061, kipriyanov et al (1999), J.mol.biol.293:41-56 and Rovers et al (2001), cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments of the invention, the scFv may form a di-scFv, which refers to two or more individual scFv connected in tandem to form an antibody. In certain embodiments of the invention, the scFv may form a (scFv) ₂ It refers to two or more individual scfvs connected in parallel to form an antibody.

Each of the above antibody fragments retains the ability to specifically bind to the same antigen to which the full length antibody binds, and/or competes with the full length antibody for specific binding to the antigen.

Antigen-binding fragments of antibodies (e.g., antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of antibodies are specifically screened for specificity in the same manner as for intact antibodies.

Herein, when the term "antibody" is referred to, it includes not only intact antibodies, but also antigen-binding fragments of antibodies, unless the context clearly indicates otherwise.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction may be the equilibrium dissociation constant (K) of the interaction _D ) And (4) showing. In the present invention, the term "K _D "refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the more tight the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

The specific binding properties between two molecules can be determined using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rate of association and dissociation (see Malmqvist M, nature,1993, 361. The ratio of kdis/kon is equal to the dissociation constant K _D (see Davies et al, annual Rev Biochem, 1990. K can be measured by any effective method _D Kon and kdis values. In certain embodiments, the dissociation constant may be measured in Biacore using Surface Plasmon Resonance (SPR). In addition to this, dissociation constants can be measured using bioluminescence interferometry or Kinexa.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection such that the genetic material element it carries is expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs), or artificial chromosomes of P1 origin (PACs); bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication origin.

As used herein, the term "host cell" refers to a cell that can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK293 cells, or human cells.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions in total match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such alignments can be performed by using, for example, needleman et al (1970) j.mol.biol.48: 443-453. The algorithm of e.meyers and w.miller (comput.appl biosci.,4, 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0) can also be used to determine percent identity between two amino acid sequences using a PAM120 weight residue table (weight residue table), a gap length penalty of 12, and a gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48:444-453 (1970)) algorithms that have been incorporated into the GAP program of the GCG software package (available at www.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix, and GAP weights (GAP weights) of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., of similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., brummell et al, biochem.32:1180-1187 (1993); kobayashi et al Protein Eng.12 (10): 879-884 (1999); and Burks et al Proc. Natl Acad. Set USA 94 412-417 (1997), which are incorporated herein by reference).

The writing of the twenty conventional amino acids referred to herein follows conventional usage. See, for example, immunology-A Synthesis (2 nd edition, E.S. Golub and D.R.Gren, eds., sinauer Associates, sunderland, mass. (1991)) which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations as is well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, which are well known in the art (see, e.g., remington's Pharmaceutical sciences. Edited by geno AR, 9th ed. Pennsylvania: pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, naCl, and the like. Agents that delay absorption include, but are not limited to, monostearate salts and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, for example, thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning generally understood by those skilled in the art to stabilize the desired activity of the active ingredient in a medicament, and include, but are not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or degradation products thereof (such as milk albumin hydrolysate), and the like. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), glucose solutions (e.g., 5% glucose), solutions containing surfactants (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), ringer's solution, and any combination thereof.

As used herein, the term "treatment" refers to a method performed in order to obtain a beneficial or desired clinical result. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Furthermore, "treatment" may also refer to prolonging survival as compared to expected survival (if not treated). For tumors, the term "treatment" may mean an increase in the life expectancy of a cancer patient, or a reduction in one or more symptoms of the disease.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, a desired effect. For example, a "therapeutically effective dose" refers to an amount of a therapeutic agent administered that will, to some extent, alleviate one or more symptoms of the disease being treated. In treating tumors, a therapeutically effective amount refers to an amount that has at least one of the following effects: tumor volume is reduced, tumor metastasis is inhibited (i.e., slowed to some extent, better stopped), tumor growth is inhibited (i.e., slowed to some extent, better stopped), and one or more symptoms associated with the tumor are alleviated (or, better eliminated) to some extent. It is well within the ability of those skilled in the art to determine such effective amounts. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, and the like.

As used herein, the term "subject" may refer to a mammal, such as a human. In certain embodiments, the subject has a tumor.

Advantageous effects of the invention

The invention provides an anti-ALK-1 antibody with optimized affinity, which can more effectively inhibit an ALK-1/TGF-beta-1/Smaddl signal pathway and block Smaddl phosphorylation and downstream transcription reaction, thereby having the effect of inhibiting tumor angiogenesis. The antibody of the invention has important clinical value for tumor angiogenesis.

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

Drawings

FIG. 1 shows the binding assay of the flow-detection mutant antibody to CHO-ALK1 cells in example 3.

FIG. 2 shows the binding assay of the flow assay mutant antibody to HUVEC cells in example 3.

FIG. 3 shows the block experiment of BMP9 induced the phosphorylation of Smad1 downstream of ALK1 by the mutant antibody in example 4.

Sequence information

Information on partial sequences to which the present invention relates is provided in table 1 below.

Table 1: description of the sequences

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, the molecular biological experimental methods and immunoassays used in the present invention are essentially described in reference to j.sambrook et al, molecular cloning: a laboratory manual, 2nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, eds. Molecular biology laboratory Manual, 3 rd edition, john Wiley & Sons, inc., 1995; the use of restriction enzymes follows the conditions recommended by the product manufacturer. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed.

Example 1: construction of affinity maturation libraries and antibody screening

Affinity maturation library construction

The synthesized GT90001 gene is used as a template, a weight variable region (VH) and a light variable region (VK) are amplified by PCR, and a yeast expression vector is constructed by a homologous recombination method according to a commercial instruction book (Vazyme). And simultaneously electrically transforming the GT90001 heavy chain and the light chain constructed into the yeast expression vector into the saccharomyces cerevisiae, plating, selecting and cloning, and verifying to obtain the correct GT90001 yeast monoclonal. At the same time, the GT90001 heavy chain and the light chain are respectively transformed into Saccharomyces cerevisiae by electric transformation, plated, selected, cloned and verified, and correct yeast monoclonals of the GT90001 heavy chain and the light chain are respectively obtained. In the first round of PCR, GT90001VH and VK are used as templates, and degenerate primers are used for mutation to obtain partial fragments of VH and VK. The first round of PCR products were subjected to agarose gel electrophoresis, and the gel was cut and recovered for the second round of Overlap PCR amplification. The desired fragment was recovered using a PCR purification kit (purchased from QIAGEN). The linearized yeast display vector and the second round of VH and VK PCR products were mixed and separately electro-transformed into GT90001 light and heavy chain yeast monoclonals, affinity maturation libraries for heavy and light chain mutations were constructed and the storage capacity was determined.

Screening of ALK-1 antibodies

The affinity maturation library constructed above was inoculated into SD-CAA amplification medium and cultured overnight. Yeast cells with 10 Xthe library volume were inoculated into SD-CAA induction medium and induced overnight. Yeast cells were harvested at 10 Xthe pool volume and centrifuged to remove the medium. The yeast cells were resuspended with 50ml of washing buffer (PBS +0.5% BSA +2mM EDTA) and the supernatant removed by centrifugation. The yeast cells were resuspended in 10ml of wash buffer. Biotin-labeled ALK-1 protein was added, incubated at room temperature for 30min, yeast cells were collected by centrifugation, and the yeast was washed 3 times with 50ml of washing buffer. The yeast cells were resuspended in 5ml of wash buffer and 200. Mu.l of SA magnetic beads (purchased from Edward and whirlpool) were added and incubated for 10min with inversion. The yeast and magnetic bead mixture was washed 3 times with wash buffer and the mixture was added to an LS column (purchased from America and whirlwind). The LS column was placed on a magnetic rack and washed with washing buffer to remove non-specifically bound yeast cells. The column was removed from the magnetic rack and the yeast was eluted by adding wash buffer. The eluted yeast was centrifuged and transferred to 20ml SD-CAA amplification medium for amplification. The MACS-enriched yeast cells were inoculated into SD-CAA amplification medium and cultured overnight. The yeast cells were resuspended in induction medium and induced overnight. Biotin-labeled ALK-1 antigen was added and incubated at room temperature for 20min. The yeast was washed 3 times with PBS, FITC-anti-c-Myc antibody and streptavidin APC conjugated fluorescent antibody (purchased from Invitrogen) were added, and incubated at 4 ℃ in the dark for 15min. Adding PBS to resuspend the cells, and using a BD FACSAraiIII instrument to sort the cells to obtain the yeast with higher binding capacity with the ALK-1 antigen.

Extraction of candidate antibody gene of ALK-1 antibody

The yeast liquid which is obtained by MACS and FACS enrichment and has higher binding capacity with ALK-1 antigen is cultured overnight in SD-CAA amplification culture medium at 30 ℃ and 225rpm, and yeast plasmids are extracted according to the operation of a yeast plasmid extraction kit (purchased from Tiangen). Plasmids were transformed into Top10 competent cells (purchased from Tiangen), plated with ampicillin and kanamycin plates, and cultured overnight at 30 ℃. And (4) selecting a monoclonal to perform sequencing to obtain a gene sequence.

Example 2: construction and expression purification of antibody

Construction of antibody Gene into pCDNA3.1 expression vector

The heavy chain mutant gene sequence and the light chain mutant gene sequence were constructed into pCDNA3.1-constant heavy chain vector (comprising the heavy chain constant region sequence shown in SEQ ID NO: 19) and pCDNA3.1-constant light chain vector (comprising the light chain constant region sequence shown in SEQ ID NO: 20), respectively, using a homologous recombinase (purchased from Vazyme), and the procedures were followed according to the commercial instructions. The homologous recombination products are transformed into Top10 competent cells, coated with ampicillin resistant plates, cultured overnight at 37 ℃, and single clone sequencing is picked.

Cell transfection, antibody purification and ALK-1 antibody affinity assay

The plasmid is transferred into an Expi-293 cell by adopting an Expi293TM expression system kit, and the transfection method is as follows: HEK293 cells were passaged according to the desired transfection volume and cell density was adjusted to 2.5-3X 10 one day before transfection ⁶ Cells/ml; the cells to be transfected were counted and the cell density was adjusted to 3X 10 with pre-warmed OPM-293CD05 Medium ⁶ Cells/ml; taking MEM medium with one tenth of cell transfection volume as transfection buffer solution, and refining according to 1LAdding 1mg of plasmid into the cells, adding the plasmid (wherein the ratio of the heavy chain to the light chain is H: L = 1; according to the mass ratio of PEI: adding PEI at the ratio of plasmid =3:1, mixing well, incubating at room temperature for 10-20 min, gently adding the mixture to HEK293F cells at 36.5 ℃,8% CO ₂ The cells were cultured in a shaker at 130rpm and fed with an Orpmeyer feed at 5% of the cell transfection volume 20-22h after transfection. The supernatant was collected after 6 days of cell culture. The antibody of interest was purified using protein A filler (purchased from Hezhou, tiandi, and Japan) and the binding affinity of the antibody to ALK-1 (Acro, AL 1-H5227) was determined using Fortebio.

ForteBio affinity assays were performed according to the current method (Estep, P et al, measurement of high throughput antibody-antigen affinity and epitope fractionation based on solutions, MAbs,2013.5 (2): p.270-8). Briefly, the sensor was equilibrated 10min under the line in assay buffer, then the baseline was established by on-line detection for 60s, and the antibody obtained as described above was loaded on-line onto the AHC sensor. The sensors were then exposed to 100nM ALK-1 antigen for 150s, after which the sensors were transferred to PBS for 450s dissociation. Kinetic analysis was performed using a 1. As shown in Table 2, 16 mutant antibodies were able to bind to human ALK-1 at the protein level with higher affinity than GT90001 binds to ALK-1.

Table 2: monovalent affinity of 16 mutant antibodies

Cloning of Cross-paired heavy and light chain mutations

And carrying out pairwise pairing transient expression on the heavy chain mutation and the light chain mutation which can be combined with the ALK-1 with high affinity, and detecting the binding affinity of the antibody and the human ALK-1 by using Fortebio. The monovalent affinity range is shown in Table 3, and the 19 cross-paired mutant antibodies have higher affinity with human ALK-1 protein.

Table 3: monovalent affinity of 19 cross-paired mutant antibodies

Example 3: flow assay cell level binding assay

CHO-S cells expressing human ALK-1 (CHO-ALK-1 cells) were generated by transfecting pCHO1.0 vector (Invitrogen) encoding cDNA encoding human ALK-1 cloned into MCS. Adjusting the cells subjected to the expanded culture to a proper cell density, adding the cells into a 96-well flow plate, detecting CHO-ALK1 cells, centrifuging, adding a sample to be detected in a gradient dilution manner, and incubating for 1 hour at 4 ℃. PBS washing twice, adding fluorescent secondary antibody (abcam, ab 98596) diluted to appropriate concentration, incubating for 30min at 4 ℃, and PBS washing twice. PBS was added to resuspend the cells, detected on a CytoFlex flow cytometer and the corresponding MFI calculated. For HUVEC cell (ATCC) assays, cells were fixed with 4% formaldehyde solution, centrifuged, and then the test samples were added in a gradient dilution and incubated at 4 ℃ for 1 hour or at room temperature for 2 hours. PBS washing twice, adding corresponding diluted fluorescent secondary antibody to proper concentration, incubating for 30min at 4 ℃ or incubating for 1 hour at room temperature, and PBS washing twice. PBS was added to resuspend the cells, detected on a CytoFlex flow cytometer and the corresponding MFI calculated. As shown in FIGS. 1 and 2, in CHO-ALK-1 cells with high expression abundance of ALK-1, the cell binding capacity of the antibodies mut-VK7, mut-VK8 and mut-VK12 after affinity maturation was at least equivalent to that of the parent antibody GT 90001; in HUVEC cells, the cell binding capacity of the antibodies mut-VK7, mut-VK8 and mut-VK12 after affinity maturation is more obviously superior to that of the parent antibody GT90001.

Example 4: BMP 9-induced ALK1 downstream Smad1 phosphorylation blocking experiment

HUVEC cells were seeded in 96-well plates, 10 ⁴ The cells/well were placed in ECM (Sciencell, 100) containing 5% FBS and ECG1) In a culture medium. Incubated overnight at 37 ℃ in 5% carbon dioxide. The medium was removed from the cell culture plate and washed twice with 200. Mu.l PBS. 100 μ l of ECM without FBS and ECG was added and the cells were then starved for 4 hours. The medium was removed from the cell culture plate, 100. Mu.l of the test article diluted in a gradient was added, and treated for 1.5 hours, and then the cells were treated with BMP9 (Biolegend, 553102) added to the medium at a final concentration of 1. Mu.g/mL for 45 minutes. The medium was removed and Smad1 phosphorylation levels were determined using an ELISA kit (Invitrogen, 85-86182-11). As shown in FIG. 3, the ALK1 antibodies mut-VK7, mut-VK8 and mut-VK12 after affinity maturation have greatly improved capability of blocking BMP 9-induced Smad1 phosphorylation compared with the parent antibody GT90001, thereby blocking ALK-1/TGF-beta-1/Smad signaling pathway more effectively and inhibiting angiogenesis.

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.

SEQUENCE LISTING

<110> Pmis biotech (Zhuhai) Inc

<120> antibody against activin receptor-like kinase 1 and use thereof

<130> IDC210248

<160> 20

<170> PatentIn version 3.5

<210> 1

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> GT90001 VH

<400> 1

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Glu Tyr Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Ser Val Ala Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 2

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<220>

<223> GT90001 VL

<400> 2

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Thr Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 3

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> mutVH-1

<400> 3

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Glu Tyr Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ala Tyr Tyr Ser Gly Ala Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Thr Val Ala Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 4

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<213> Artificial sequence

<220>

<223> mutVH-2

<400> 4

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Glu Tyr Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Val Tyr Tyr Ser Gly Ser Ala Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Ser Val His Gly Phe Asp Pro Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 5

<211> 118

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<213> Artificial sequence

<220>

<223> mutVH-4

<400> 5

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Glu Tyr Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Ala Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Asn Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Ser Val Glu Gly Phe Ser Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 6

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<220>

<223> mutVH-5

<400> 6

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Glu Tyr Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ala Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Ser Val Ala Gly Phe Glu Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 7

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> mutVH-7

<400> 7

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Glu Tyr Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Gly Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Ser Val Gln Gly Phe Val Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 8

<211> 118

<212> PRT

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<220>

<223> mutVH-8

<400> 8

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Glu Tyr Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ala Tyr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Ser Val Met Gly Phe Asp Pro Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 9

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> mutVH-9

<400> 9

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Glu Tyr Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Ala Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Thr Val Gln Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 10

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> mutVH-10

<400> 10

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Glu Tyr Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Val Phe Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Ser Val Glu Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 11

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> mutVH-13

<400> 11

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Glu Tyr Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ala Lys Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Val Arg Glu Ser Val Ala Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 12

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> mutVK-1

<400> 12

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Thr Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Phe Pro

85 90 95

Ile Leu Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 13

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> mutVK-2

<400> 13

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Thr Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Phe Pro

85 90 95

Ile Pro Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 14

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> mutVK-4

<400> 14

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Thr Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Asp

85 90 95

Thr Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 15

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> mutVK-7

<400> 15

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Thr Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Val Tyr Gly Ser Ser Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 16

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> mutVK-8

<400> 16

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Thr Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Leu Tyr Gly Ser Phe Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 17

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> mutVK-11

<400> 17

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Thr Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Phe Pro

85 90 95

Ile Val Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 18

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> mutVK-12

<400> 18

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Thr Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Leu Tyr Gly Glu Ser Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 19

<211> 329

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain constant region sequence

<400> 19

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 20

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> light chain constant region sequence

<400> 20

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to activin receptor-like kinase 1 (ALK-1), said antibody or antigen-binding fragment thereof comprising:

(i) A heavy chain variable region (VH) comprising the sequence shown in any one of SEQ ID NOs:1, 3-11; and, a light chain variable region (VL) comprising the sequence set forth in any one of SEQ ID NOs: 12-18; or the like, or, alternatively,

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 1; and, a light chain variable region (VL) comprising the sequence set forth in any one of SEQ ID NOs: 12-18;

preferably, the antibody or antigen-binding fragment thereof comprises:

(a) A heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 1; and, a light chain variable region (VL) comprising the sequence set forth in SEQ ID NO: 15; or the like, or, alternatively,

(b) A heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 1; and, a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 16; or the like, or, alternatively,

(c) A heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 1; and a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 18.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

a heavy chain variable region (VH) comprising the sequence set forth in any one of SEQ ID NOs: 3-11; and a light chain variable region (VL) comprising the sequence set forth in SEQ ID NO: 2.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

a heavy chain variable region (VH) comprising the sequence set forth in any one of SEQ ID NOs: 3-9; and, a light chain variable region (VL) comprising the sequence set forth in any one of SEQ ID NOs:12, 13, 16-18;

preferably, the antibody or antigen-binding fragment thereof comprises:

(a) A heavy chain variable region (VH) comprising the sequence set forth in any one of SEQ ID NOs:3, 4, 6, 7, 9; and, a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 18; or the like, or, alternatively,

(b) A heavy chain variable region (VH) comprising the sequence shown in any one of SEQ ID NOs:3, 5, 6, 9; and, a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 17; or the like, or, alternatively,

(c) A heavy chain variable region (VH) comprising the sequence set forth in any one of SEQ ID NOs: 3-9; and, a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 16; or the like, or a combination thereof,

(d) A heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO:7 or 8; and, a light chain variable region (VL) comprising the sequence set forth in SEQ ID NO 13; or the like, or, alternatively,

(e) A heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO 6; and a light chain variable region (VL) comprising the sequence set forth in SEQ ID NO: 12.

5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of IgG, igM, igE, igD or IgA, such as IgG1, igG2, igG3 or IgG4.

6. The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of scFv, di-scFv, (scFv) ₂ 、Fab、Fab’、(Fab’) ₂ Fv, disulfide-linked Fv.

7. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of any one of claims 1-6.

8. The isolated nucleic acid molecule of claim 7, comprising a first nucleotide sequence encoding the heavy chain or heavy chain variable region of the antibody or antigen-binding fragment thereof of any one of claims 1-6 and a second nucleotide sequence encoding the light chain or light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different isolated nucleic acid molecules.

9. A vector comprising the isolated nucleic acid molecule of claim 7 or 8;

preferably, the vector comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of the antibody or antigen-binding fragment thereof of any one of claims 1-6 and a second nucleotide sequence encoding a light chain or light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors.

10. A host cell comprising the isolated nucleic acid molecule of claim 7 or 8, or the vector of claim 9.

11. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-6, comprising culturing the host cell of claim 10 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

12. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-6, or the isolated nucleic acid molecule of claim 7 or 8, or the vector of claim 9, or the host cell of claim 10, and a pharmaceutically acceptable carrier and/or excipient.

13. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-6, or the isolated nucleic acid molecule of claim 7 or 8, or the vector of claim 9, or the host cell of claim 10, or the pharmaceutical composition of claim 12, in the manufacture of a medicament for inhibiting tumor angiogenesis, inhibiting tumor growth, inhibiting tumor metastasis, and/or treating a tumor in a subject (e.g., a human).

14. The use of claim 13, wherein the tumor is selected from a solid tumor, such as hepatobiliary cancer, renal cell carcinoma, colorectal cancer, mesothelioma or urinary tract cancer.