WO2026008055A1 - Anti-kit antibody and pharmaceutical use thereof - Google Patents

Abstract

The present invention relates to an anti-KIT antibody and pharmaceutical use thereof. Specifically, the present invention relates to an anti-KIT antibody and use thereof in preparing a medicament for preventing or treating a disease such as an inflammatory disease and autoimmune disease.

Description

Anti-KIT antibodies and their pharmaceutical uses Technical Field

This disclosure pertains to the field of biotechnology, and more specifically, to anti-KIT antibodies and their pharmaceutical uses.

Background Technology

The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

KIT, also known as CD117, specifically binds to the stem cell factor SCF. The KIT structure includes an extracellular region, a transmembrane region, and an intracellular region. The extracellular region consists of five immunoglobulin-like domains; the first three domains participate in SCF binding, while the fourth and fifth domains participate in receptor dimerization. The intracellular region possesses receptor tyrosine kinase activity. Under normal conditions, KIT exists as a monomer. After binding to its ligand SCF, it dimers and autophosphorylates, thereby activating downstream signaling pathways and participating in cellular processes such as cell proliferation, differentiation, apoptosis, and migration.

Mast cells are widely distributed around microvessels in the skin and visceral mucosa. They secrete various cytokines, release allergy mediators, and play an immunomodulatory role. Mast cells are unique tissue-resident immune cells and important effector cells that cause many allergies and autoimmune diseases. KIT is one of the important receptors on the surface of mast cells, and KIT activation is closely related to mast cell growth, survival, differentiation, maturation, and homing. The binding of SCF to KIT receptors can enhance mast cell degranulation and cytokine release induced by antigens. Therefore, KIT, which is associated with mast cell activation, can serve as a drug target for the treatment of certain allergic or autoimmune diseases, such as urticaria.

Existing patents such as WO2007127317A1, WO2014018625A1, WO2021107566A1, WO2022159737A1, and WO2020112687 disclose anti-KIT antibodies.

Summary of the Invention

This disclosure provides an anti-KIT antibody comprising a heavy chain variable region and a light chain variable region, wherein:

I, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with the same amino acid sequence as the heavy chain variable region shown in SEQ ID NO: 26, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with the same amino acid sequence as the light chain variable region shown in SEQ ID NO: 27; or

II, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with the same amino acid sequence as the heavy chain variable region shown in SEQ ID NO: 14, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with the same amino acid sequence as the light chain variable region shown in SEQ ID NO: 15; or

III, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with the same amino acid sequence as the heavy chain variable region shown in SEQ ID NO: 32, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with the same amino acid sequence as the light chain variable region shown in SEQ ID NO: 33; or

IV. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with the same amino acid sequence as the heavy chain variable region shown in SEQ ID NO: 16, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with the same amino acid sequence as the light chain variable region shown in SEQ ID NO: 17.

This disclosure also provides an anti-KIT antibody comprising a heavy chain variable region and a light chain variable region, wherein:

V, wherein the heavy chain variable region HCDR1 contains the amino acid sequence shown in SEQ ID NO: 2, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 3, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 4; the light chain variable region LCDR1 contains the amino acid sequence shown in SEQ ID NO: 38, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 39, and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 7; wherein

SEQ ID NO: 38 is LCDR1 as shown in X ₁ ASSSVSYMH, where X ₁ is R or S;

SEQ ID NO: 39 is LCDR2 as shown in STSNLAX ₂ , where X ₂ is D or S;

or

VI. The heavy chain variable region HCDR1 contains the amino acid sequence shown in SEQ ID NO: 8, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 9, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 10; the light chain variable region LCDR1 contains the amino acid sequence shown in SEQ ID NO: 11, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 12, and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 13.

In some embodiments, the anti-KIT antibody, as described in any of the preceding embodiments, comprises a heavy chain variable region and a light chain variable region, wherein:

I, wherein the heavy chain variable region HCDR1 contains the amino acid sequence shown in SEQ ID NO: 2, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 3, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 4; the light chain variable region LCDR1 contains the amino acid sequence shown in SEQ ID NO: 36, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 37, and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 7; or

II, wherein the heavy chain variable region HCDR1 contains the amino acid sequence shown in SEQ ID NO: 2, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 3, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 4; the light chain variable region LCDR1 contains the amino acid sequence shown in SEQ ID NO: 5, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 6, and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 7; or

III. The heavy chain variable region HCDR1 contains the amino acid sequence shown in SEQ ID NO: 8, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 9, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 10; the light chain variable region LCDR1 contains the amino acid sequence shown in SEQ ID NO: 11, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 12, and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 13.

In some embodiments, such as the anti-KIT antibody described in any of the preceding embodiments, the anti-KIT antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

In some implementations, such as the anti-KIT antibody described in any of the preceding embodiments, the anti-KIT antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

In some implementations, such as the anti-KIT antibody described in the preceding one, wherein the anti-KIT antibody is a humanized antibody.

In some embodiments, the anti-KIT antibody, as described in any of the preceding embodiments, comprises a heavy chain variable region and a light chain variable region, wherein:

I, wherein the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 26 or an amino acid sequence having at least 75% sequence identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% sequence identity with it; or

II, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having at least 75% sequence identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 15 or an amino acid sequence having at least 75% sequence identity with it; or

III, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 32 or an amino acid sequence having at least 75% sequence identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 33 or an amino acid sequence having at least 75% sequence identity with it; or

IV. The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% sequence identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 17 or an amino acid sequence having at least 75% sequence identity with it.

The above-mentioned sequences have at least 75% sequence identity with the variable region of the antibody heavy chain or the variable region of the light chain, including but not limited to having at least 75%, 77%, 80%, 82%, 84%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity in the frame region of the antibody.

In some embodiments, the anti-KIT antibody, as described in any of the preceding embodiments, comprises a heavy chain variable region and a light chain variable region, wherein:

I, wherein the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 26 or an amino acid sequence having at least 90% sequence identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 27 or an amino acid sequence having at least 90% sequence identity with it; or

II, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 15 or an amino acid sequence having at least 90% sequence identity with it; or

III, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 32 or an amino acid sequence having at least 90% sequence identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 33 or an amino acid sequence having at least 90% sequence identity with it; or

IV. The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 16 or an amino acid sequence having at least 90% sequence identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 17 or an amino acid sequence having at least 90% sequence identity with it.

The above-mentioned regions have at least 90% sequence identity with the variable regions of the antibody heavy chain or light chain, including but not limited to having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity in the frame region of the antibody.

In some implementations, such as the anti-KIT antibody described in the preceding one, the anti-KIT antibody is an antibody fragment.

In some embodiments, such as the anti-KIT antibody described in any of the preceding embodiments, the antibody fragment of the anti-KIT antibody is selected from Fab, Fab′, F(ab′)2, Fd, Fv, scFv, dsFv or dAb.

In some implementations, such as the anti-KIT antibody described in the preceding one, the antibody comprises a heavy chain constant region and a light chain constant region.

In some embodiments, such as the anti-KIT antibody described in any of the preceding embodiments, the antibody comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region being derived from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, and the light chain constant region being the light chain constant region of human κ or λ.

In some embodiments, such as the anti-KIT antibody described in any of the preceding embodiments, wherein the antibody comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region being derived from the human IgG1 heavy chain constant region and the light chain constant region being the human κ or λ light chain constant region.

In some embodiments, such as the anti-KIT antibody described in any of the preceding embodiments, wherein the antibody comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region being derived from the human IgG1 heavy chain constant region and the light chain constant region being the human κ light chain constant region; the human IgG1 heavy chain constant region variant contains mutations at positions 234, 235, 237, 428 and 434 (in accordance with EU numbering rules, the same below).

In some embodiments, such as the anti-KIT antibody described in any of the preceding embodiments, wherein the antibody comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region being derived from the human IgG1 heavy chain constant region, and the light chain constant region being the human κ light chain constant region; the human IgG1 heavy chain constant region contains mutations of L234A, L235A, G237A, M428L, and N434S (in accordance with EU numbering rules, the same below).

In some embodiments, such as the anti-KIT antibody described in any of the preceding claims, wherein the antibody comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region comprising an amino acid sequence as shown in SEQ ID NO: 18, and the light chain constant region comprising an amino acid sequence as shown in SEQ ID NO: 19.

In some embodiments, such as the anti-KIT antibody described in any of the preceding embodiments, wherein the anti-KIT antibody comprises a heavy chain and a light chain, wherein:

I, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 28 or an amino acid sequence having at least 85% sequence identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 29 or an amino acid sequence having at least 85% sequence identity with it; or

II, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 20 or an amino acid sequence having at least 85% sequence identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 21 or an amino acid sequence having at least 85% sequence identity with it; or

III, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 34 or an amino acid sequence having at least 85% sequence identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 35 or an amino acid sequence having at least 85% sequence identity with it; or

IV. The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 22 or an amino acid sequence having at least 85% sequence identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 23 or an amino acid sequence having at least 85% sequence identity with it.

The above have at least 85% sequence identity with the antibody heavy or light chain, including but not limited to having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

On the other hand, this disclosure also provides a multispecific antibody comprising the anti-KIT antibody as described in the previous one.

On the other hand, this disclosure provides an anti-KIT antibody that competes with the anti-KIT antibody as described in any of the preceding claims or the multispecific antibody as described above for binding to human KIT.

On the other hand, this disclosure provides an anti-KIT antibody that binds to the same KIT antigenic epitope as described in any of the preceding anti-KIT antibodies or as described above for multispecific antibodies.

On the other hand, this disclosure provides a pharmaceutical composition comprising an anti-KIT antibody as described in any of the preceding claims or a multispecific antibody as described above, and one or more pharmaceutically acceptable carriers, diluents or excipients.

On the other hand, this disclosure provides an isolated nucleic acid that encodes an anti-KIT antibody as described in any of the preceding claims or a multispecific antibody as described above.

On the other hand, this disclosure provides a carrier containing the isolated nucleic acid as described above.

On the other hand, this disclosure provides a host cell containing the isolated nucleic acid as described above.

On the other hand, this disclosure provides a method for producing an anti-KIT antibody as described in any of the preceding claims or a multispecific antibody as described above, the method comprising culturing host cells as described above in a culture medium to form and accumulate an anti-KIT antibody as described in any of the preceding claims or a multispecific antibody as described above, and recovering the antibody from the culture.

On the other hand, this disclosure provides the use of the anti-KIT antibody as described in any of the preceding claims, or the multispecific antibody as described above, or the pharmaceutical composition as described above, in the preparation of a medicament for the prevention or treatment of diseases or conditions related to KIT.

On the other hand, this disclosure provides a method for preventing or treating diseases or conditions related to KIT, the method comprising administering to a subject an anti-KIT antibody as described in any of the preceding claims, or a multispecific antibody as described above, or a pharmaceutical composition as described above.

On the other hand, this disclosure provides an anti-KIT antibody as described in any of the preceding claims, or a multispecific antibody as described above, or a pharmaceutical composition as described above, for use as a medicament.

In some implementations, the diseases or conditions associated with KIT, as described in any of the preceding embodiments, are selected from inflammatory diseases, eye diseases, cancer, blood diseases, allergies, and autoimmune diseases.

In some implementations, the disease or condition associated with KIT, as described in any of the preceding embodiments, is selected from nodular prurigo, esophagitis, asthma, atopic dermatitis, urticaria, myeloma, connective tissue tumors, leukemia, lung cancer, gastrointestinal stromal tumors, hemoglobinopathies, retinal disorders, diabetic macular edema, and wet age-related macular degeneration.

In some implementations, the disease or condition associated with KIT, as described in any of the preceding embodiments, is urticaria.

In some implementations, the disease or condition associated with KIT as described in any of the preceding embodiments is chronic urticaria; preferably, the chronic urticaria is selected from chronic spontaneous urticaria, chronic induced urticaria, and cold contact urticaria.

On the other hand, this disclosure provides a method for preventing or treating a disease or condition, the method comprising administering to a subject an anti-KIT antibody as described in any of the preceding claims, or a multispecific antibody as described above, or a pharmaceutical composition as described above.

On the other hand, this disclosure provides the use of the anti-KIT antibody as described in any of the preceding claims, or the multispecific antibody as described above, or the pharmaceutical composition as described above, in the preparation of a medicament for the prevention or treatment of a disease or condition.

On the other hand, this disclosure provides the use of the anti-KIT antibody as described in any of the preceding claims, or the multispecific antibody as described above, or the pharmaceutical composition as described above, in the preparation of a medicament for the prevention or treatment of a disease or condition selected from inflammatory diseases, eye diseases, cancer, blood diseases, allergies, and autoimmune diseases.

On the other hand, this disclosure provides anti-KIT antibodies as described in any of the preceding claims, or multispecific antibodies as described above, or pharmaceutical compositions as described above for the preparation of treatments or preventions of nodular prurigo, esophagitis, asthma, atopic dermatitis, urticaria, myeloma, connective tissue tumors, leukemia, lung cancer, gastrointestinal stromal tumors, hemoglobinopathies, retinal diseases, diabetic macular edema, and wet age-related macular degeneration.

On the other hand, this disclosure provides the use of the anti-KIT antibody as described in any of the preceding claims, or the multispecific antibody as described above, or the pharmaceutical composition as described above, in the preparation of a medicament for the prevention or treatment of urticaria or other diseases or conditions.

In some embodiments, the urticaria is chronic urticaria; preferably, the chronic urticaria is selected from chronic spontaneous urticaria, chronic induced urticaria, and cold contact urticaria.

In some implementations, the inflammatory condition described in any of the preceding embodiments is selected from nodular prurigo, esophagitis, asthma, atopic dermatitis, and chronic urticaria; the cancer is selected from myeloma, connective tissue tumors, leukemia, lung cancer, and gastrointestinal stromal tumors; and the hematologic disease is hemoglobinopathies.

In some embodiments, the hemoglobinopathies are selected from sickle cell anemia and β-thalassemia; the chronic urticaria is selected from chronic spontaneous urticaria, chronic induced urticaria, and cold contact urticaria; the lung cancer is small cell lung cancer; the leukemia is chronic myeloid leukemia or acute myeloid leukemia; and the esophagitis is eosinophilic esophagitis.

In some implementations, the disease or condition described in any of the preceding embodiments is selected from retinal disorders, diabetic macular edema, and wet age-related macular degeneration.

Attached Figure Description

Figure 1A: Comparison of the inhibitory activities of humanized antibody 531-Hu and control antibody CDX-0159 on mast cell GM-CSF secretion.

Figure 1B: Comparison of the inhibitory activities of humanized antibody 4A11-Hu and control antibody CDX-0159 on mast cell secretion of GM-CSF.

Figure 2: Pharmacokinetic comparison of humanized antibody 531-Hu and control antibody CDX-0159 in clearing mast cells from canine skin.

Detailed Implementation

the term

To facilitate understanding of this disclosure, certain technical and scientific terms are described below. Unless otherwise expressly defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

The singular forms “a,” “an,” and “the” used in the specification and claims include plural references unless the context clearly indicates otherwise.

Unless the context clearly requires otherwise, the words “comprising,” “having,” “including,” etc., in the patent specification and claims should be understood as “including but not limited to,” rather than as exclusive or exhaustive.

The term "and/or" implies both "and" and "or". For example, the phrase "A, B and/or C" is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

The term "amino acid mutation" includes amino acid substitution (also known as amino acid replacement), deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be performed to achieve the final construct, provided that the final construct possesses the desired properties, such as reduced or absent binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino and/or carboxyl ends of the polypeptide chain. A specific amino acid mutation can be an amino acid substitution. In some embodiments, an amino acid mutation is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid that has a different structure and/or chemical properties. Amino acid substitution includes substitution by non-naturally occurring amino acids or by derivatives of 20 naturally occurring amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. Methods other than genetic engineering that alter amino acid side chain groups, such as chemical modification, are also expected to be available. Various names may be used herein to refer to the same amino acid mutation. In this document, the amino acid residue at a specific site can be represented by the format "position + amino acid residue". For example, 428L indicates that the amino acid residue at position 428 is L. M428L indicates that the amino acid residue at position 428 has mutated from M to L. It should be understood that when the amino acid sequence is defined by the format "position + residue" in the claims, the amino acid before the mutation at that site does not constitute a limitation on the technical solution. In this document, "the Fc region contains the amino acid mutations of 428L and 434S" means that the amino acid mutation in the Fc region includes a mutation at position 428 to lysine (L) and a mutation at position 434 to serine (S).

The terms “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. The term applies to amino acid polymers, where one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to both naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise stated, a particular peptide sequence also implicitly encompasses variants with conserved modifications.

The term “antibody” is used in the broadest sense and covers a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies); full-length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.

The term "antigen-binding fragment" encompasses full-length antibodies, Fab, modified Fab, Fab', Fab'-SH, modified Fab', F(ab')2, Fv, dsFv, Fab-Fv, Fab-dsFv, Fd, single-domain antibodies (sdAb, e.g., VH, VL, or VHH), single-chain Fab (scFab), single-chain antibodies (e.g., scFv, sc(Fv)2), biantibodies, linear antibodies, bivalent, trivalent, or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above. Methods for generating and preparing these antigen-binding fragments are well known in the art.

"Natural antibodies" refer to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetraglycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N to the C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy domain or heavy chain variable region, followed by a heavy chain constant region. The natural IgG heavy chain constant region typically contains three constant domains (CH1, CH2, and CH3). Similarly, from the N to the C-terminus, each light chain has a variable region (VL), also known as a variable light domain or light chain variable domain, followed by a constant light domain (light chain constant region, CL).

The terms "full-length antibody," "intact antibody," and "complete antibody" are used interchangeably in this document, referring to antibodies with a structure substantially similar to that of natural antibodies or with a heavy chain containing the Fc region as defined herein. The light chain of a natural intact antibody includes a variable region (VL) and a constant region (CL), with VL located at the amino terminus of the light chain. The constant region includes the κ and λ chains. The heavy chain includes a variable region (VH) and constant regions (CH1, CH2, and CH3), with VH located at the amino terminus of the heavy chain and the constant region located at the carboxyl terminus. CH3 is closest to the carboxyl terminus of the polypeptide. The heavy chain can belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including both native and modified Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. In some embodiments, the Fc region of a human IgG heavy chain is defined as an amino acid residue extending from the Cys226 position or from Pro230 to its carboxyl terminus. Suitable Fc regions for the antibodies described herein include the Fc regions of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some embodiments, the boundaries of the Fc region may also vary, for example, by omitting the C-terminal lysine (residue 447 according to the EU numbering system) or omitting both the C-terminal glycine and lysine (residues 446 and 447 according to the EU numbering system). Unless otherwise stated, the Fc region is numbered according to the EU numbering system, also known as the EU index.

The Fc region can be appropriately obtained by partially digesting IgG monoclonal antibodies with proteolytic enzymes such as pepsin, followed by eluting the components adsorbed on the protein A or protein G column. As the proteolytic enzyme, any enzyme capable of restrictively digesting full-length antibodies to produce Fab and F(ab')2 by appropriately setting the enzyme reaction conditions such as pH is acceptable; there is no particular limitation, and examples include pepsin and papain.

The term "variable region" or "variable domain" in an antibody refers to the domain in the antibody heavy or light chain involved in antibody binding to the antigen. In this paper, the antibody heavy chain variable region (VH) and light chain variable region (VL) each contain four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region within the variable domain that primarily facilitates antigen binding; "frame" or "FR" refers to the variable domain residues other than the CDR residues. The VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; the VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus (also called the N-terminus) to the carboxyl terminus (also called the C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The amino acid sequence boundaries of CDRs can be determined using various well-known schemes, such as the "Kabat" numbering rule, the "Chothia" numbering rule, the "ABM" numbering rule, and the "contact" numbering rule; the correspondence between various numbering systems is well known to those skilled in the art.

Unless otherwise stated, the variable areas and CDRs in this disclosure embodiment are subject to the "Kabat" numbering rule.

The term "antibody fragment" refers to a molecule that is distinct from the intact antibody but contains a portion of the intact antibody that binds to the antigen to which the intact antibody is bound. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab′, Fab′-SH, F(ab′)2, single-domain antibodies, single-chain Fab (scFab), biantibodies, linear antibodies, single-chain antibodies (e.g., scFv); and multispecific antibodies formed from antibody fragments.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a specific source or species, while the remaining portion of the heavy and/or light chain is derived from another different source or species.

The term "humanized" antibody refers to an antibody that retains the reactivity of a non-human antibody while exhibiting lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR region and replacing the rest of the antibody with its human counterpart (i.e., the frame region portion of the constant region and the variable region).

The terms "human antibody," "humanized antibody," "fully human antibody," and "completely human antibody" are used interchangeably, referring to antibodies whose variable and constant regions are human sequences. This term encompasses antibodies derived from human genes but with sequence alterations, such as reduced potential immunogenicity, increased affinity, or the elimination of cysteine or glycosylation sites that might cause undesirable folding. This term also covers antibodies recombined in non-human cells (which may confer glycosylations not characteristic of human cells). The term also includes antibodies produced in transgenic mice containing some or all human immunoglobulin heavy and light chain loci. The term "human antibody" explicitly excludes humanized antibodies.

The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, binding "affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its ligand Y can typically be represented by the dissociation constant (KD). Affinity can be measured using conventional methods known in the art, including those described herein.

As used herein, the term "kassoc" or "ka" refers to the association rate of a specific antibody-antigen interaction, and the term "kdis" or "kd" refers to the dissociation rate of a specific antibody-antigen interaction. The term "KD" refers to the dissociation constant, which is derived from the ratio of kd to ka (i.e., kd/ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods known in the art. For example, it can be measured using a biosensing system such as a system for measuring surface plasmon resonance (e.g., Biacore), or by measuring affinity in solution using solution equilibrium titration (SET).

The term “surface plasmon resonance” refers to the analysis of optical phenomena involving real-time interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore system (Biacore LifeSciences division of GE Healthcare, Piscataway, NJ).

The term "effector function" refers to biological activities attributable to the antibody's Fc region (either the native Fc region or the Fc region with amino acid sequence mutations) and that vary across antibody isotypes. Examples of antibody effector functions include, but are not limited to: C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

The term "monoclonal antibody" refers to a group of substantially homogeneous antibodies, meaning that the antibody molecules contained in this group have the same amino acid sequence, except for the possible small number of naturally occurring mutations. In contrast, polyclonal antibodies typically comprise a variety of different antibodies with different amino acid sequences in their variable domains, and they generally specifically target different epitopes. "Monoclonal" should not be construed as requiring the antibody to be produced by any particular method. In some embodiments, the antibodies provided in this disclosure are monoclonal antibodies.

The term "bispecific antibody" refers to an antibody (including the antibody or its antigen-binding fragment, such as a single-chain antibody) capable of specifically binding to two different antigens or at least two different antigenic epitopes of the same antigen. Various structures of bispecific antibodies have been disclosed in the prior art. Based on the integrity of the IgG molecule, they can be classified into IgG-like bispecific antibodies and antibody fragment-based bispecific antibodies. Based on the number of antigen-binding regions, they can be classified into bivalent, trivalent, tetravalent, or more bispecific antibodies. Based on structural symmetry, they can be classified into symmetrical and asymmetrical bispecific antibodies. Among these, bispecific antibodies based on antibody fragments, such as Fab fragments lacking the Fc fragment, form bispecific antibodies by combining two or more Fab fragments into one molecule. They exhibit lower immunogenicity, smaller molecular weight, and higher tumor tissue penetration. IgG-like bispecific antibodies (e.g., those with an Fc fragment) have a relatively larger molecular weight. The Fc fragment facilitates antibody purification and improves its solubility and stability. The Fc portion may also bind to the receptor FcRn, increasing the antibody's serum half-life.

The term "antigen" refers to a molecule or molecular part that can be selectively bound by antigen-binding proteins, including, for example, antibodies. An antigen may have one or more epitopes that can interact with different antigen-binding proteins, such as antibodies.

The term "epitope" refers to a region on an antigen that can specifically bind to an antibody or its antigen-binding fragment. Epitopes can be formed from a continuous string of amino acids (linear epitopes) or contain non-continuous amino acids (conformal epitopes), such as those spatially close due to antigen folding. The difference between conformational and linear epitopes is that antibody binding to a conformational epitope is lost in the presence of a denaturing solvent. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, Western blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen, and cross-blocking.

The terms "capable of specific binding,""specificbinding," or "binding" refer to the ability of an antibody to bind to an antigen or epitope with a higher affinity than to other antigens or epitopes. Typically, antibodies bind to antigens or epitopes with an equilibrium dissociation constant (KD) of about 1 × ^10⁻⁶ M or less (e.g., about 1 × ^10⁻⁷ M, 1 × ^10⁻⁸ M, 1 × ^10⁻⁹ M or less). In some embodiments, the KD of antibody binding to an antigen is 10% or less (e.g., 1%) of the KD of antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, such as by... The surface plasmon resonance assay is used to measure this. However, antibodies that specifically bind to an antigen or its epitope do not preclude cross-reactivity with other related antigens, such as cross-reactivity with corresponding antigens from other species (homologous) (e.g., humans or monkeys, such as the cynomolgus (cyno), the chimpanzee (chimp), or the common marmoset (marmoset)).

The terms “antibody-dependent cell cytotoxicity,” “antibody-dependent cell-mediated cytotoxicity,” or “ADCC” refer to mechanisms that induce cell death that rely on the interaction between antibody-coated target cells and lytic effector cells (such as natural killer (NK) cells, monocytes, macrophages, and neutrophils) via Fcγ receptors (FcγR) expressed on the effector cells. For example, NK cells express FcγRIIIa, while monocytes express FcγRI, FcγRII, and FcγRIIIa. The ADCC activity of the antibodies described herein can be assessed in vitro using cells expressing the antigen as target cells and NK cells as effector cells. Cell lysis is detected based on the release of markers (e.g., radioactive substrates, fluorescent dyes, or native intracellular proteins) from lysed cells.

The term "antibody-dependent phagocytosis (ADCP)" refers to the mechanism by which antibody-coated target cells are eliminated through internalization by phagocytes (such as macrophages or dendritic cells).

The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism that induces cell death in which the Fc effector domain of a target-binding antibody binds to and activates the complement component C1q. C1q then activates the complement cascade, leading to target cell death. Activation of complement can also result in the deposition of complement components on the surface of target cells, which promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.

The term "nucleic acid" is used interchangeably with the term "polynucleotide" herein and refers to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-natural, have similar binding properties to a reference nucleic acid, and are metabolized in a manner similar to that of a reference nucleotide. Examples of such analogs include, but are not limited to, phosphate thioesters, aminophosphate esters, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs).

"Separated" nucleic acids refer to nucleic acid molecules that have been separated from their components in their natural environment. Separated nucleic acids encoding polypeptides refer to one or more nucleic acid molecules encoding polypeptides, including one or more such nucleic acid molecules in a single vector or separate vectors, and one or more such nucleic acid molecules present at one or more locations in the host cell. Unless otherwise stated, a specific nucleic acid sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly stated sequences. Specifically, as detailed below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and/or deoxyinosine residues.

The term "sequence identity" refers to the degree (percentage) to which two sequences share the same amino acids/nucleic acids at equivalent positions when optimally aligned; gaps may be introduced, where necessary, to obtain the maximum percentage of sequence identity, without considering any conserved substitutions as part of sequence identity. To determine the percentage of sequence identity, alignment can be performed using techniques known in the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide linked to it. One type of vector is a "plasmid," which is a circular double-stranded DNA loop in which an additional DNA segment can be attached. Another type of vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), in which an additional DNA segment can be attached to the viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome. The term "expression vector" or "expression construct" refers to a vector capable of transforming host cells and containing a nucleic acid sequence that directs and/or controls (alongside the host cell) the expression of one or more heterologous coding regions operatively linked to it. Expression constructs can include, but are not limited to, sequences that affect or control transcription, translation, and, in the presence of introns, influence RNA splicing of coding regions operatively linked to them.

The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of the number of passages. Progeny may not be identical to parental cells in their nucleic acid contents and may contain mutations. Mutant progeny are included herein, which have the same function or biological activity as cells screened or selected in the initial transformed cells. Host cells include prokaryotic and eukaryotic host cells, wherein eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells. Fungal cells include yeast and filamentous fungal cells, such as *Pichia pastoris*, *Pichia finlandica*, *Pichia trehalophila*, *Pichia koclamae*, *Pichia membranaefaciens*, *Pichia minuta* (*Ogataea minuta*, *Pichia lindneri*), *Pichia xiaopuntiae*, *Pichia thermomotolerans*, *Pichia salictaria*, *Pichia guercuum*, *Pichia pijperi*, *Pichia stiptis*, *Pichia methanolica*, *Pichia* genus, and *Saccharomyces*. *C. cerevisiae*, *Saccharomyces*, *Hansenula polymorpha*, *Kluyveromyces*, *Kluyveromyces lactis*, *Candida albicans*, *Aspergillus*, *Aspergillus nidulans*, *Aspergillus niger*, *Aspergillus oryzae*, *Trichoderma reesei*, *Chrysosporium lucknowense*, *Fusarium* sp., *Fusarium gramineum*, *Fusarium venenatum*, *Physcomitrella patens*, *Neurospora crassa*, and *Yarrowia lipolytica*.

"Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

The term "pharmaceutical composition" refers to a mixture containing one or more antibodies described herein along with other chemical components, such as physiological/pharmaceutical carriers and excipients.

The term "pharmaceutically acceptable carrier, diluent, or excipient" refers to a component in a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers, diluents, or excipients include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

The terms “subject” or “individual” include both humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise specified, the terms “patient” or “subject” are used interchangeably herein. In some embodiments, the individual or subject is a human being.

"Administration" or "giving," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and the animal, human, subject, cell, tissue, organ, or biological fluid.

The term "sample" refers to a collection (such as fluid, cells, or tissue) separated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary samples include biological fluids such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cystic fluid, tears, excretions, sputum, mucosal secretions of secretory tissues or organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids in the abdominal cavity and other body cavities, fluids collected by bronchoalveolar lavage fluid, synovial fluid, liquid solutions in contact with the subject or biological sources, such as culture media (including conditioned media), lavage fluids, tissue biopsy samples, fine-needle aspiration, surgically removed tissue, organ cultures, or cell cultures.

"Treatment" and "treatment" (and their grammatical variations) refer to a clinical intervention on the individual being treated, and may be administered for prevention or during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing/decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and resolving or improving prognosis. In some implementations, the antibodies disclosed herein are used to delay the onset of disease or slow its progression.

An "effective amount" is generally an amount sufficient to reduce the severity and/or frequency of symptoms, eliminate these symptoms and/or underlying causes, prevent the occurrence of symptoms and/or underlying causes, and/or improve or mitigate damage caused by or associated with a disease state (e.g., lung disease). In some embodiments, an effective amount is a therapeutically effective amount or a preventatively effective amount.

A “therapeutic effective dose” is a dose sufficient to treat a disease state or symptom, especially a state or symptom associated with that disease state, or otherwise prevent, inhibit, delay, or reverse the progression of the disease state or any other undesirable symptom associated with that disease. A “preventive effective dose” is a dose that, when administered to a subject, will have a predetermined preventive effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms. Complete treatment or prevention may not occur after a single dose, but may occur after a series of doses. Therefore, a therapeutic or preventive effective dose may be administered in a single or multiple-dose manner. Both “therapeutic effective doses” and “preventive effective doses” can vary depending on a variety of factors, such as an individual’s disease state, age, sex, and weight, and the ability of the therapeutic agent or combination of therapeutic agents to elicit the desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status in the patient.

antibody structure

In some implementations, the antibodies provided herein are full-length antibodies.

In some implementations, the antibodies provided herein are antibody fragments.

In some embodiments, the antibody fragment is a Fab, Fab′, Fab′-SH, or F(ab′)2 fragment, particularly a Fab fragment. “Fab” is a monovalent fragment consisting of VL, VH, CL, and CH1 domains. A “Fab fragment” can be generated by cleavage of an antibody with papain. “Fab′” contains VL, CL, VH, and CH1, and also contains a region between the CH1 and CH2 domains, allowing interchain disulfide bonds to form between the two heavy chains of two Fab′ fragments to form an F(ab′)2 molecule. “Fab′-SH” is a Fab′ fragment in which the cysteine residues in the constant region have free thiol groups. “F(ab′)2” is a divalent fragment comprising two Fab fragments linked by disulfide bonds in the hinge region.

In some implementations, the antibody fragment is a biantibody, triantibody, or tetraantibody. A biantibody is an antibody fragment with two antigen-binding sites, containing linked VH and VL domains within the same polypeptide chain (VH-VL). By using a short linker that prevents pairing between two domains on the same chain, these domains are forced to pair with complementary domains on another chain, thereby creating two antigen-binding sites. The two antigens can be the same or different.

In some embodiments, the antibody fragment is a single-chain Fab fragment. A “single-chain Fab fragment” or “scFab” is a polypeptide consisting of VH, CH1, VL, CL, and a linker, wherein the antibody domain and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. In some embodiments, the linker is a polypeptide having at least 30 amino acids. In some embodiments, the linker is a polypeptide having between 32 and 50 amino acids. The single-chain Fab fragment is stabilized via a native disulfide bond between CL and CH1. Additionally, these single-chain Fab molecules can be further stabilized by inserting cysteine residues (e.g., at position 44 in the heavy chain variable region and position 100 in the light chain variable region, according to Kabat numbering) to create interchain disulfide bonds.

In some implementations, the antibody fragment is an Fv fragment composed of the VH and VL domains of a single arm of the antibody.

In some embodiments, the antibody fragment is a single-chain variable fragment (scFv). An “scFv” is a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are sequentially linked by a short, flexible peptide linker, capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, the scFv may have VL and VH variable regions in any order herein; for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv may contain VL-linker-VH or may contain VH-linker-VL.

In some embodiments, the antibody fragment is dsFv, which is obtained by linking polypeptides in which one amino acid residue in each VH and VL is replaced by a cysteine residue via disulfide bonds between cysteine residues. The amino acid residues to be replaced by cysteine residues can be selected based on known methods according to predictions of the antibody's three-dimensional structure.

In some implementations, the antibody fragment is a single-domain antibody (dAb). A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain.

In some embodiments, the antibodies provided herein are chimeric antibodies. In some embodiments, chimeric antibodies comprise a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In some embodiments, chimeric antibodies are "class-switched" antibodies, wherein the class or subclass has been changed from the class or subclass of the parent antibody.

In some embodiments, the antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody contains one or more variable regions, wherein the CDR or a portion thereof is derived from the non-human antibody, and the FR or a portion thereof is derived from the human antibody. Optionally, the humanized antibody may also contain a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody may be replaced with corresponding residues from the non-human antibody (e.g., an antibody providing the CDR sequence).

Humanized antibodies and their generation methods are reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5,821,337,7,527,791,6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describes specificity-determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describes “resurfuacing”); Dall’Acqua et al., Methods 36:43-60 (2005) (describes “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (describes the “guided selection” method for FR shuffling).

Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a "best-fit" method (see, for example, Sims et al., J. Immunol. 151:2296 (1993)); frame regions of the common sequence of human antibodies derived from specific subgroups of light chain variable regions or heavy chain variable regions (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623). (1993)); human mature (somatic mutation) framework regions or human germline framework regions (see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions obtained by screening FR libraries (see, for example, Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

Variants of anti-KIT antibodies

In some embodiments, amino acid sequence variants of the anti-KIT antibodies provided herein are included. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletion, and/or insertion, and/or substitution of residues within the amino acid sequence of the anti-KIT antibody. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as antigen-binding properties.

Replace, insert, and delete variants

In some embodiments, antibody variants with one or more amino acid substitutions are provided. Substitution mutagenesis sites of interest include CDR and FR. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for desired activities, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

Based on common side-chain characteristics, amino acids can be grouped as follows:

(1) Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile;

(2) Neutral and hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) Acidic: Asp, Glu;

(4) Alkaline: His, Lys, Arg;

(5) Residues that affect chain orientation: Gly, Pro;

(6) Aromatic: Trp, Tyr, Phe.

Non-conservative replacement would require replacing a member of one of these categories with a member of another category.

One class of substitution variants involves replacing one or more CDR residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further research will have alterations (e.g., improvements) to certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody, and/or will substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques (such as those described herein). In short, one or more CDR residues are mutated, and the variant antibody is displayed on a phage and screened for specific biological activities (e.g., binding affinity). CDRs can be altered (e.g., substituted), for example, to improve antibody affinity. Such alterations can be made to CDR “hotspots,” residues encoded by codons that undergo mutations at a high frequency during somatic maturation, and/or residues that contact the antigen, while testing the binding affinity of the resulting variant VH or VL. In some implementations of affinity maturation, diversity is introduced into the selected variant gene for maturation using any of a variety of methods, such as error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis. A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves CDR-directed approaches, where several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scan mutagenesis or modeling. In particular, HCDR3 and LCDR3 are frequently targeted.

In some embodiments, substitution, insertion, or deletion may occur within one or more CDRs, provided that such changes do not materially reduce the antibody's ability to bind to the antigen. For example, conserved changes (e.g., conserved substitutions, as provided herein) may be made to the CDRs that do not materially reduce binding affinity. Such changes may, for example, be external to the antigen-contacting residues in the CDR. In some embodiments of the variant VH and VL sequences provided above, each CDR is unchanged or contains no more than one, two, or three amino acid substitutions.

One method for identifying residues or regions in an antibody that can serve as mutagenic targets is called "alanine scan mutagenesis." In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., Ala or polyalanine) to determine if the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Furthermore, the contact points between the antibody and antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine if they contain the desired properties.

Amino acid sequence insertions include fusion of the amino and/or carboxyl ends of peptides ranging in length from 1 residue to 100 or more residues, and intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N- or C-terminus of the antibody with an enzyme or a peptide that extends the serum half-life of the antibody.

Recombination method

Anti-KIT antibodies can be generated using recombinant methods. For these methods, one or more isolated nucleic acids encoding the anti-KIT antibody are provided.

In some embodiments, this disclosure provides isolated nucleic acids encoding the anti-KIT antibodies as described above. Such nucleic acids can be derived from independently encoding any of the aforementioned polypeptide chains. In another aspect, this disclosure provides one or more vectors (e.g., expression vectors) comprising such nucleic acids. In yet another aspect, this disclosure provides host cells comprising such nucleic acids. In some embodiments, a method for preparing a polypeptide or fusion protein is provided, wherein the method includes culturing host cells comprising nucleic acids encoding the polypeptide or fusion protein, as provided above, under conditions suitable for expression, and optionally recovering the anti-KIT antibody from the host cells (or host cell culture medium).

To generate anti-KIT antibodies through recombinant synthesis, the nucleic acid encoding the protein is isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced using standard procedures, or generated via recombinant methods or obtained through chemical synthesis.

Suitable host cells for cloning or expressing vectors encoding anti-KIT antibodies include prokaryotic or eukaryotic cells as described herein. For example, they can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. After expression, the expression can be separated from the bacterial cell paste in a soluble fraction and further purified.

Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding fusion proteins, including fungal and yeast strains. Suitable host cells for expressing fusion proteins can also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of fall armyworm (Spodoptera frugiperda) cells; plant cell cultures can also be used as hosts, such as US5959177, US6040498, US6420548, US7125978, and US6417429; and vertebrate cells, such as mammalian cell lines adapted for growth in suspension, can also be used as hosts. Other examples of suitable mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (293 or 293T cells); young hamster kidney cells (BHK); mouse seltoli cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines such as Y0, NSO, and Sp2/0. For reviews of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (eds.), Humana Press, Totowa, NJ (2004), pp. 255-268.

Measurement

The anti-KIT antibodies provided herein can be identified, screened, or characterized by their physical/chemical properties and/or biological activities using a variety of assays known in the art. In some embodiments, the activity of the disclosed anti-KIT antibodies is tested, for example, by known methods such as ELISA, Western blotting, etc.

Treatment methods and routes of administration

Any anti-KIT antibody provided in this disclosure may be used for treatment methods. In some embodiments, the anti-KIT antibody provided in this disclosure is used in the manufacture or preparation of a medicament. In some embodiments, the disease is a KIT-related disease or condition.

In some embodiments, a pharmaceutical composition comprising the anti-KIT antibody is provided, for example, for any of the pharmaceutical uses or treatments described above. In some embodiments, the pharmaceutical composition comprises any anti-KIT antibody provided herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises at least one additional therapeutic agent.

The anti-KIT antibody disclosed herein can be used alone or in combination with other agents for treatment. For example, the antibody disclosed herein can be administered co-administered with at least one other therapeutic agent.

The anti-KIT antibody (and any other therapeutic agents) disclosed herein may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration may be carried out via any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules are considered herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulsatile infusion.

The anti-KIT antibody disclosed herein will be formulated, administered, and applied in accordance with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to a medical practitioner. The anti-KIT antibody may be formulated with or without one or more agents currently used for the prevention or treatment of the stated condition. The effective amount of such other agents depends on the amount present in the pharmaceutical composition, the type of condition or treatment, and other factors. These are generally used at the same dosage and route of administration as described herein, or at approximately 1% to 99% of the dosage described herein, or at other dosages, and in any route determined empirically/clinically as appropriate.

For the prevention or treatment of disease, the appropriate dosage of the disclosed anti-KIT antibody (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of therapeutic molecule, the severity and duration of the disease, whether it is administered for prophylactic or therapeutic purposes, prior treatment, the patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician. The therapeutic molecule is appropriately administered to the patient either as a single dose or after a series of treatments.

Products

In another aspect of this disclosure, an article of manufacture is provided comprising materials that can be used to treat, prevent, and/or diagnose the aforementioned conditions. The article of manufacture comprises a container and a label or package insert on or in conjunction with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from various materials such as glass or plastic. The container contains a composition, alone or in combination with another composition, that is effective in treating, preventing, and/or diagnosing the condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper puncturable by a hypodermic needle). At least one active agent in the composition is the anti-KIT antibody of this disclosure. The label or package insert indicates that the use of the composition is for the treatment of a selected condition. Furthermore, the article of manufacture may comprise: (a) a first container containing the composition, wherein the composition comprises the anti-KIT antibody of this disclosure; and (b) a second container containing the composition, wherein the composition comprises additional cytotoxic agents or other therapeutic agents. The article of manufacture in this embodiment of the present disclosure may further include a packaging insert indicating that the composition can be used to treat a specific condition. Alternatively, or additionally, the article of manufacture may further include a second (or third) container containing a pharmaceutically acceptable buffer solution. From a commercial and user perspective, it may further include other materials as desired, including other buffers, diluents, filters, needles, and syringes.

The anti-KIT antibody disclosed herein

This disclosure provides an anti-KIT antibody with many advantageous properties, such as good affinity, therapeutic activity, safety, pharmacokinetic properties, and drug-likeness.

Exemplary anti-KIT antibody

This disclosure provides an anti-KIT antibody comprising a heavy chain variable region and a light chain variable region, wherein:

I, wherein the heavy chain variable region HCDR1 contains the amino acid sequence shown in SEQ ID NO: 2, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 3, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 4; the light chain variable region LCDR1 contains the amino acid sequence shown in SEQ ID NO: 36, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 37, and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 7; or

II, wherein the heavy chain variable region HCDR1 contains the amino acid sequence shown in SEQ ID NO: 2, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 3, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 4; the light chain variable region LCDR1 contains the amino acid sequence shown in SEQ ID NO: 5, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 6, and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 7; or

III. The heavy chain variable region HCDR1 contains the amino acid sequence shown in SEQ ID NO: 8, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 9, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 10; the light chain variable region LCDR1 contains the amino acid sequence shown in SEQ ID NO: 11, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 12, and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 13.

In some embodiments, the anti-KIT antibody, as described in any of the preceding embodiments, comprises a heavy chain variable region and a light chain variable region, wherein:

I, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 26, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27; or

II, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15; or

III, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 32, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 33; or

IV. The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17.

In some embodiments, such as the anti-KIT antibody described in any of the preceding embodiments, wherein the antibody comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region being a human IgG1 heavy chain constant region variant, and the light chain constant region being a human κ or λ light chain constant region; preferably, the human IgG1 heavy chain constant region variant contains mutations of L234A, L235A, G237A, M428L, and N434S, and the light chain constant region is a human κ light chain constant region.

In some embodiments, the anti-KIT antibody as described in any of the preceding embodiments, wherein the antibody comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region comprising an amino acid sequence as shown in SEQ ID NO: 18, and the light chain constant region comprising an amino acid sequence as shown in SEQ ID NO: 19.

In some embodiments, such as the anti-KIT antibody described in any of the preceding claims, wherein the anti-KIT antibody comprises a heavy chain and a light chain, wherein:

I, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 28, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 29; or

II, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 20, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 21; or

III, the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 34, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 35; or

IV. The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 22, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 23.

On the other hand, this disclosure provides the use of the anti-KIT antibody as described in any of the preceding claims in the preparation of medicaments for the prevention or treatment of urticaria; particularly in the preparation of medicaments for the treatment and prevention of chronic urticaria.

In some implementations, the chronic urticaria is selected from chronic spontaneous urticaria, chronic induced urticaria, and cold contact urticaria.

Detailed Implementation

Example

The following examples and test cases further describe the invention, and these examples and test cases should not be construed as limiting the scope of the invention. The embodiments or test cases of the present invention do not include detailed descriptions of conventional methods. Experimental methods without specific conditions are generally performed under conventional conditions, as such methods are well known to those skilled in the art and described in many publications, such as *Molecular Cloning* (Green M R, Sambrook J. *A Laboratory Manual*, 4th, 2012) published by Cold Spring Harbor Laboratory, or *Antibody Engineering: Methods and Protocols* (*Antibody Engineering: Methods and Protocols* (*Humana Press*, 2018) published by Springer Protocols), or according to the conditions recommended by the manufacturer of the experimental materials. Some experimental materials without a specific source are obtained commercially.

Example 1. Preparation and screening of anti-KIT hybridoma monoclonal antibodies

This disclosure describes the preparation of a monoclonal antibody targeting human KIT using hybridoma technology. Recombinant human KIT extracellular fragment protein (SEQ ID NO: 1) or M07e cells (purchased from Peking Union Medical College) were used as antigens. Gold Adjuvant (Sigma, T2684) or Alum (Thermo, 77161) was used as an immune adjuvant. Complexes were prepared by combining antigens with different immune adjuvants and mice were immunized alternately. After primary and booster immunizations, mice with high antibody titers in their serum were selected, and B cells from the spleen of these mice were collected and fused with myeloma cells to prepare hybridoma cells.

The corresponding gene was constructed into an expression vector using conventional molecular cloning techniques, transiently expressed in 293 cells, and the recombinant human KIT extracellular fragment protein (SEQ ID NO: 1) was obtained by Ni-NTA affinity chromatography. Its amino acid sequence is shown below:

Human KIT extracellular recombinant protein:

Based on the growth of monoclonal hybridoma cells, cell culture supernatants were collected for analysis. Hybridoma monoclonal antibodies that effectively bound human KIT-ECD (ACROBiosystems, catalog number: CD7-H52H4) and cynomolgus monkey KIT-ECD (ACROBiosystems, catalog number: CD7-C52H9) were screened using ELISA. Hybridoma monoclonal antibodies that effectively bound M07e cells were screened using flow cytometry. Monoclonal antibodies that effectively inhibited M07e cell proliferation were screened using bioactivity analysis. Through these multiple rounds of screening, murine monoclonal anti-KIT antibodies 531 and 4A11, with strong binding affinity and good functional activity, were obtained. The corresponding monoclonal hybridoma cell lines were amplified. Logarithmically growing cells were collected, and RNA was extracted from the cells using TRIzol (Thermo Fisher Scientific). cDNA was obtained by reverse transcription of RNA using PrimeScript ^™ Reverse Transcriptase (Takara, 2680A). PCR was performed using a specific primer combination reported in the literature (the relevant primer sequences are from page 323 of the second edition of *Antibody Engineering Volume 1*, authored by Roland Kontermann and Stefan Dübel) to amplify the cDNA. The PCR products were collected and sequenced, and the resulting nucleotide sequences were translated into the amino acid sequence of the murine antibody variable region. The preferred monoclonal hybridoma cell lines have the following CDR and amino acid sequences of the variable region:

The heavy and light chain CDR region sequences of murine anti-KIT antibodies 531 and 4A11 are shown in Table 1 below:

Table 1. CDR sequences of murine anti-KIT antibodies

Note: The complementarity determining region (CDR) and framework region (FR) of each antibody heavy chain and light chain variable region are determined according to the Kabat numbering scheme.

The heavy chain variable region is denoted as VH, and the light chain variable region as VL. The VH/VL amino acid sequences of the above-mentioned murine antibody are as follows:

>Rat monoclonal antibody 531-VH amino acid sequence

>Rat monoclonal antibody 531-VL amino acid sequence

>Rat monoclonal antibody 4A11-VH amino acid sequence

>Amino acid sequence of murine monoclonal antibody 4A11-VL

Note: The primary structure of the above sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, with the underlined part being the CDR determined according to the Kabat numbering scheme, and the ununderlined part being FR.

The heavy chain variable region and light chain variable region of the murine 531 and murine 4A11 anti-KIT antibodies were recombined with the heavy chain constant region IgG1-AAA+LS and the light chain constant region human κ chain CL, respectively, to obtain full-length chimeric antibodies 531-Chi and 4A11-Chi. IgG1-AAA+LS contains the L234A+L235A+G237A mutation (abbreviated as AAA) and the M428L+N434S mutation (abbreviated as LS mutation combination).

>IgG1-AAA+LS amino acid sequence:

Human κ chain CL amino acid sequence:

>531-Chi heavy chain:

>531-Chi Light Chain:

>4A11-Chi heavy chain:

>4A11-Chi Light Chain:

Note: Single underlined sequences correspond to CDR, and italicized sequences correspond to light chain constant regions or heavy chain constant regions.

Example 2. Humanization of the anti-KIT antibody of the present invention

2.1. Humanization of the 531 antibody

Using the IgBlast tool, the murine 531-VH and VL were compared with the NCBI human antibody variable region germline gene database. Through homology analysis, IGHV4-59*01 and IGKV3-11*01 were selected as templates for the humanization of 531-VH and VL, respectively. The CDRs of the murine 531-VH and VL were transplanted into the corresponding human templates, and WGQGTLVTVSS (SEQ ID NO: 24) and FGQGTKVEIK (SEQ ID NO: 25) were selected as FR4 for VH and VL, respectively, forming CDR-grafted VH and VL with the regional structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Here, reverse mutations are performed on the amino acid residues of CDR-grafted VH and VL (amino acid residues at specific positions in the frame region play an important role in maintaining the correct conformation and affinity of VH and VL; the process of mutating such amino acid residues in CDR-grafted VH and VL to the corresponding amino acid residues in murine antibodies is called reverse mutation) or other mutations (mutating and substituting specific amino acid residues in CDR/FR to improve the physicochemical properties of the antibody or to reduce the risk of immunogenicity). Preferably, amino acid residues at positions 1, 37, 48, 67, 71, and 73 of CDR-grafted VH are mutated, with specific mutation combinations of Q1E, I37V, I48L, V67L, V71R, and T73N, to obtain humanized VH. Preferably, amino acid residues at positions 24, 36, 47, 56, and 58 of the CDR-grafted VL are mutated, specifically in the combinations S24R, Y36F, L47W, S56D, and I58V, to obtain humanized VL. During antibody humanization, the Kabat coding scheme is used to determine the positions of the amino acid residues in VH and VL. Q1E indicates that the Q at position 1 is mutated to E, and the meanings of other mutations are deduced accordingly.

The humanized VH and VL sequences of 531 are as follows:

>531-Hu-VH

>531-Hu-VL

Note: In the above sequence, the order of the regions VH and VL is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The ununderlined part is FR, the underlined part is CDR determined according to the Kabat coding scheme, and the double underlined and bolded part indicates reversion mutation or other mutation.

The aforementioned humanized VH and VL were recombined with the heavy chain constant region IgG1-AAA+LS (SEQ ID NO: 18) and the light chain constant region CL (SEQ ID NO: 19), respectively, to obtain the complete 531 humanized antibody 531-Hu.

The full-length sequence of the 531 humanized antibody (denoted as 531-Hu) is as follows:

Heavy chain amino acid sequence of >531-Hu humanized antibody

Light chain amino acid sequence of >531-Hu humanized antibody

Note: In the above sequence, the underlined part is the CDR determined according to the Kabat coding scheme, and the dotted underlined part is the antibody constant region.

2.2. Humanization of the 4A11 antibody

Using the IgBlast tool, the murine 4A11-VH and VL were compared with the NCBI human antibody variable region germline gene database. Through homology analysis, IGHV1-46*01 and IGKV6-21*01 were selected as templates for the humanization of 4A11-VH and VL, respectively. The CDRs of the murine 4A11-VH and VL were transplanted into the corresponding human templates, and WGQGTTVTVSS (SEQ ID NO: 30) and FGGGTKLEIK (SEQ ID NO: 31) were selected as FR4 for VH and VL, respectively, forming CDR-grafted VH and VL with the regional structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Here, reverse mutations or other mutations are performed on the amino acid residues of CDR-grafted VH and VL. Preferably, reverse mutations or other mutations are performed on amino acid residues at positions 1, 48, 49, 67, 69, 71, 73, and 78 of CDR-grafted VH, specifically the mutation combinations Q1E, M48I, G49A, V67A, M69L, R71A, T73K, and V78A, to obtain humanized VH. Preferably, reverse mutations or other mutations are performed on the amino acid residue at position 58 of CDR-grafted VL, specifically the mutation V58I, to obtain humanized VL.

The humanized VH and VL sequences of 4A11 are as follows:

>4A11-Hu-VH

>4A11-Hu-VL

Note: In the above sequence, the order of the regions VH and VL is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The ununderlined part is FR, the underlined part is CDR determined according to the Kabat coding scheme, and the double underlined and bolded part indicates reversion mutation or other mutation.

The aforementioned humanized VH and VL were recombined with the heavy chain constant region IgG1-AAA+LS (SEQ ID NO: 18) and the light chain constant region CL (SEQ ID NO: 19), respectively, to obtain the complete 4A11 humanized antibody 4A11-Hu.

The full-length sequence of the 4A11 humanized antibody (denoted as 4A11-Hu) is as follows:

Heavy chain amino acid sequence of the 4A11-Hu humanized antibody

Light chain amino acid sequence of the 4A11-Hu humanized antibody

Note: In the above sequence, the underlined part is the CDR determined according to the Kabat coding scheme, and the dotted underlined part is the antibody constant region.

Table 2. CDR sequences of humanized antibodies

Note: The CDR and FR of the variable regions of the heavy and light chains of each antibody are determined according to the Kabat numbering scheme.

The positive control molecule used in this disclosure is CDX-0159 (Barzolvolimab). The heavy and light chain amino acid sequences of Barzolvolimab are derived from WHO Drug Information, Vol. 36, No. 1, 2022.

Barzolvolimab heavy chain amino acid sequence:

Barzolvolimab light chain amino acid sequence:

Note: In the above sequence, the underlined part is the CDR determined according to the Kabat coding scheme, and the dotted underlined part is the antibody constant region.

DNA encoding the above-mentioned amino acid sequences was synthesized by Suzhou Genewiz Biotechnology Co., Ltd. or Shanghai Sangon Biotech Co., Ltd. The heavy and light chain genes were cloned into expression vectors pcDNA3.4 using conventional methods. The corresponding heavy and light chain expression vectors were simultaneously transfected into Expi293F ^™ cells (Thermo Fisher Scientific, catalog number: A14527) using PEI (Polyethylenimine) to express the antibody. The 293F cells were cultured in serum-free medium for 5-6 days, and the cell supernatant was collected. The antibody was purified using Protein A affinity chromatography.

The purification steps are described as follows: High-speed centrifugation was used to remove impurities from the cell culture supernatant. Antibodies in the supernatant were captured using MabSelect Sure affinity chromatography (Cytiva, catalog number: 17543801). The MabSelect Sure affinity column was first washed with 0.2M NaOH, rinsed with pure water, and then equilibrated with PBS. The supernatant was then passed through the affinity column, and the column was washed with PBS until the A280 level dropped to baseline. The target protein was eluted with 0.1M acetate buffer (pH 3.5), and the antibody solution was neutralized with 1M Tris-HCl (pH 8.0). The antibody solution was appropriately concentrated using ultrafiltration, and then further purified using a HiLoad Superdex 200 gel chromatography column (Cytiva, catalog number: 28989335). The antibody concentration was determined by UV spectrophotometry. The solution was filtered for sterilization and stored at 4°C. This method is used to purify related monoclonal antibodies, and it can also be used to purify other antibodies or recombinant proteins disclosed herein.

Test case

Test Example 1: ELISA detection of the binding affinity of humanized anti-KIT antibody to KIT.

The ELISA detection steps used in this example are described below. Human KIT-ECD (ACROBiosystems, catalog number: CD7-H52H4) was diluted to 1 μg/mL with sodium carbonate buffer (pH 9.0), and this solution was transferred to 96-well microplates (100 μL per well) and incubated overnight at 4°C. After washing, phosphate buffer containing 1% bovine serum albumin and 0.05% Tween-20 was transferred to the microplates (200 μL per well) and incubated at room temperature for 1 hour to block the microplates. The antibody to be tested was serially diluted and transferred to the above microplates (100 μL per well), and incubated at room temperature for 1 hour. After washing, 100 μL of moderately diluted HRP-goat anti-human Fc secondary antibody (Jackson, catalog number: 109-035-088) was added to each well, and incubated at room temperature for half an hour. After washing the plate, add 100 μL of colorimetric reagent (KPL, catalog number: 5120-0077) with TMB (3,3′,5,5′-Tetramethylbenzidine) as the substrate to each well and incubate at room temperature for 1–5 minutes. When the color development reaches an appropriate level, stop the reaction by adding 50 μL _of 2M _H₂SO₄ solution to each well. OD450 was measured using a microplate reader (Molecular Devices). Data analysis was performed using a GraphPad Prism 10. The results are shown in Table 3.

Table 3. Binding ability of anti-KIT antibodies to recombinant human KIT protein

Experimental results showed that the humanized antibodies 531-Hu and 4A11-Hu, along with their corresponding chimeric antibodies, had good binding ability to human KIT recombinant protein.

Test Example 2: Biacore detects the affinity of anti-KIT antibody for KIT.

In this embodiment, the recombinant KIT extracellular fragment proteins from humans, cynomolgus monkeys, and dogs are designated as human KIT-ECD (ACROBiosystems, catalog number: CD7-H52H4), cynomolgus monkey KIT-ECD (ACROBiosystems, catalog number: CD7-C52H9), and canine KIT-ECD, respectively. [The sequences are derived from UniProt (Entry: O97799), with amino acid sequences 28-527 truncated, a signal peptide added to the N-terminus, a 6*His tag added to the C-terminus, and expression vectors constructed using conventional molecular cloning methods. After transient expression of the recombinant proteins in HEK293 cells, the proteins were purified by Ni-NTA affinity chromatography.] The affinity of the humanized antibody disclosed herein for the recombinant KIT proteins from multiple species was tested using a Biacore T200. The antibody was captured using a Protein A biosensor chip (Cytiva, 29127556), and the recombinant KIT proteins were then passed through the chip surface. The reaction signals were detected in real time to obtain binding and dissociation curves. After dissociation in each experimental cycle, the sensor chip was cleaned and regenerated using 10 mM Glycine-HCl pH 1.5 (Cytiva, BR-1003-54). A 1:1 model was used for data fitting. The binding, dissociation, and equilibrium dissociation constants of each antibody are shown in Table 4.

Table 4. Binding, dissociation, and equilibrium dissociation constants of anti-KIT humanized antibodies to KIT

Experimental results show that the humanized antibodies 531-Hu and 4A11-Hu disclosed herein have high affinity for recombinant KIT proteins from multiple species.

Test Example 3: FACS detection of the binding of anti-KIT antibody to KIT on cell surface

The binding ability of the humanized antibody disclosed herein to M07e cells was tested using FACS. M07e cell cultures were collected, centrifuged at 300g for 5 minutes to collect cells, washed twice with PBS containing 0.5% BSA and resuspended. After counting, 5 μL of Fc receptor blocking solution (BioLegend, catalog number: 422303) was added per million cells, and 100,000 cells were transferred to each well of a round-bottom 96-well plate. The plates were blocked at room temperature for 10 minutes. The humanized antibody of this invention was serially diluted in PBS containing 0.5% BSA in the 96-well plates. The serially diluted antibody was transferred to the above-mentioned round-bottom 96-well plates containing M07e cells, and the cells and antibody were mixed thoroughly using a multichannel pipette. The plates were incubated at 4°C for 1 hour. Centrifuge 96-well round-bottom plates at 300g for 5 minutes, discard the supernatant, wash cells twice with PBS, add 100 μL of Alexa Fluor ^™ 488-labeled Goat-anti-human IgG (Thermo Fisher Scientific, catalog number: A11013) diluted with PBS containing 1% BSA to each well, and incubate at 4°C in the dark for 30 minutes. Centrifuge 96-well round-bottom round-bottom plates at 300g for 5 minutes, discard the supernatant, wash cells twice with PBS, and fix cells with Fix Buffer I (BD Biosciences, catalog number: 557870) (at room temperature for 5 minutes). Centrifuge 96-well round-bottom round-bottom round-bottom plates at 300g for 5 minutes, discard the supernatant, wash cells twice with PBS, and measure cell fluorescence intensity on an Attune NxT Cytometer. Analyze FACS data using Flowjo 10.9, and process and analyze the data using Graphpad Prism 10. The results are shown in Table 5.

Table 5. Binding ability of anti-KIT humanized antibodies to M07e cells

Experimental results show that the humanized antibodies 531-Hu and 4A11-Hu disclosed herein have a high binding capacity with M07e cells.

Test Example 4: Proliferation Inhibition Activity of Anti-KIT Antibody

Stem cell factor (SCF), as a ligand of KIT, can induce KIT dimerization and downstream signal transduction, stimulating M07e cell proliferation. Anti-KIT antibodies can block SCF-induced KIT downstream signal transduction, thereby inhibiting M07e cell proliferation. The method for determining the proliferation-inhibiting activity of anti-KIT antibodies is described below. M07e cells were routinely cultured and passaged according to the supplier's recommended method. M07e cells in the logarithmic growth phase were seeded into 96-well plates, with 50 μL of cell suspension (containing 10,000 cells) per well. SCF (PeproTech; catalog number: 300-07) was diluted to 200 ng/mL with medium containing 2% FBS. The antibody was serially diluted with this solution, and the SCF and antibody mixture was then transferred into the cell-seedled 96-well plates (50 μL per well). After mixing, the 96-well plates were incubated in a CO2 cell culture incubator for 72 hours. Cell viability was assessed using the Luminescent Cell Viability Assay (Promega, catalog number: G7572, abbreviated as CTG). Luminescence was measured using a microplate reader (PerkinElmer). Data analysis was performed using a GraphPad Prism10. The results are shown in Table 6.

Table 6. Inhibitory activity of anti-KIT humanized antibody against M07e cell proliferation

Experimental results show that the humanized antibodies 531-Hu and 4A11-Hu disclosed herein have high proliferative inhibitory activity against M07e cells.

Test Example 5: Inhibitory activity of anti-KIT antibody against induced mast cells

This embodiment measures the activity of anti-KIT antibody in inhibiting the release of inflammatory factors from mast cells. In vitro, human peripheral blood-derived CD34 ⁺ hematopoietic stem cells were induced into mature human mast cells using human interleukin-6 (IL-6, Peprotech, catalog number: 96-200-06-50) and stem cell factor [SCF, sequence from UniProt (Entry: P21583)]. The maturation process of mast cells was accelerated by adding human low-density lipoprotein (LDL, Absin, catalog number: abs47014900). The degree of mast cell maturation was determined by the expression of KIT and FceRI. Take an appropriate amount of mature mast cells (20,000 cells per well in a 96-well plate), centrifuge at 300g for 8 minutes to collect the cells, and resuspend them in experimental medium [the experimental medium was a self-prepared IMDM medium (Thermofisher Scientific, 12440053) containing 1×GlutaMAX ^™ -I (Thermo, 35050061), 1× Sodium Pyruvate (Gibco, 11360070), 50 μM β-mercaptoethanol (Invitrogen, 21985023), 0.5% Bovine Serum Albumin (Solarbio, A8020), 1× Insulin-Transferrin-Selenium (Gibco, 41400-045), and antibiotics (Gibco, 15240062)]. Wash the cells twice and resuspend them at a concentration of 5× ^10⁵ /mL. Add 100 ng/mL to the suspension. IgE (Sigma-Aldrich, AG30P) was incubated overnight at 37°C. The next day, cells were collected by centrifugation, washed twice with IMDM medium, and resuspended in experimental medium at a density of 2 × ^10⁵ /mL. 100 μL of mast cells were seeded per well in a 96-well cell culture plate (Thermofisher Scientific, 062096). Anti-KIT antibody was serially diluted with experimental medium and added to the cell culture, mixed well, and incubated at 37°C for 4 hours. Then, 100 ng/mL SCF and anti-IgE (Bethyl Laboratories, A80-109A) were added to the cell culture, and the plate was incubated overnight at 37°C (approximately 16 hours). The next day, the cell supernatant was collected by centrifugation at 300g for 8 minutes, and the GM-CSF level in the cell culture supernatant was measured using a GM-CSF ELISA kit (Dakewei, 1117302). The results are shown in Figures 1A and 1B.

Figures 1A and 1B show the experimental results, which indicate that the humanized antibodies 531-Hu and 4A11-Hu disclosed herein have a stronger inhibitory effect on the secretion of GM-CSF by mast cells than the control antibody CDX-0159.

Test Example 6: Pharmacokinetic Effect of Anti-KIT Antibody on Canine Skin Mast Cells

Two groups of beagles, four in each group (half male and half female), were used. Skin tissue samples were collected one week prior to drug administration, sectioned, stained, and the mast cell count was determined. Anti-KIT antibody was administered via subcutaneous injection at a single point in the neck at a volume of 0.6 mL/kg and a dose of 3 MPa. The day of administration was designated as day 0. Skin tissue samples were collected on days 7 and 14 post-administration to determine the mast cell count. The mast cell count one week prior to administration was normalized to 100%, and the relative mast cell count was the percentage of mast cells in the skin after administration compared to the initial mast cell count. The experimental results are shown in Figure 2.

The experimental results showed that, compared with before administration, on days 7 and 14, CDX-0159 reduced the number of mast cells in the skin of beagle dogs by 4.5% and 8.3%, respectively, while 531-Hu reduced the number of mast cells in the skin by 39.8% and 50.3%, respectively. The effect of 531-Hu in clearing skin mast cells was significantly stronger than that of the control antibody CDX-0159. On day 14, there was a significant difference in the pharmacokinetic effects of 531-Hu and CDX-0159 on the number of mast cells (t-test P value 0.013 < 0.05).

Although the invention has been described in detail with the aid of examples for clarity of understanding, the description and examples should not be construed as limiting the scope of this disclosure. All patent and scientific literature disclosures cited herein are clearly and fully incorporated by reference.

Claims (11)

An anti-KIT antibody comprising a heavy chain variable region and a light chain variable region, wherein: I, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with the same amino acid sequence as the heavy chain variable region shown in SEQ ID NO: 26, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with the same amino acid sequence as the light chain variable region shown in SEQ ID NO: 27; or II, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with the same amino acid sequence as the heavy chain variable region shown in SEQ ID NO: 14, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with the same amino acid sequence as the light chain variable region shown in SEQ ID NO: 15; or III, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with the same amino acid sequence as the heavy chain variable region shown in SEQ ID NO: 32, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with the same amino acid sequence as the light chain variable region shown in SEQ ID NO: 33; or IV. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with the same amino acid sequence as the heavy chain variable region shown in SEQ ID NO: 16, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with the same amino acid sequence as the light chain variable region shown in SEQ ID NO: 17. or V, wherein the heavy chain variable region HCDR1 contains the amino acid sequence shown in SEQ ID NO: 2, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 3, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 4; the light chain variable region LCDR1 contains the amino acid sequence shown in SEQ ID NO: 38, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 39, and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 7; wherein SEQ ID NO: 38 is LCDR1 as shown in X ₁ ASSSVSYMH, where X ₁ is R or S; SEQ ID NO: 39 is LCDR2 as shown in STSNLAX ₂ , where X ₂ is D or S; or VI. The heavy chain variable region HCDR1 contains the amino acid sequence shown in SEQ ID NO: 8, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 9, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 10; the light chain variable region LCDR1 contains the amino acid sequence shown in SEQ ID NO: 11, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 12, and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 13. The anti-KIT antibody according to claim 1 comprises a heavy chain variable region and a light chain variable region, wherein: I, wherein the heavy chain variable region HCDR1 contains the amino acid sequence shown in SEQ ID NO: 2, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 3, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 4; the light chain variable region LCDR1 contains the amino acid sequence shown in SEQ ID NO: 36, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 37, and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 7; or II, wherein the heavy chain variable region HCDR1 contains the amino acid sequence shown in SEQ ID NO: 2, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 3, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 4; the light chain variable region LCDR1 contains the amino acid sequence shown in SEQ ID NO: 5, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 6, and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 7; or III. The heavy chain variable region HCDR1 contains the amino acid sequence shown in SEQ ID NO: 8, HCDR2 contains the amino acid sequence shown in SEQ ID NO: 9, and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 10; the light chain variable region LCDR1 contains the amino acid sequence shown in SEQ ID NO: 11, LCDR2 contains the amino acid sequence shown in SEQ ID NO: 12, and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 13. The anti-KIT antibody according to claim 1 or 2, wherein the anti-KIT antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; Preferably, the anti-KIT antibody is a murine antibody, a chimeric antibody, or a humanized antibody; More preferably, the anti-KIT antibody is a humanized antibody. The anti-KIT antibody according to any one of claims 1 to 3, wherein: I, wherein the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 26 or an amino acid sequence having at least 75% sequence identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% sequence identity with it; or II, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having at least 75% sequence identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 15 or an amino acid sequence having at least 75% sequence identity with it; or III, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 32 or an amino acid sequence having at least 75% sequence identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 33 or an amino acid sequence having at least 75% sequence identity with it; or IV. The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% sequence identity with it, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 17 or an amino acid sequence having at least 75% sequence identity with it. The anti-KIT antibody according to any one of claims 1 to 4, wherein the anti-KIT antibody is an antibody fragment; preferably, the antibody fragment is selected from Fab, Fab′, F(ab′)2, Fv, scFv or dsFv. The anti-KIT antibody according to any one of claims 1 to 4, wherein the antibody comprises a heavy chain constant region and a light chain constant region; Preferably, the heavy chain constant region is derived from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, and the light chain constant region is derived from the light chain constant region of human κ or λ; more preferably, the heavy chain constant region is derived from the heavy chain constant region of human IgG1, and the light chain constant region is derived from the light chain constant region of human κ or λ. Most preferably, the heavy chain constant region contains the amino acid sequence shown in SEQ ID NO: 18, and the light chain constant region contains the amino acid sequence shown in SEQ ID NO: 19. The anti-KIT antibody according to any one of claims 1 to 4 and 6, wherein the anti-KIT antibody comprises a heavy chain and a light chain, wherein: I, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 28 or an amino acid sequence having at least 85% sequence identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 29 or an amino acid sequence having at least 85% sequence identity with it; or II, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 20 or an amino acid sequence having at least 85% sequence identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 21 or an amino acid sequence having at least 85% sequence identity with it; or III, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 34 or an amino acid sequence having at least 85% sequence identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 35 or an amino acid sequence having at least 85% sequence identity with it; or IV. The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 22 or an amino acid sequence having at least 85% sequence identity with it, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 23 or an amino acid sequence having at least 85% sequence identity with it. A pharmaceutical composition comprising the anti-KIT antibody according to any one of claims 1 to 7, and one or more pharmaceutically acceptable carriers, diluents, or excipients. An isolated nucleic acid encoding the anti-KIT antibody according to any one of claims 1 to 7. A host cell comprising the isolated nucleic acid as described in claim 9. A method for preventing or treating a disease or condition, the method comprising administering to a subject the anti-KIT antibody of any one of claims 1 to 7, or the pharmaceutical composition of claim 8; The diseases or conditions mentioned therein are selected from inflammatory diseases, eye diseases, cancer, blood diseases, allergies, and autoimmune diseases; Preferably, the disease or condition is selected from nodular prurigo, esophagitis, asthma, atopic dermatitis, urticaria, myeloma, connective tissue tumor, leukemia, lung cancer, gastrointestinal stromal tumor, hemoglobinopathies, retinal diseases, diabetic macular edema, and wet age-related macular degeneration. More preferably, the hemoglobinopathies are selected from sickle cell anemia and β-thalassemia; the urticaria is selected from chronic urticaria, chronic spontaneous urticaria, chronic induced urticaria, and cold contact urticaria; the lung cancer is small cell lung cancer; the leukemia is chronic myeloid leukemia or acute myeloid leukemia; and the esophagitis is eosinophilic esophagitis.