Recombinant antibodies and their uses

Abstract

The present specification discloses a recombinant antibody or a fragment thereof. According to some embodiments of the present disclosure, the recombinant antibody comprises a VHH domain, and CDR-1, CDR-2, and CDR-3 comprise the amino acid sequences of SEQ ID NOs: 1-3, respectively. The present specification also discloses a pharmaceutical composition comprising the recombinant antibody, and a method for treating cancer by using the recombinant antibody.

Description

[Technical Field]

[0001] The present disclosure relates generally to the field of disease treatment. More specifically, the present disclosure relates to recombinant antibodies specific for carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) and their use in the treatment of cancer. [Background technology]

[0002] Cancer is a group of diseases characterized by the development of abnormal cells that divide uncontrollably and have the ability to invade and destroy normal body tissues. More than 100 different types of cancer have been identified in humans, with lung, prostate, and colorectal cancer being the most common in men, and breast, lung, and colorectal cancer being the most common in women.

[0003] The main treatments for cancer include surgery, radiation therapy, chemotherapy, hormone therapy, and targeted therapy. Generally, treatments vary depending on the type, location, and grade of cancer, as well as the health and wishes of the patient. However, most of these treatments cannot provide satisfactory results to cancer patients due to limitations such as the development of drug resistance, low specificity, low therapeutic efficiency, and / or adverse side effects such as pain, anemia, bleeding, lymphedema, diarrhea, constipation, fatigue, loss of appetite, infection, neuropathy, and memory impairment.

[0004] CEACAM6 (also known as CD66c) is a member of the carcinoembryonic antigen (CEA) family. CEACAM6 is overexpressed in approximately 70% of solid tumors, including colorectal, gastric, endometrial, breast, ovarian, cervical, pancreatic, and lung cancers, and head and neck squamous cell carcinoma (SCCC). It is known to promote cancer progression by inducing epithelial-mesenchymal transition (EMT). Based on its role in cancer development and progression, CEACAM6 could serve as a target for cancer therapy. However, the efficacy of anti-CEACAM6 agents against cancer remains unproven and requires experimental evaluation.

[0005] In view of the foregoing, there is a need in the art for new agents and / or methods for treating cancer, particularly cancers that express CEACAM6. Summary of the Invention

[0006] The following presents a simplified summary of the disclosure in order to provide the reader with a basic understanding. This summary is not an extensive overview of the disclosure and does not identify key / critical elements or delineate the scope of the invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

[0007] As embodied and broadly described herein, one aspect of the present disclosure is directed to a recombinant antibody or fragment thereof. According to embodiments of the present disclosure, the recombinant antibody comprises a variable heavy chain domain (VHH domain) and a fragment crystallizable region of an immunoglobulin (Fc region) fused to the VHH domain. The VHH domain comprises a first chain complementarity determining region (CDR-1), a second CDR (CDR-2), and a third CDR (CDR-3). According to some embodiments of the present disclosure, the first, second, and third CDRs comprise the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively.

[0008] According to some preferred embodiments, the VHH domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 4. In one particular embodiment, the VHH domain comprises an amino acid sequence that is 100% identical to SEQ ID NO: 4.

[0009] According to a particular embodiment, the VHH domain is derived from a camelid heavy chain antibody.

[0010] According to particular embodiments, the immunoglobulin is human immunoglobulin G (IgG) or immunoglobulin A (IgA). In one particular embodiment, the immunoglobulin is human IgG1. According to another embodiment, the immunoglobulin is mouse IgG or IgA. In one exemplary embodiment, the immunoglobulin is mouse IgG2.

[0011] A second aspect of the present disclosure is directed to a pharmaceutical composition comprising a recombinant antibody or antibody fragment of the present disclosure and a pharmaceutically acceptable excipient.

[0012] Also disclosed herein is a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of a recombinant antibody or antibody fragment of the present disclosure.

[0013] Depending on the desired purpose, the cancer may be any cancer that expresses CEACAM6, such as gastric cancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer, kidney cancer, colorectal cancer, cervical cancer, ovarian cancer, brain cancer, prostate cancer, hepatocellular carcinoma, melanoma, esophageal cancer, multiple myeloma, or head and neck squamous cell carcinoma.

[0014] Optionally, the method further comprises administering to the subject an effective amount of an anti-cancer agent prior to, concurrently with, or after administration of the recombinant antibody or antibody fragment. According to some embodiments, the anti-cancer agent is cetuximab.

[0015] The subject is a mammal, preferably a human.

[0016] Many of the attendant features and advantages of the present disclosure will become better understood by reference to the following detailed description considered in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0017] The present disclosure will be better understood from the following detailed description when read in conjunction with the accompanying drawings, in which: [Figure 1] FIG. 1 is a schematic diagram showing the structure of a recombinant antibody according to an embodiment of the present disclosure. [Figure 2A] 1 shows histograms showing the effect of the recombinant antibodies on the migration and invasion ability of tumor cells according to Example 2 of the present disclosure, where NS is not significant, P>0.05, *P<0.05, **P<0.01, and ***P<0.001. [Figure 2B] 1 shows histograms showing the effect of the recombinant antibodies on the migration and invasion ability of tumor cells according to Example 2 of the present disclosure, where NS is not significant, P>0.05, *P<0.05, **P<0.01, and ***P<0.001. [Figure 3A] 10 is a line graph showing the anti-tumor effect of the recombinant antibodies in an animal model according to Example 3 of the present disclosure. [Figure 3B] 10 is a line graph showing the anti-tumor effect of the recombinant antibodies in an animal model according to Example 3 of the present disclosure.

[0018] In accordance with common practice, the various features / elements described are not drawn to scale, but are drawn to best illustrate the particular features / elements relevant to the present invention, and reference numerals and characters in the various figures are used to indicate elements / portions. DETAILED DESCRIPTION OF THE INVENTION

[0019] The detailed description provided below in connection with the accompanying drawings is intended as a description of embodiments of the invention and is not intended to represent the only manner in which the embodiments of the invention may be constructed or utilized. The description sets forth functions of the embodiments and the sequence of steps for constructing and operating the embodiments. However, the same or equivalent functions and sequences may be accomplished by different embodiments.

[0020] I. Definition

[0021] For convenience, certain terms used in the specification, examples, and appended claims are collected here. Unless otherwise defined herein, scientific and technical terms employed in this disclosure shall have the meanings commonly understood and used by those of ordinary skill in the art. It is also understood that singular terms include the plural of the same, and plural terms include the singular, unless otherwise required by context. Specifically, as used in this specification and claims, the singular forms "a" and "an" include the plural, unless the context clearly dictates otherwise. Also, as used in this specification and claims, the terms "at least one" and "one or more" have the same meaning and include one, two, three, or more.

[0022] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Also, as used herein, the term "about" generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term "about" means within an acceptable standard error of the mean as considered by one of ordinary skill in the art. Except in the operating / working examples, or unless otherwise expressly stated, all numerical ranges, amounts, values, and percentages, such as amounts of materials, durations, temperatures, operating conditions, ratios of amounts, etc., disclosed herein should be understood in all instances to be modified by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in this disclosure and the appended claims are approximations that can be varied as necessary. At the very least, each numerical parameter should be construed in light of at least the number of reported significant digits and by applying ordinary rounding techniques.

[0023] The term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific or multivalent antibodies (e.g., bispecific antibodies), chimeric antibodies, heavy-chain antibodies (HCAbs), and antibody fragments, so long as they exhibit the desired biological activity. The term "antibody fragment" or "fragment of an antibody" refers to a portion of a full-length antibody, typically the antigen-binding or variable domains (i.e., the VH and / or VL domains) of a full-length antibody. Examples of antibody fragments include VHH domains (also known as single-domain antibodies (sdAbs) or nanobodies (Nbs)), VHH dimers, fragment antigen-binding (Fab), Fab', F(ab')2, single-chain variable region fragments (scFv), diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. According to some embodiments of the present disclosure, the term "antibody fragment" refers to the VHH domain of an HCAb or a VHH dimer.

[0024] As used herein, the term "heavy chain antibody" (HCAb) refers to an antibody consisting of two heavy chains and lacking light chains (see, e.g., Hamers-Casterman, et al. Nature. 1993; 363: 446-448). Specifically, HCAbs are composed of two heavy chains linked by a covalent disulfide bond, and each heavy chain in an HCAb has a variable domain at one end. The variable domain of the heavy chain of an HCAb is called a "VHH domain" to distinguish it from the variable domain of the heavy chain (VH domain) of a "traditional" antibody, which has two heavy chains and two light chains. HCAbs naturally present in camelids and sharks can bind to antigens via the VHH domain.

[0025] As used herein, the term "VHH domain" of an HCAb refers to the amino-terminal region of the antibody heavy chain. These regions are generally the most variable parts of an antibody and contain the antigen-binding site. The term "variable" refers to the fact that certain portions of the VHH domain vary significantly in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not uniformly distributed throughout the VHH domain of an antibody. It is concentrated in three segments of the VHH domain called complementarity-determining regions (CDRs) or hypervariable regions. The more highly conserved portions of the VHH domain are called the framework (FR). Each VHH domain of a native heavy chain contains four FR regions, primarily adopting a β-sheet structure connected by three CDRs, which form loops and, in some cases, form part of the β-sheet structure. The CDRs in each chain are held in close proximity to each other by the FR regions and contribute to the formation of the antigen-binding site of the antibody. The constant region is not involved directly in binding an antibody to an antigen, but exhibits various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0026] As used herein, the term "complementarity determining region" (CDR) refers to the hypervariable regions of a VHH domain that form a surface complementary to the three-dimensional surface of a bound antigen. From the N- to C-terminus, each VHH of an antibody contains three CDRs (i.e., CDR-1, CDR-2, and CDR-3) that define the binding affinity and specificity of the VHH domain.

[0027] The term "fragment crystallizable region" or "Fc region," as used herein, refers to the tail region of an antibody that interacts with cell surface receptors called Fc receptors and / or proteins that are part of the complement system. Structurally, the Fc region includes, from the N-terminus to the C-terminus, at least the hinge region of the heavy chain (a short sequence connecting the CH1 and CH2 domains), the CH2 domain (the second constant domain of the heavy chain), and the CH3 domain (the third constant domain of the heavy chain). The Fc region of an IgG1 antibody can be generated, for example, by digesting an IgG1 antibody with papain.

[0028] "Percent sequence identity" with respect to an amino acid sequence identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a particular reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering conservative substitutions as part of the sequence identity. Alignment for determining percentage sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including the algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. Herein, sequence comparison between two amino acid sequences was performed using the computer program Blastp (protein-protein BLAST), available online from the National Center for Biotechnology Information (NCBI). The percentage sequence identity of a given sequence A to a subject sequence B (alternatively, it can be expressed as a given sequence A having a particular % sequence identity to a given sequence B) is calculated by the following formula: JPEG2026506269000002.jpg13170X is the number of amino acid residues scored as perfect matches by the sequence alignment program BLAST in the alignment of programs A and B, and Y is the total number of amino acid residues in the subject sequence B.

[0029] As discussed herein, minor variations in the amino acid sequence of an antibody are considered to be encompassed within the inventive concepts disclosed and claimed herein, provided that the variations in the amino acid sequence maintain at least 85% sequence identity, e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity. The antibodies of the present disclosure may be specifically modified to alter characteristics unrelated to the biological activity of the peptide. For example, certain amino acids can be altered and / or deleted without affecting the biological activity of the antibody in this study (i.e., its ability to bind to coronavirus). Conservative amino acid substitutions are particularly contemplated. Conservative substitutions are those made within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally classified into the following families: (1) acidic = aspartate, glutamate, (2) basic = lysine, arginine, histidine, (3) nonpolar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More preferred families are as follows: serine and threonine are the aliphatic hydroxy family, asparagine and glutamine are the amide-containing family, alanine, valine, leucine, and isoleucine are the aliphatic family, and phenylalanine, tryptophan, and tyrosine are the aromatic family. For example, it is reasonable to expect that singular substitutions of leucine with isoleucine or valine, aspartate with glutamate, threonine with serine, or similar substitutions of amino acids with structurally related amino acids will not significantly affect the binding or properties of the resulting molecule, particularly if the substitutions do not involve amino acids within framework sites. Whether an amino acid change results in a functional peptide can be readily determined by measuring the specific activity of the peptide derivative. Antibody fragments or analogs can be readily prepared by one of skill in the art.Preferred amino- and carboxyl-termini of fragments or analogs occur near boundaries of functional regions.

[0030] The term "subject" refers to a mammal, including a human, that is the target of the recombinant antibody, pharmaceutical composition and / or method of the present invention. The term "subject" refers to both males and females, unless either gender is specifically indicated. II. DESCRIPTION OF THE INVENTION

[0031] The present disclosure relates to recombinant antibodies specific for CEACAM6 and their use in the treatment of cancer, particularly cancers in which CEACAM6 is overexpressed on the surface and / or within the cancer.

[0032] Accordingly, a first aspect of the present disclosure relates to a recombinant antibody or fragment thereof (e.g., a VHH domain). Reference is made to FIG. 1, which is a schematic diagram illustrating a recombinant antibody according to a specific embodiment of the present disclosure. Structurally, the recombinant antibody comprises a pair of VHH domains and an immunoglobulin Fc region linked to the pair of VHH domains. As known in the art, an Fc region comprises two identical protein fragments linked by disulfide bonds, each protein fragment comprising, from N- to C-terminus, a hinge region, a CH2 domain, and a CH3 domain. The pair of VHH domains are linked to the N-terminus of the protein fragment. As shown in FIG. 1, the recombinant antibody is in the form of an HCAb (i.e., a homodimeric VHH-Fc complex) comprising two heavy chains linked via disulfide bonds at the hinge region, each heavy chain comprising, from N- to C-terminus, a VHH domain, a hinge region, a CH2 domain, and a CH3 domain.

[0033] Alternatively, a pair of VHH domains are linked to the N-terminus of the protein fragment via a linker, in which case the recombinant antibody is in the form of an HCAb (i.e., a homodimeric VHH-linker-Fc complex) comprising two heavy chains linked via disulfide bonds at the hinge region, and each heavy chain comprises, from N- to C-terminus, a VHH domain, a linker, a hinge region, a CH2 domain, and a CH3 domain.

[0034] It is widely accepted that the binding properties (including binding affinity and specificity) of an antibody are determined by its variable domains (i.e., the VH and VL domains of a conventional antibody, or the VHH domain of an HCAb), more specifically, by the CDR sequences of the variable domains (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of a conventional antibody, or CDR-1, CDR-2, and CDR-3 of an HCAb). According to some embodiments of the present disclosure, each VHH domain of the recombinant antibody comprises three CDRs (i.e., CDR-1, CDR-2, and CDR-3), wherein CDR-1 comprises the amino acid sequence of SEQ ID NO: 1, CDR-2 comprises the amino acid sequence of SEQ ID NO: 2, and CDR-3 comprises the amino acid sequence of SEQ ID NO: 3.

[0035] According to some embodiments, each VHH domain of the recombinant antibody comprises an amino acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 4. As will be understood, the sequence of the VHH domain (e.g., framework sequence) may be altered (e.g., substituted with conserved or non-conserved amino acid residues) without affecting the binding affinity and / or specificity of the antibody. Preferably, the sequence of the VHH domain is conservatively substituted with one or more suitable amino acids having similar properties. Examples include substitution of leucine (a non-polar amino acid residue) with isoleucine, alanine, valine, proline, phenylalanine, or tryptophan (another non-polar amino acid residue), substitution of aspartate (an acidic amino acid residue) with glutamate (another acidic amino acid residue), or substitution of lysine (a basic amino acid residue) with arginine or histidine (another basic amino acid residue). According to some preferred embodiments, the VHH domain of the recombinant antibody comprises an amino acid sequence at least 90% identical to SEQ ID NO: 4. More preferably, the VHH domain of the recombinant antibody comprises an amino acid sequence at least 95% identical to SEQ ID NO: 4. In one example of the present disclosure, the VHH domain of the recombinant antibody has the amino acid sequence of SEQ ID NO: 4 (i.e., has 100% identical amino acid sequence to SEQ ID NO: 4).

[0036] Depending on the desired purpose, the immunoglobulin may be human immunoglobulin G1 (IgG1), immunoglobulin G2 (IgG2), immunoglobulin G3 (IgG3), immunoglobulin G4 (IgG4), or immunoglobulin A (IgA). According to some exemplary embodiments, the immunoglobulin is human IgG1. In one particular embodiment, a pair of VHH domains are linked to the N-terminus of the Fc region of human IgG1, and each heavy chain of the recombinant antibody comprises the amino acid sequence of SEQ ID NO:5.

[0037] Alternatively, the immunoglobulin may be mouse IgG1, IgG2, IgG3, or IgA. According to a specific embodiment, the immunoglobulin is mouse IgG2. In one specific embodiment, the pair of VHH domains is linked to the N-terminus of the Fc region of mouse IgG2, and each heavy chain of the recombinant antibody comprises the amino acid sequence of SEQ ID NO:6.

[0038] According to certain embodiments of the present disclosure, recombinant antibodies are produced by ribosome display and DNA cloning.

[0039] Ribosome display is a cell-free system for in vitro selection of proteins and peptides from large libraries. Methods for constructing ribosome display systems are known in the art. Generally, ribosome display systems are established by immunizing a host animal (e.g., a camelid, preferably an alpaca) with a peptide (e.g., a CEACAM6 polypeptide) according to commonly employed procedures, for example, every two or three weeks until a desired antibody titer is reached. After the final immunization, peripheral lymphocytes from the immunized animal are collected and total RNA is isolated therefrom. Corresponding cDNA fragments are synthesized, and a cDNA library is therefore constructed by amplifying cDNA fragments encoding VHH domains and subsequently ligating the PCR products into an expression vector. VHH-encoding cDNAs are useful for transcribing mRNA and translating proteins in a ribosome display format (i.e., protein-ribosome-mRNA (PRM) complex). In the selection step (also known as the "biopanning step"), the PRM complexes are added to an immobilized ligand (e.g., an immobilized CEACAM6 polypeptide), and complexes that exhibit binding affinity to the immobilized ligand are then eluted by salt concentrations that allow dissociation of the mRNA, chelators, or mobile ligands that complex with the protein's binding motif. The mRNA can then be reverse transcribed into cDNA for the next selection round. The selection step may be repeated multiple times (e.g., 3-5 times) to obtain cDNAs encoding VHH domains specific to the peptide (e.g., a CEACAM6 polypeptide).

[0040] According to some embodiments of the present disclosure, the cDNA thus obtained is then cloned into an expression vector containing the Fc region of a human immunoglobulin (i.e., an IgG1 vector). After introduction into a suitable host cell, the expression vector is useful for expressing a recombinant antibody in the host cell, and the recombinant antibody thus produced contains, in its structure, a human-derived Fc region and a camelid-derived VHH domain linked to the N-terminus of the Fc region. The host cell is preferably a mammalian cell, such as a Chinese hamster ovary (CHO) cell or an embryonic kidney 293 cell. In one preferred embodiment, the host cell is an embryonic kidney 293 cell.

[0041] Alternatively, the recombinant antibody may be produced directly by DNA cloning. DNA encoding the recombinant antibody can be readily isolated and sequenced using conventional procedures, such as using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the mAb. Once isolated, the DNA is placed into an expression vector, which is then introduced into an appropriate host cell as described above, allowing the synthesis of the desired antibody in the host cell.

[0042] Recombinant antibodies can be purified according to standard procedures in the art, such as cross-flow filtration, affinity column chromatography, gel filtration, and the like.

[0043] Depending on the desired purpose, the recombinant antibodies of the present disclosure can be conjugated to a desired drug (i.e., in the form of an antibody-drug conjugate, ADC, e.g., an anti-cancer drug) or therapeutic agent (e.g., a therapeutic peptide) to improve their therapeutic effect.

[0044] According to certain embodiments of the present disclosure, recombinant antibody treatment inhibits tumor growth. Accordingly, also disclosed herein are pharmaceutical compositions or medicaments for treating cancer. The pharmaceutical composition or medicament comprises an effective amount of a recombinant antibody or fragment thereof according to any embodiment of the present disclosure, and optionally a pharmaceutically acceptable excipient.

[0045] Generally, the recombinant antibody or antibody fragment of the present invention is present at a level of about 0.1% to 99% by weight based on the total weight of the pharmaceutical composition or medicament. In some embodiments, the recombinant antibody or antibody fragment of the present invention is present at a level of at least 1% by weight based on the total weight of the pharmaceutical composition or medicament. In certain embodiments, the recombinant antibody or antibody fragment is present at a level of at least 5% by weight based on the total weight of the pharmaceutical composition or medicament. In yet other embodiments, the recombinant antibody or antibody fragment is present at a level of at least 10% by weight based on the total weight of the pharmaceutical composition or medicament. In yet other embodiments, the recombinant antibody or antibody fragment is present at a level of at least 25% by weight based on the total weight of the pharmaceutical composition or medicament.

[0046] The pharmaceutical composition or drug product may be prepared according to the methods described in Remington's Pharmaceutical Sciences, 17 th Pharmaceutically acceptable excipients prepared in accordance with acceptable pharmaceutical procedures, as described in "Pharmaceutical Preparations for the Preparation of Pharmaceuticals," ed. Alfonoso R. Gennaro, Mack Publishing Company, Easton, Pa. (1985), are compatible with the other ingredients in the formulation and are biologically acceptable.

[0047] The pharmaceutical compositions or medicaments can be formulated into solid, semi-solid, or liquid forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, and injectables. Thus, administration of the recombinant antibody or antibody fragment can be achieved in a variety of ways, including oral, buccal, rectal, parenteral, intravenous, and intraperitoneal administration. In pharmaceutical dosage forms, the recombinant antibody or antibody fragment can be administered alone or in combination with other known pharmaceutically active agents for treating cancer. Those skilled in the art are familiar with the various dosage forms suitable for use by each route. It should be noted that the most appropriate administration route in a particular case will depend on the nature and severity of the disease or condition being treated.

[0048] Preferably, the pharmaceutical composition or medicament is formulated into a liquid form, such as a sterile solution or suspension, that can be administered by, for example, intravenous, intraarterial, intramuscular, subcutaneous, intrathecal, intraperitoneal, or intratumoral injection. The pharmaceutical composition or medicament can be formulated as an isotonic suspension, solution, or emulsion in an oily or aqueous vehicle and may contain formulating agents such as suspending agents, stabilizers, or dispersing agents. Alternatively, the pharmaceutical composition or medicament can be provided in a dry form, such as a powder, crystal, or lyophilized solid, with sterile pyrogen-free water or isotonic saline before use. They can be provided in sterile ampoules or vials.

[0049] When the recombinant antibody or antibody fragment is formulated for administration by intravenous, cutaneous, or subcutaneous injection, the antibody is in the form of a pyrogen-free, parenterally acceptable aqueous solution. Preparation of such parenterally acceptable polypeptide solutions, with due consideration given to pH, isotonicity, stability, and the like, is within the skill of those in the art. Preferred pharmaceutical compositions or medicaments for intravenous, cutaneous, or subcutaneous injection should contain, in addition to the recombinant antibody or antibody fragment, an isotonic vehicle such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection, or other vehicles known to those skilled in the art. The pharmaceutical compositions or medicaments of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those skilled in the art. The duration of intravenous therapy using the pharmaceutical compositions or medicaments of the present invention will vary depending on the severity of the disease being treated, as well as the condition and potential idiosyncratic responses of each individual subject. It is contemplated that the duration of each application of the antibody will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately, the attending physician will determine the appropriate duration of intravenous therapy.

[0050] Another aspect of the present disclosure relates to a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of a recombinant antibody, antibody fragment, pharmaceutical composition or medicament of the present disclosure.

[0051] The effective dosage administered to a subject is about 0.01 to 1,000 mg per kg of the subject's body weight, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 1 9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 , 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,000 mg per kg of subject body weight, preferably about 0.1 to 100 mg per kg of subject body weight. The dosage can be administered in a single aliquot or multiple aliquots. A skilled technician or clinician can adjust the dosage or administration schedule depending on the patient's physical condition or the severity of the disease.

[0052] According to some embodiments of the present disclosure, administration of the recombinant antibody inhibits tumor growth in a subject through downregulation of epidermal growth factor receptor (EGFR) signaling and the self-renewal and invasive capacity of cancer / tumor cells.

[0053] Optionally, the method further comprises administering to the subject an effective amount of an anti-cancer agent before, simultaneously with, or after administration of the recombinant antibody. Non-limiting examples of anti-cancer agents include alkylating agents (drugs that prevent cell replication by damaging DNA, e.g., altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine). , ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa, and trabectedin), antimetabolites (drugs that interfere with DNA and RNA by acting as substitutes for the normal building blocks of RNA and DNA, e.g., azacitidine zacitidine, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cladribine, clofarabine, cytarabine (Ara-C), decitabine, floxuridine, fludarabine, gemcitabine (g emcitabine, hydroxyurea, methotrexate, nelarabine, pemetrexed, pentostatin, pralatrexate, and thioguanine), anthracyclines or antibiotics (drugs that interfere with enzymes involved in replicating DNA during the cell cycle, e.g., daunorubicin,doxorubicin, epirubicin, idarubicin, valrubicin, bleomycin, dactinomycin, mitomycin-C, and mitoxantrone), mitotic inhibitors (drugs derived from natural products that stop cells from dividing and forming new cells, such as cabazitaxel, docetaxel, nab-paclitaxel, paclitaxel, vinblastine, vincristine, and vinorelbine), corticosteroids (natural hormones) and steroids, including, but not limited to, steroid or hormone-like drugs such as prednisone, methylprednisolone, and dexamethasone, and antibodies (e.g., rituximab, trastuzumab, gemtuzumab, alemtuzumab, tositumomab, cetuximab, ibritumomab, bevacizumab, panitumumab, catumaxomab, ofatumumab, ipilimumab, and brentuximab). According to some preferred embodiments, the anti-cancer agent is an anti-EGFR antibody such as cetuximab, panitumumab, nimotuzumab, necitumumab, duligotuzumab, depatuxizumab, matuzumab, or zalutumumab. According to one specific example, the anti-cancer agent is cetuximab. In this example,Administration of the recombinant antibody and cetuximab additively or synergistically inhibits tumor growth by downregulating EGFR signaling and the self-renewal and invasive capacity of cancer / tumor cells.

[0054] Subjects treatable by the present method are mammals, such as humans, mice, rats, guinea pigs, hamsters, monkeys, pigs, dogs, cats, horses, sheep, goats, cows, and rabbits. Preferably, the subject is a human.

[0055] The recombinant antibodies of the present disclosure can be administered to a subject by a suitable route, such as oral, enteral, nasal, topical, transmucosal, or parenteral administration. Depending on the intended purpose, parenteral administration can be intratumoral, intramuscular, intravenous, or intraperitoneal injection.

[0056] The following examples are provided to clarify certain aspects of the present invention and to assist those skilled in the art in practicing the present invention. These examples should not be construed as limiting the scope of the present invention in any way. Without further explanation, it is believed that those skilled in the art can utilize the present invention to its fullest extent based on the description herein. All publications cited herein are incorporated by reference in their entirety. Example

[0057] Materials and Methods

[0058] cell culture

[0059] HT-29 and Caco-2 cells (CEACAM6-expressing cells) were obtained from the American Type Culture Collection (ATCC). Both cell lines were cultured in RPMI 1640 (Roswell Park Memorial Institute 1640) medium supplemented with 10% fetal bovine serum (FBS).

[0060] Preparation of recombinant antibodies (hereinafter referred to as "HCAb")

[0061] HCAb is EXPI293FTM and EXPICHO-S TM The EXPI293F cells were expressed and purified according to the manufacturer's instructions. TM Cells are exposed to TM 293 and EXPIFECTAMINE TM The HCAb construct was transfected using CHO reagents, the cell culture medium was collected, and the HCAb was purified using a Protein G column.

[0062] The HCAb thus generated contained a homodimer of a camelid-derived VHH domain fused to a human-derived Fc region. Analysis revealed that each VHH domain contained the amino acid sequence of SEQ ID NO: 4, CDR-1, CDR-2, and CDR-3 contained the amino acid sequences of SEQ ID NOs: 1 to 3, respectively (Table 1), and the human-derived Fc region contained the amino acid sequence of SEQ ID NO: 5.

[0063] Table 1. Amino acid sequence of this HCAb JPEG2026506269000003.jpg26170 The CDR sequences in the VHH domain are shown in bold in order from the N-terminus to the C-terminus, including CDR-L1, CDR-L2 and CDR-L3.

[0064] binding affinity

[0065] The binding affinity of recombinant HCAb to recombinant CEACAM6 was measured by surface plasmon resonance (SPR). TM The binding kinetics of HCAb to CEACAM6 was analyzed using the instrument. Recombinant CEACAM6 protein was immobilized on a sensor chip according to the manufacturer's instructions. Five serial dilutions of HCAb (20, 10, 5, 2.5, and 1 nM) were injected at time zero over a chip containing 3,000 resonance units (RU) of CEACAM6 in HBS-P buffer at a flow rate of 10 μl / min. The resulting sensorgrams were overlaid, and the binding kinetics from two independent experiments were averaged and analyzed using software.

[0066] Immunohistochemistry (IHC)

[0067] The binding affinity and specificity of recombinant HCAb to tumor and normal tissues were measured by immunohistochemistry. Specifically, for tissue microarrays, serial 5 μm tissue sections were deparaffinized in xylene and rehydrated. After blocking endogenous peroxidase by incubation with 3% hydrogen peroxide, the slides were incubated with HCAb overnight at 4°C. CEACAM6 expression was detected by applying streptavidin-biotin-peroxidase complex (DAKO). Peroxidase activity was visualized with 3,3'-diaminobenzidine (DAB). Slides were counterstained with hematoxylin and mounted with mounting medium.

[0068] Immunoblotting

[0069] Cell protein lysates were collected by scraping cells in cold mammalian protein extraction reagent buffer containing phosphatase and protease inhibitors. Protein concentration was measured. Approximately 20 μg of protein was resolved on a 10% sodium dodecyl sulfate polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane. Primary antibodies were diluted in Tris buffer containing 3% BSA and incubated with the membrane overnight at 4°C. Blots were visualized using horseradish peroxidase (HRP) substrate with a gel documentation system.

[0070] Invasion and migration assays

[0071] Cell invasion and migration assays were performed using 24-well transwell polycarbonate filters (pore size, 8 μm) coated or uncoated with Matrigel. Briefly, cancer cells were seeded in the upper chamber, and 1% FBS or epidermal growth factor (EGF) was added to the lower chamber. After 24 hours of incubation, the cells were individually counted for invasion and migration. Nonpenetrating cells were removed from the upper surface of the filter using a cotton swab. Penetrating cells were fixed and stained using a staining kit according to the manufacturer's instructions. For quantification, all cells that had invaded or migrated to the lower surface were stained and counted under a light microscope.

[0072] animal research

[0073] 1 x 10 cells mixed in 0.1 ml of Matrigel 7 HT-29 cells were subcutaneously injected into 8-week-old severe combined immunodeficient (SCID) male mice. Seven days after cell injection, the mice were divided into four groups and treated with phosphate-buffered saline (PBS), cetuximab (10 mg / kg), HCAb (10 mg / kg), and cetuximab (10 mg / kg) + HCAb (10 mg / kg) by intravenous injection (10 mg / kg) every seven days for a total of six doses. Tumor volume was measured weekly with a vernier caliper and calculated using the following formula: (length × width) 2 ×0.45).

[0074] statistical analysis

[0075] All statistical analyses were performed using software. Results were analyzed using an unpaired Student's t-test to compare two independent groups, or a one-way ANOVA test to compare two or more comparisons. For each result, all data were presented as the mean ± SD from three independent experiments. *P<0.05, **P<0.01, ***P<0.001; ns indicates no significance at the 95% two-sided confidence interval.

[0076] Example 1 Characterization of HCAbs

[0077] IHC staining and SPR results demonstrated that this HCAb recognized glycosylated CEACAM6 protein, and its binding affinity (KD) for recombinant CEACAM6 was less than 1 nM (data not shown). The IHC data demonstrated that this HCAb exhibited binding affinity and specificity for cancer tissues (including rectal adenocarcinoma, descending colon adenocarcinoma, metastatic colorectal adenocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, and cholangiocarcinoma) compared with normal tissues (including skin, breast, lymph node, stomach, gastric muscularis, lung, liver, colon, testis, prostate, kidney, esophagus, cerebellum, cerebrum, adrenal gland, placenta, heart, spleen, skeletal muscle, appendix, salivary gland, gallbladder, pancreas, and tonsil) in which no significant antibody signal was detected (data not shown).

[0078] These data demonstrated that the present HCAb can specifically target cancer cells.

[0079] Example 2 Tumor cell inhibitory effect of HCAb

[0080] Immunoblotting and invasion assay data showed that CEACAM6 protein was overexpressed in tumor cells and promoted tumor progression through its interaction with EGFR (data not shown). Downregulation of CEACAM6 significantly inhibited the invasive ability of tumor cells (data not shown). Compared with the control group, treatment with this HCAb significantly inhibited the phosphorylation levels of extracellular signal-regulated protein kinases 1 and 2 (ERK1 / 2), thereby inhibiting activation of the ERK1 / 2 MAP kinase pathway (data not shown). The data in Figures 2A and 2B further demonstrated that treatment with this HCAb reduced the spheroid formation and invasive ability of tumor cells. Combination treatment with this HCAb and cetuximab was confirmed to achieve an additive effect in inhibiting tumor cells (Figure 2B).

[0081] The data suggest that the present HCAbs may function as anti-cancer agents that can be administered therapeutically either alone or in combination with other active agents (eg, cetuximab).

[0082] Example 3 In vivo studies

[0083] The activity of this HCAb to inhibit tumor growth in animals was investigated in this example. The data in Figure 3A demonstrate that administration of HCAb significantly inhibited tumor growth in the animal model compared to the PBS control group.

[0084] Cetuximab is a monoclonal chimeric antibody that serves as a first-line treatment for metastatic colorectal cancer (RAS-wild-type) and advanced head and neck cancer. Cancer patients typically acquire resistance to cetuximab 3–12 months after treatment. In this study, CEACAM6 was upregulated in cetuximab-resistant head and neck squamous cell carcinoma (HNSCC) cell lines (data not shown). To evaluate whether this HCAb could enhance the therapeutic effect of cetuximab against cancer, this HCAb and cetuximab were administered to tumor-bearing mice according to the procedures described in Materials and Methods. The data in Figure 3B demonstrated that coadministration of this HCAb and cetuximab significantly suppressed tumor growth compared with the control group. Therefore, this HCAb can improve therapeutic efficacy when used sequentially or in combination with first-line anticancer drugs (e.g., cetuximab).

[0085] In conclusion, the present disclosure provides a novel anti-CEACAM6 antibody. According to examples of the present disclosure, the anti-CEACAM6 antibody exhibits binding affinity and specificity to tumor cells and can inhibit tumor growth through downregulation of CEACAM6 expression and the ERK1 / 2 MAP kinase pathway. Therefore, the present anti-CEACAM6 antibody can serve as a potential antibody for treating cancer.

[0086] It will be understood that the above description of the embodiments is for purposes of illustration only, and that various modifications may be made by those skilled in the art. The above specification, examples, and data provide a complete description of the structure and use of exemplary embodiments of the invention. While various embodiments of the invention have been described above with a certain degree of particularity, or with reference to one or more specific embodiments, those skilled in the art could make many modifications to the disclosed embodiments without departing from the spirit or scope of the invention.

Claims

1. A recombinant antibody or fragment thereof, comprising a variable heavy domain (VHH domain) and a fragment crystallizable region of an immunoglobulin (Fc region) fused to said VHH domain, said VHH domain comprising: a first strand complementarity determining region (CDR-1) comprising the amino acid sequence of SEQ ID NO: 1; a second CDR (CDR-2) comprising the amino acid sequence of SEQ ID NO: 2; and a third CDR (CDR-3) comprising the amino acid sequence of SEQ ID NO:

3.

2. The recombinant antibody of claim 1, wherein the VHH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO:

4.

3. The recombinant antibody of claim 2, wherein the VHH domain comprises an amino acid sequence 100% identical to SEQ ID NO:

4.

4. 2. The recombinant antibody of claim 1, wherein the VHH domain is derived from a camelid heavy chain antibody.

5. 2. The recombinant antibody of claim 1, wherein the immunoglobulin is human immunoglobulin G (IgG) or immunoglobulin A (IgA).

6. The recombinant antibody of claim 5, wherein the immunoglobulin is IgG1.

7. A pharmaceutical composition comprising the recombinant antibody of claim 1 and a pharmaceutically acceptable excipient.

8. A method for treating cancer in a subject, comprising administering to the subject an effective amount of the recombinant antibody of claim 1.

9. 9. The method of claim 8, wherein the cancer is gastric cancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer, kidney cancer, colorectal cancer, cervical cancer, ovarian cancer, brain cancer, prostate cancer, hepatocellular carcinoma, melanoma, esophageal cancer, multiple myeloma, or head and neck squamous cell carcinoma.

10. 10. The method of claim 8, further comprising administering to the subject an effective amount of an anti-cancer agent.

11. 11. The method of claim 10, wherein the anti-cancer agent is cetuximab.

12. 9. The method of claim 8, wherein the subject is a human.

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