Recombinant antibodies and uses thereof

Abstract

Disclosed herein are a recombinant antibody or the fragment thereof. According to some embodiments of the present disclosure, the recombinant antibody comprises a VHH domain, in which the CDR-1, CDR-2 and CDR-3 respectively comprise the amino acid sequences of SEQ ID NOs: 1-3. Also disclosed herein are a pharmaceutical composition comprising the recombinant antibody, and methods of treating cancers by using the recombinant antibody.

Description

RECOMBINANT ANTIBODIES AND USES THEREOF BACKGROUND OF THE INVENTION

[0001] 1. FIELD OF THE INVENTION

[0002] The present disclosure in general relates to the field of disease treatment. More particularly, the present disclosure relates to a recombinant antibody specific to carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), and uses thereof in treating cancers.

[0003] 2. DESCRIPTION OF RELATED ART

[0004] Cancer is a class of diseases characterized by the development of abnormal cells that divide uncontrollably and have the ability to infiltrate and destroy normal body tissues. More than 100 different types of cancer have been identified in humans, in which lung cancer, prostate cancer and colorectal cancer are prevalent in male subjects, while breast cancer, lung cancer and colorectal cancer prevail in female subjects.

[0005] The major treatments for cancers include surgery, radiation therapy, chemotherapy, hormonal therapy and targeted therapy. In general, the treatment may vary with the type, location and grade of the cancer as well as the patient’s health and preferences. However, most of these treatments cannot produce a satisfactory effect on cancer patients due to the limitations of, for example, the development of drug-resistance, low-specificity, low-efficiency, and / or adverse side-effects, such as pain, anemia, bleeding, lymphedema, diarrhea, constipation, fatigue, appetite loss, infection, neuropathy and memory problems.

[0006] CEACAM6 (also known as CD66c) is a member of the carcinoembryonic antigen (CEA) family. It is known that CEACAM6 is overexpressed in nearly 70% of solid tumors, including colorectal, gastric, endometrial, breast, ovarian, cervical, pancreatic and lung tumors, and head and neck squamous cell carcinoma; and would promote cancer progression via inducing epithelial-mesenchymal transition (EMT). Based on the role of CEACAM6 in cancer development and progression, it may serve as a target for cancer treatment. Unfortunately, the effect of anti-CEACAM6 agents on cancers has not been proven yet and needs to be experimentally evaluated.

[0007] In view of the forging, there exists in the related art a need for a novel agent and / or a method for treating cancers, especially CEACAM6-expressing cancers. SUMMARY

[0008] The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key / critical elements of the present invention or delineate the scope of the present 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.

[0009] As embodied and broadly described herein, one aspect of the disclosure is directed to a recombinant antibody or the 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 (Fc region) of an immunoglobulin fused to the VHH domain, in which 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 respectively comprise the amino acid sequences of SEQ ID NOs: 1, 2 and 3.

[0010] According to some preferred embodiments, the VHH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 4. In one specific embodiment, the VHH domain comprises an amino acid sequence 100% identical to SEQ ID NO: 4.

[0011] According to certain embodiments, the VHH domain is derived from a camelid heavy-chain antibody.

[0012] According to certain embodiments, the immunoglobulin is human immunoglobulin G (IgG) or immunoglobulin A (IgA). In one specific embodiment, the immunoglobulin is human IgG1. According to alternative embodiments, the immunoglobulin is mouse IgG or IgA. In one exemplary embodiment, the immunoglobulin is mouse IgG2.

[0013] The second aspect of the present disclosure is directed to a pharmaceutical composition, which comprises the recombinant antibody or the antibody fragment of the present disclosure, and a pharmaceutically acceptable excipient.

[0014] Also disclosed herein is a method of treating a cancer in a subject. The method comprises administering to the subject an effective amount of the recombinant antibody or antibody fragment of the present disclosure.

[0015] Depending on desired purposes, the cancer may be any CEACAM6-expressing cancers, for example, gastric cancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer, renal cancer, colorectal cancer, cervical cancer, ovarian cancer, brain tumor, prostate cancer, hepatocellular carcinoma, melanoma, esophageal carcinoma, multiple myeloma, or head and neck squamous cell carcinoma.

[0016] Optionally, the method further comprises administering to the subject an effective amount of an anti-cancer agent prior to, concurrently with, or after the administration of the present recombinant antibody or antibody fragment. According to some embodiments, the anti-cancer agent is cetuximab.

[0017] The subject is a mammal; preferably, a human.

[0018] Many of the attendant features and advantages of the present disclosure will become better understood with reference to the following detailed description considered in connection with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present description will be better understood from the following detailed description read in light of the accompanying drawings, where:

[0020] Fig. 1 is a schematic diagram depicting the structure of the recombinant antibody according to embodiments of the present disclosure;

[0021] Figs. 2A and 2B are histograms respectively depicting the effect of the present recombinant antibody on migration and invasion ability of tumor cells according to Example 2 of the present disclosure; N.S.: not significant, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001; and

[0022] Figs. 3A and 3B are line charts respectively depicting the anti-tumor effect of the present recombinant antibody in animal models according to Example 3 of the present disclosure.

[0023] In accordance with common practice, the various described features / elements are not drawn to scale but instead are drawn to best illustrate specific features / elements relevant to the present invention. Also, reference numerals and designations in the various drawings are used to indicate elements / parts. DETAILED DESCRIPTION OF THE INVENTION

[0024] The detailed description provided below in connection with the appended drawings is intended as a description of the present examples and is not intended to represent the only forms in which the present example may be constructed or utilized. The description sets forth the functions of the example and the sequence of steps for constructing and operating the example. However, the same or equivalent functions and sequences may be accomplished by different examples.

[0025] I. DEFINITION

[0026] For convenience, certain terms employed in the specification, examples and appended claims are collected here. Unless otherwise defined herein, scientific and technical terminologies employed in the present disclosure shall have the meanings that are commonly understood and used by one of ordinary skill in the art. Also, unless otherwise required by context, it will be understood that singular terms shall include plural forms of the same and plural terms shall include the singular. Specifically, as used herein and in the claims, the singular forms “a” and “an” include the plural reference unless the context clearly indicates otherwise. Also, as used herein and in the claims, the terms “at least one” and “one or more” have the same meaning and include one, two, three, or more.

[0027] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in the 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 when considered by one of ordinary skill in the art. Other than in the operating / working examples, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages such as those for quantities of materials, durations of times, temperatures, operating conditions, ratios of amounts, and the likes thereof disclosed herein should be understood as modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and attached claims are approximations that can vary as desired. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0028] The term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multi-specific or multivalent antibodies (e.g., bi-specific antibodies), chimeric antibodies, heavy chain antibodies (HCAbs) and antibody fragments so long as they exhibit the desired biological activity. The term “antibody fragment” or “the fragment of an antibody” refers to a portion of a full-length antibody, generally the antigen binding or variable domain (i.e., VH and / or VL domains) of a full-length antibody. Examples of the antibody fragment include VHH domain (also known as single domain antibody (sdAb) or nanobody (Nb)), VHH dimer, fragment antigen-binding (Fab), Fab’, F(ab’)2, single-chain variable fragment (scFv), diabody, linear antibody, single-chain antibody molecule, and multi-specific antibody formed from antibody fragments. According tosome embodiments of the present disclosure, the term “antibody fragment” refers to the VHH domain or VHH dimer of a HCAb.

[0029] As used herein, the term “heavy-chain antibody” (HCAb) refers to an antibody which consists of two heavy chains and is devoid of light chains (See, for example, Hamers-Casterman, et al. Nature. 1993; 363: 446-448). Specifically, the HCAbs are composed of two heavy chains linked by a covalent disulfide bond, in which each heavy chain in the HCAb has a variable domain at one end. The variable domain of the heavy chain of the HCAb is referred to as “VHH domain” in order to distinguish it from the variable domain (VH domain) of the heavy chain of “conventional” antibody that has two heavy chains and two light chains. HCAbs, which naturally occur in camelids and sharks, can bind antigens via the VHH domains.

[0030] As used herein, the term “VHH domain” of an HCAb refers to the amino-terminal regions of heavy chains of the antibody. These regions are generally the most variable parts of an antibody and contain the antigen-binding sites. The term “variable” refers to the fact that certain portions of the VHH domains differ extensively in sequence among antibodies, and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the VHH domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the VHH domains. The more highly conserved portions of VHH domains are called the framework (FR). Each VHH domain of native heavy chains comprises four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions, and contribute to the formation of the antigen-binding site of antibodies. The constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular toxicity.

[0031] The term “complementarity determining region” (CDR) used herein refers to the hypervariable region of the VHH domain that forms a surface complementary to the 3-dimensional surface of a bound antigen. Proceeding from N-terminus to C-terminus, each VHH of the antibody comprises three CDRs (i.e., CDR-1, CDR-2, and CDR-3), which define the binding affinity and specificity of the VHH domains.

[0032] 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 some proteins of the complement system. In structure, the Fc region comprises, from N-terminus to C-terminus, at least a hinge region (a short sequence of the heavy chain that linksthe CH1 and CH2 domains), a CH2 domain (the second constant domain of the heavy chain) and a CH3 domain (the third constant domain of the heavy chain). An Fc region of an IgGl antibody can, for example, be generated by digestion of an IgG1 antibody with papain.

[0033] “Percentage (%) sequence identity” with respect to any amino acid sequence identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the specific reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining the percentage of sequence identity can be achieved in various ways that are within the skill in the art, for instance, 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 any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, sequence comparison between two amino acid sequences was carried out by computer program Blastp (protein-protein BLAST) provided online by Nation Center for Biotechnology Information (NCBI). The percentage sequence identity of a given sequence A to a subject sequence B (which can alternatively be phrased as a given sequence A that has a certain % sequence identity to a given sequence B) is calculated by the formula as follows: X u 100 % Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program BLAST in that program's alignment of A and B, and where Y is the total number of amino acid residues in the subject sequence B.

[0034] As discussed herein, minor variations in the amino acid sequences of antibodies are contemplated as being encompassed by the presently disclosed and claimed inventive concept(s), providing that the variations in the amino acid sequence maintain at least 85% sequence identity, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity. Antibodies of the present disclosure may be modified specifically to alter a feature of the peptide unrelated to its physiological activity. For example, certain amino acids can be changed and / or deleted without affecting the physiological activity of the antibody in this study (i.e., the ability to bind to coronavirus). In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into 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: serine and threonine are aliphatic-hydroxy family; asparagine and glutamine are an amide-containing family; alanine, valine, leucine and isoleucine are an aliphatic family; and phenylalanine, tryptophan, and tyrosine are an aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the peptide derivative. Fragments or analogs of antibodies can be readily prepared by those of ordinary skill in the art. Preferred amino- and carboxyl-termini of fragments or analogs occur near the boundaries of functional regions.

[0035] The term “subject” refers to a mammal including the human species that can be subjected to the recombinant antibody, pharmaceutical composition and / or method of the present invention. The term “subject” is intended to refer to both the male and female gender unless one gender is specifically indicated.

[0036] II. DESCRIPTION OF THE INVENTION

[0037] The present disclosure is directed to a recombinant antibody specific to CEACAM6, and uses of the recombinant antibody in treating cancers, especially the cancers having CEACAM6 overexpressed thereon and / or therein.

[0038] Accordingly, the first aspect of the present disclosure is directed to a recombinant antibody or the fragment thereof (e.g., the VHH domain). Reference is now made to Fig. 1, which is a schematic diagram depicting a recombinant antibody according to certain embodiments of the present disclosure. In structure, the present recombinant antibody comprises a pair of VHH domains, and an Fc region of an immunoglobulin linked to the pair of VHH domains. As known in the art, the Fc region comprises two identical protein fragments linked by a disulfide bond, in which each protein fragment comprises, from N-terminus to C-terminus, a hinge region, a CH2 domain and a CH3 domain. The pair of VHH domains is linked to the N-termini of the protein fragments. As depicted in Fig. 1, the present recombinant antibody is in the form of an HCAb (i.e., a homodimeric VHH-Fc complex) that comprises two heavy chains linked via the disulfide bond in the hinge region, in which each heavy chain comprises, from N-terminus to C-terminus, a VHH domain, a hinge region, a CH2 domain and aCH3 domain.

[0039] Alternatively, the pair of VHH domains are linked to the N-termini of the protein fragments via a linker. In this case, the present recombinant antibody is in the form of an HCAb (i.e., a homodimeric VHH-linker-Fc complex) that comprises two heavy chains linked via the disulfide bond in the hinge region, in which each heavy chain comprises, from N-terminus to C-terminus, a VHH domain, a linker, a hinge region, a CH2 domain and a CH3 domain.

[0040] It is well-accepted that the binding properties (including the binding affinity and specificity) of an antibody are determined by its variable domain(s) (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 domain(s) (i.e., the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 of a conventional antibody, or the 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), in which the CDR-1 comprises the amino acid sequence of SEQ ID NO: 1, the CDR-2 comprises the amino acid sequence of SEQ ID NO: 2, and the CDR-3 comprises the amino acid sequence of SEQ ID NO: 3.

[0041] According to some embodiments, each VHH domain of the recombinant antibody comprises an amino acid sequence of 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 would be appreciated, the sequence (e.g., the framework sequence) of the VHH domain may vary (e.g., being substituted by conserved or non-conserved amino acid residues) without affecting the binding affinity and / or specificity of the present antibody. Preferably, the sequence of the VHH domain is conservatively substituted by one or more suitable amino acid(s) with similar properties; for example, the substitution of leucine (a nonpolar amino acid residue) by isoleucine, alanine, valine, proline, phenylalanine, or tryptophan (another nonpolar amino acid residue); the substitution of aspartate (an acidic amino acid residue) by glutamate (another acidic amino acid residue); or the substitution of lysine (a basic amino acid residue) by 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 working example of the present disclosure, the VHH domain of the recombinant antibody has the amino acid sequence of SEQ ID NO: 4 (i.e., having an amino acid sequence 100% identical to SEQ ID NO: 4).

[0042] Depending on desired purposes, the immunoglobulin may be a humanimmunoglobulin 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 specific embodiment, the pair of VHH domains is linked to the N-termini of the Fc region of human IgG1, in which each heavy chain of the present recombinant antibody comprises the amino acid sequence of SEQ ID NO: 5.

[0043] Alternatively, the immunoglobulin may be mouse IgG1, IgG2, IgG3 or IgA. According to certain embodiments, the immunoglobulin is mouse IgG2. In one specific embodiment, the pair of VHH domains is linked to the N-termini of the Fc region of mouse IgG2, in which each heavy chain of the present recombinant antibody comprises the amino acid sequence of SEQ ID NO: 6.

[0044] According to certain embodiments of the present disclosure, the recombinant antibody is produced by ribosome display and DNA cloning.

[0045] Ribosome display is a cell-free system for in vitro selection of proteins and peptides from large libraries. The methods of producing a ribosome display system are known in the art. In general, the ribosome display system is established by immunizing a host animal (e.g., a camelid, preferably a Lama Pacos (alpaca)) with a peptide (e.g., a CEACAM6 polypeptide) in accordance with commonly adopted procedures; for example, immunizing the host animal with the peptide every two or three weeks, until a desired antibody titer is reached. After the final immunization, peripheral lymphocytes of the immunized animal are collected, and total RNAs are isolated therefrom. Corresponding cDNA fragments are synthesized, and a cDNA library is constructed accordingly via amplifying the cDNA fragment encoding the VHH domains followed by ligating the PCR products into an expression vector. The VHH-encoding cDNAs are useful in transcribing mRNAs and translating proteins in ribosome display format (i.e., protein-ribosome-mRNA (PRM) complex). In the selection stage (also known as the “biopanning stage”), the PRM complexes are added to an immobilized ligand (e.g., an immobilized CEACAM6 polypeptide), and the complexes exhibiting binding affinity to the immobilized ligand are then eluted by salt concentrations, chelating agents, or mobile ligands which complex with the binding motif of the protein allow dissociation of the mRNA. The mRNA can then be reverse transcribed back into cDNA for the next round of selection. The selection stage may be repeated several times (for example, 3-5 times) so as to obtain the cDNA encoding VHH domains specific to the peptide (e.g., the CEACAM6 polypeptide)

[0046] According to some embodiments of the present disclosure, the thus-obtained cDNA is then cloned into an expression vector (i.e., IgG1 vector), which comprises the Fc region of a human immunoglobulin. After introducing a suitable host cell, the expression vector is usefulin expressing the recombinant antibody in the host cell; the thus-produced recombinant antibody in structure comprises a human-derived Fc region, and camelid-derived VHH domains linked to the N-terminus of the Fc region. The host cell is preferably a mammalian cell, for example, Chinese hamster ovary (CHO) cells or embryonic kidney 293 cells. According to one preferred embodiment, the host cell is an embryonic kidney 293 cells.

[0047] Alternatively, the present recombinant antibody may be directly produced by DNA cloning. DNA encoding the present recombinant antibody may be easily isolated and sequenced by use of conventional procedures, such as using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the mAb. Once isolated, the DNA may be placed into an expression vector, which is then transfected into a suitable host cell as described above, so as to synthesize the desired antibody in the host cell.

[0048] The recombinant antibody may be purified according to standard procedures in the art, such as cross-flow filtration, affinity column chromatography, gel filtration and the like.

[0049] Depending on desired purposes, the recombinant antibody of the present disclosure may be conjugated with a desired drug (i.e., in the form of the antibody-drug conjugate, ADC; e.g., an anti-cancer agent) or a therapeutic agent (e.g., a therapeutic peptide) for improving its therapeutic effect.

[0050] According to certain embodiments of the present disclosure, the treatment of the recombinant antibody inhibits tumor growth. Accordingly, also disclosed herein is a pharmaceutical composition or a medicament for treating cancers. The pharmaceutical composition or medicament comprises an effective amount of the recombinant antibody or the fragment thereof in accordance with any embodiments of the present disclosure; and optionally, a pharmaceutically acceptable excipient.

[0051] Generally, the recombinant antibody or antibody fragment of this 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 this 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 still 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 still 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.

[0052] The pharmaceutical composition or medicament is prepared in accordance with acceptable pharmaceutical procedures, such as described in Remington’s Pharmaceutical Sciences, 17thedition, ed. Alfonoso R. Gennaro, Mack Publishing Company, Easton, Pa (1985). Pharmaceutically acceptable excipients are those that are compatible with other ingredients in the formulation and biologically acceptable.

[0053] The present pharmaceutical composition or medicament may be formulated into solid, semi-solid, or liquid forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, and injections. As such, administration of the present recombinant antibody or antibody fragment can be achieved in various ways, including oral, buccal, rectal, parenteral, intravenous, intraperitoneal, and etc. administration. In pharmaceutical dosage forms, the present recombinant antibody or antibody fragment may be administered alone or in combination with other known pharmaceutically active agents to treat cancers. One of skilled people in the art is familiar with the various dosage forms that are suitable for use in each route. It is to be noted that the most suitable route in any given case would depend on the nature or severity of the disease or condition being treated.

[0054] Preferably, the pharmaceutical composition or medicament is formulated into liquid forms, e.g., sterile solutions or suspensions that can be administered by, for example, intravenous, intraarterial, intramuscular, subcutaneous, intrathecal, intraperitoneal or intratumoral injection. The pharmaceutical composition or medicament may be formulated as isotonic suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulary agents such as suspending, stabilizing or dispersing agents. Alternatively, the pharmaceutical composition or medicament may be provided in dry forms such as powders, crystallines or freeze-dried solids with sterile pyrogen-free water or isotonic saline before use. They may be presented in sterile ampoules or vials.

[0055] When the present recombinant antibody or antibody fragment is formulated to be administered by intravenous, cutaneous or subcutaneous injection, the antibody will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable polypeptide solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition or medicament for intravenous, cutaneous, or subcutaneous injection should contain, in addition to the present 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 as known in the art. The pharmaceutical composition or medicament of the invention may also contain stabilizers, preservatives, buffers,antioxidants, or other additives known to those of skill in the art. The duration of intravenous therapy using the pharmaceutical composition or medicament of the invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual subject. It is contemplated that the duration of each application of the present antibody will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy.

[0056] Another aspect of the present disclosure pertains to a method of treating a cancer in a subject. The method comprises administering to the subject an effective amount of the recombinant antibody, antibody fragment, pharmaceutical composition or medicament of the present disclosure.

[0057] The effective dose administered to the subject is from about 0.01 to 1,000 mg / Kg body weight of the subject, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.30.4, 0.5, 0.6, 0.7, 0.8, 0.9, 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 / Kg body weight of the subject; preferably, about 0.1 to 100 mg / Kg body weight of the subject. The dose can be administered in a single aliquot, or alternatively in more than one aliquot. The skilled artisan or clinical practitioner may adjust the dosage or regime in accordance with the physical condition of the patient or the severity of the diseases.

[0058] According to some embodiments of the present disclosure, the administration of the recombinant antibody inhibits tumor growth in the subject via down-regulating epidermal growth factor receptor (EGFR) signaling, and self-renewal and invasion abilities of cancer / tumor cells.

[0059] Optionally, the method further comprises administering to the subject an effective amount of an anti-cancer agent prior to, concurrently with, or after the administration of the present recombinant antibody. Non-limiting examples of anti-cancer agent include, but are not limited to, alkylating agents (agents that keep cells from replication by damaging their DNA; e.g., altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa and trabectedin), antimetabolites (agents that interfere with DNA and RNA by acting as a substitute for the normal building blocks of RNA and DNA; e.g., azacitidine, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cladribine, clofarabine,cytarabine (Ara-C), decitabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, nelarabine, pemetrexed, pentostatin, pralatrexate and thioguanine), anthracyclines or antibiotics (agents that interfere with enzymes involved in copying DNA during the cell cycle; e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, bleomycin, dactinomycin, mitomycin-C and mitoxantrone), mitotic inhibitors (agents derived from nature products, and stopping cells from deviding to form new cells; e.g., cabazitaxel, docetaxel, nab-paclitaxel, paclitaxel, vinblastine, vincristine and vinorelbine), corticosteroids (natural hormones or hormone-like drugs; e.g., 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, the administration of the recombinant antibody and cetuximab additively or synergistically inhibits tumor growth via down-regulating EGFR signaling, and self-renewal and invasion abilities of cancer / tumor cells.

[0060] The subject treatable by the present method is a mammal, for example, a human, mouse, rat, guinea pig, hamster, monkey, swine, dog, cat, horse, sheep, goat, cow, and rabbit. Preferably, the subject is a human.

[0061] The recombinant antibody of the present disclosure may be administered to the subject by an appropriate route, such as oral, enteral, nasal, topical, transmucosal, or parenteral administration. Depending on intended purposes, the parenteral administration may be intrautumoral, intramuscular, intravenous or intraperitoneal injection.

[0062] The following Examples are provided to elucidate certain aspects of the present invention and to aid those of skilled in the art in practicing this invention. These Examples are in no way to be considered to limit the scope of the invention in any manner. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety. EXAMPLE

[0063] Materials and Methods

[0064] Cell culture

[0065] HT-29 and Caco-2 cells (CEACAM6-expressing cells) were obtained from ATCC (American Type Culture Collection). Both cell lines were cultured in RPMI 1640 (Roswell Park Memorial Institute 1640) media supplemented with 10% fetal bovine serum (FBS).

[0066] Preparation of recombinant antibody (hereinafter as “HCAb”)

[0067] The HCAb was expressed using EXPI293F™ and EXPICHO-S™ cells and purified according to the manufacturer’s instructions. Briefly, EXPI293F™ cells were transfected with the HCAb construct using the EXPIFECTAMINE™ 293 and EXPIFECTAMINE™ CHO reagents. The cell culture medium was collected, and the HCAb was purified using a Protein G column.

[0068] The thus-produced HCAb comprised a homodimer of camelid-derived VHH domain fused with a human-derived Fc region. According to the analytic results, each VHH domain comprised the amino acid sequence of SEQ ID NO: 4, in which the CDR-1, CDR-2 and CDR-3 respectively comprised the amino acid sequences of SEQ ID NOs: 1-3 (Table 1); and the human-derived Fc region comprised the amino acid sequence of SEQ ID NO: 5.

[0069] Table 1 Amino acid sequence of the present HCAbThe CDR sequences in VHH domain were marked in boldface, including CDR-L1, CDR-L2 and CDR-L3, from N-terminus to C-terminus, in sequence.

[0070] Binding affinity

[0071] The binding affinity of the recombinant HCAb to the recombinant CEACAM6 was determined by surface plasmon resonance (SPR). Specifically, BIACORETMmachine was used to analyze the binding kinetics of HCAb to CEACAM6. A recombinant CEACAM6 protein was immobilized on sensor chips according to the manufacturer’s instructions. Five serially diluted HCAb (20, 10, 5, 2.5 and 1 nM) were injected at a flow rate of 10 μl / min in HBS-P buffer onto a chip containing 3000 resonance units (RU) of CEACAM6 at time zero. The resulting sensorgrams were superimposed, and the binding kinetics from two independent experiments were averaged and analyzed using software

[0072] Immunohistochemistry (IHC)

[0073] The binding affinity and specificity of the recombinant HCAb to the tumor and normal tissues were determined by immunohistochemistry. Specifically, in a tissue microarray, serial 5 μm histologic sections were deparaffinized in xylene and rehydrated. After blocking endogenous peroxidase by incubating with 3% hydrogen peroxide, the slides were incubatedwith the HCAb overnight at 4ƱC. The expression of CEACAM6 was detected by applying streptavidin-biotin-peroxidase complex (DAKO), and the peroxidase activity was revealed by 3,3'-Diaminobenzidine (DAB). The slides were counterstained with hematoxylin and mounted with a mounting solution.

[0074] Immunoblotting

[0075] Cellular protein lysates were collected by scraping the cells in a cold buffer of mammalian protein extraction reagent containing phosphatase and protease inhibitors. Protein concentration was determined. Approximately 20 μg of protein were resolved on 10% sodium dodecyl sulfate polyacrylamide gel followed by transferring it onto 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 gel documentation system.

[0076] Invasion and migration assay

[0077] Cell invasion and migration assays were done in 24-well transwell polycarbonate filters (pore size, 8 μm) coated with or without matrigel. Briefly, cancer cells were plated in the upper chamber, and the 1% FBS or epidermal growth factor (EGF) was added to the lower chamber. The invaded and migrated cells were counted after the cells have been individually incubated for 24 hours. Nonpenetrating cells were removed from the upper surface of the filter with a cotton swab. Penetrating cells were fixed and stained with a stain kit according to the manufacturer’s instructions. For quantification, all of the cells invaded or migrated into the lower surface were stained and counted under a light microscope.

[0078] Animal study

[0079] 1×107HT-29 cells mixed in 0.1 ml of matrigel were subcutaneously injected into 8-week-old severe combined immunodeficient (SCID) male mice. Seven days after cell injection, the mice was divided into four groups and respectively treated with phosphate buffered saline (PBS), cetuximab (10 mg / kg), HCAb (10 mg / kg), and cetuximab (10 mg / kg) + HCAb (10 mg / kg) via intravenous injection (10mg / kg) every 7 days for a total of 6 doses. Tumor volumes were measured every week using calipers, and were calculated using the following equation: (length × width2× 0.45).

[0080] Statistical analysis

[0081] All statistical analyses were performed using software. The results were analyzed using the unpaired Student’s t-test or one-way ANOVA test to compare two independent groups or more than two comparisons, respectively. For each result, all data are presented as themeans ± S.D. from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ns indicates no significance for 95% two-tail confidence intervals.

[0082] Example 1 Characterization of HCAb

[0083] According to the IHC staining and SPR results, the present HCAb recognized glycosylated CEACAM6 protein, and the binding affinity (KD) of the present HCAb to the recombinant CEACAM6 was less than 1 nM (data not shown). The IHC data demonstrated that compared to normal tissues (including skin, breast, lymph node, stomach, muscle layer of stomach, 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 obvious antibody signal was detected, the present HCAb exhibited binding affinity and specificity to cancerous tissues (including rectal adenocarcinoma, descending colon adenocarcinoma, metastatic colorectal adenocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma and cholangiocarcinoma) (data not shown).

[0084] These data indicated that the present HCAb was capable of specifically targeting cancer cells.

[0085] Example 2 Effect of HCAb on inhibiting tumor cells

[0086] According to the data of immunoblotting and invasion assay, CEACAM6 protein was overexpressed in tumor cells, and promoted tumor progression through interaction with EGFR (data not shown); down-regulating the expression of CEACAM6 significantly inhibited the invasion ability of tumor cells (data not shown). Compared to the control group, the treatment of the present HCAb obviously inhibited the phosphorylation level of extracellular signal-regulated protein kinases 1 and 2 (ERK1 / 2), i.e., inhibiting the activation of ERK1 / 2 MAP kinase pathway (data not shown). The data of Figs. 2A and 2B further demonstrated that the treatment of the present HCAb decreased the sphere formation and invasion ability of tumor cells. It was noted that co-treatment of the present HCAb and cetuximab would achieve an additive effect on inhibiting tumor cells (Fig. 2B).

[0087] The data suggested that the present HCAb may server as an anti-cancer agent, which may be administrated alone or in combination with other active agents (e.g., cetuximab) for the treatment purpose.

[0088] Example 3 In vivo study

[0089] The activity of the present HCAb to inhibit tumor growth in animals was examined in this example. The data of Fig. 3A demonstrated that compared to the PBS control group, the administration of HCAb greatly inhibited tumor growth in the animal model.

[0090] Cetuximab is a chimeric monoclonal antibody serving as a first-line treatment for metastatic colorectal cancer (RAS-wild type) and advanced head and neck cancer. It is known that cancer patients usually develop resistance to cetuximab in 3-12 months post-treatment. According to the present study, CEACAM6 was up-regulated in a cetuximab-resistant head and neck squamous cell carcinoma (HNSCC) cell line (data not shown). For the purpose of evaluating whether the present HCAb may enhance the therapeutic effect of cetuximab on cancers, the present HCAb and cetuximab are administered to tumor-bearing mice in accordance with the procedures described in Materials and Methods. The data of Fig. 3B demonstrated that co-administration of the present HCAb and cetuximab significantly inhibited tumor growth as compared to the control group. Thus, the present HCAb may be used with the first-line anti-cancer agent (e.g., cetuximab), either in sequence or in combination, to improve the therapeutic efficacy.

[0091] In conclusion, the present disclosure provides a novel anti-CEACAM6 antibody. According to the examples of the present disclosure, the anti-CEACAM6 antibody exhibits binding affinity and specificity to tumor cells, and is capable of inhibiting tumor growth via down-regulating CEACAM6 expression and ERK1 / 2 MAP kinase pathway. Accordingly, the present anti-CEACAM6 antibody may serve as a potential antibody for treating cancers.

[0092] It will be understood that the above description of embodiments is given by way of example only and that various modifications may be made by those with ordinary skill in the art. The above specification, examples and data provide a complete description of the structure and use of exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those with ordinary skill in the art could make numerous alterations to the disclosed embodiments without departing from the spirit or scope of this invention.

Claims

WHAT IS CLAIMED IS:

1. A recombinant antibody or the fragment thereof, comprising a variable heavy chain domain (VHH domain), and a fragment crystallizable region (Fc region) of an immunoglobulin fused to the VHH domain, wherein the VHH domain comprises, a first chain 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. The recombinant antibody of claim 1, wherein the VHH domain is derived from a camelid heavy-chain antibody.

5. 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 of treating a cancer in a subject, comprising administering to the subject an effective amount of the recombinant antibody of claim 1.

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

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

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

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

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