TWI886446B - Recombinant antibodies and uses thereof - Google Patents

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 their uses

The present disclosure relates to the field of treating diseases. More specifically, the present disclosure relates to a recombinant antibody specific for carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) and its use in treating cancer.

Cancer is a disease associated with abnormal cell growth that divides uncontrollably and has the ability to infiltrate and destroy normal body tissues. More than 100 different types of cancer have been identified in humans. The most common cancers in men include lung cancer, prostate cancer and colorectal cancer, while the most common cancers in women include breast cancer, lung cancer and colorectal cancer.

The main treatments for cancer include surgery, radiation therapy, chemotherapy, hormone therapy, and targeted therapy. In general, treatments vary depending on the type, location, and severity of the cancer, as well as the patient's health and preferences. However, most treatments have limitations that do not produce satisfactory results for cancer patients, such as drug resistance, low specificity, poor efficacy, and/or adverse side effects such as pain, anemia, bleeding, lymphedema, diarrhea, constipation, fatigue, loss of appetite, infection, neuropathy, and memory problems.

CEACAM6 (also known as CD66c) is a member of the carcinoembryonic antigen (CEA) family. It is known that nearly 70% of solid tumors (including colorectal cancer, gastric cancer, endometrial cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, lung cancer and head and neck squamous cell carcinoma) overexpress CEACAM6, and CEACAM6 promotes cancer progression by inducing epithelial-mesenchymal transition (EMT). Based on the importance of CEACAM6 in cancer formation and progression, it can be used as a target for cancer treatment. However, the efficacy of anti-CEACAM6 agents in cancer has not yet been confirmed and needs to be verified experimentally.

In view of this, the relevant field is in urgent need of a novel drug and/or method for treating cancer (especially cancer expressing CEACAM6).

The content of the invention is intended to provide a simplified summary of the present disclosure so that readers can have a basic understanding of the present disclosure. This content of the invention is not a complete overview of the present disclosure, and it is not intended to point out the important/key elements of the embodiments of the present invention or to define the scope of the present invention.

The first aspect of the present disclosure is about a recombinant antibody or a fragment thereof. According to an embodiment of the present disclosure, the recombinant antibody comprises a variable heavy chain (VHH) domain and an immunoglobulin crystallizable fragment region (Fc region), wherein the Fc region is fused with the VHH domain, and the VHH domain comprises a first complementarity determining region (CDR-1), a second CDR (CDR-2) and a third CDR (CDR-3). According to certain embodiments of the present disclosure, the first, second and third CDRs comprise the amino acid sequences of sequence numbers: 1, 2 and 3, respectively.

According to certain preferred embodiments, the VHH domain comprises an amino acid sequence having at least 85% sequence similarity to SEQ ID NO: 4. In a specific embodiment, the VHH domain comprises an amino acid sequence having 100% sequence similarity to SEQ ID NO: 4.

According to certain embodiments, the VHH domain is a heavy chain antibody derived from a camelid animal.

According to certain embodiments, the immunoglobulin is human immunoglobulin G (IgG) or immunoglobulin A (IgA). In a specific embodiment, the immunoglobulin is human IgG1. According to certain embodiments, the immunoglobulin is mouse IgG or IgA. In an exemplary embodiment, the immunoglobulin is mouse IgG2.

The second aspect of the present disclosure is a pharmaceutical composition comprising the recombinant antibody or antibody fragment of the present disclosure and a pharmaceutically acceptable excipient.

The present disclosure also relates to a method for treating cancer in a subject. The method of the present invention comprises administering to the subject an effective amount of a recombinant antibody or antibody fragment of the present invention.

Depending on the desired purpose, the cancer can be any cancer that expresses CEACAM6; for example, gastric cancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer, kidney cancer, colorectal cancer, cervical cancer, ovarian cancer, brain tumor, prostate cancer, liver cancer, melanoma, esophageal cancer, multiple myeloma, or head and neck squamous cell carcinoma.

Optionally, the method of the present invention further comprises administering an effective amount of an anticancer agent to the individual before, simultaneously with, or after administering the recombinant antibody or antibody fragment of the present invention. According to certain embodiments, the anticancer agent is cetuximab.

The individual is a mammal; preferably a human.

After reading the following implementation methods, a person with ordinary knowledge in the technical field to which the present invention belongs can easily understand the basic spirit and other invention purposes of the present invention, as well as the technical means and implementation modes adopted by the present invention.

In order to make the description of the disclosure more detailed and complete, the following provides an illustrative description of the implementation and specific embodiments of the present invention; however, this is not the only form of implementing or using the specific embodiments of the present invention. The implementation covers the features of multiple specific embodiments and the method steps and their sequence for constructing and operating these specific embodiments. However, other specific embodiments can also be used to achieve the same or equal functions and step sequences.

I. Definition

Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate, the relevant numerical values in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Here, "about" generally refers to the actual value within plus or minus 10%, 5%, 1% or 0.5% of a particular value or range. Alternatively, the word "about" means that the actual value falls within the acceptable standard error of the mean, depending on the consideration of a person with ordinary knowledge in the technical field to which the present invention belongs. Except for the experimental examples, or unless otherwise expressly stated, it is understood that all ranges, quantities, values and percentages used herein (for example, to describe the amount of material used, the length of time, temperature, operating conditions, quantity ratios and other similar persons) are modified by "about". Therefore, unless otherwise stated, the numerical parameters disclosed in this specification and the attached patent application are approximate values and can be changed as needed. At least these numerical parameters should be understood as the indicated significant digits and the values obtained by applying the general rounding method. Herein, the numerical range is expressed from one end point to another or between two end points; unless otherwise stated, the numerical range described herein includes the end points.

Unless otherwise defined in this specification, the scientific and technical terms used herein have the same meanings as those understood and used by persons of ordinary skill in the art to which the present invention belongs. In addition, singular terms used in this specification include plural forms of the terms, and plural terms also include singular forms of the terms, unless otherwise conflicting with the context.

The term "antibody" in this disclosure refers to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multi- or multivalent antibodies (e.g., bi-antibodies), chimeric antibodies, heavy chain antibodies (HCAbs), and antibody fragments that can produce specific biological activities. The term "antibody fragment" in this disclosure includes a portion of a full-length antibody, which is usually the antigen-binding site or variable region (e.g., VH and/or VL domain) in the full-length antibody. Exemplary antibody fragments include VHH domains (also known as single domain antibodies (sdAb) or nanobodies (Nb)), VHH dimers, fragment antigen-binding (Fab), Fab', F(ab')2, single-chain variable fragments (scFv), bi-chain antibodies (diabody), linear antibodies (linear antibody), single-chain antibody molecules (single-chain antibody molecules) and multi-antibodies formed by antibody fragments. According to certain embodiments of the present disclosure, the term "antibody fragment" refers to the VHH domain or VHH dimer of HCAb.

In the present disclosure, the term "heavy-chain antibody" (HCAb) refers to an antibody composed of two heavy chains without light chains (for example, see Hamers-Casterman, et al. Nature. 1993; 363: 446-448). Specifically, HCAb is composed of two heavy chains connected by a covalent disulfide bond, wherein each heavy chain has a variable domain at one end. In order to distinguish it from the variable domain (VH domain) of the heavy chain of a conventional antibody (having two heavy chains and two light chains), the variable domain of the heavy chain of HCAb is referred to as the "VHH domain". HCAbs naturally present in camels and sharks can bind to antigens through the VHH domain.

In the present disclosure, the "VHH domain" of a HCAb refers to the amino-terminal region of the antibody heavy chain. These regions are usually the most variable parts of the antibody and contain the antigen binding site. The word "variable" refers to the fact that certain parts of the VHH domain have extensive sequence differences between antibodies and are used for the binding and specificity of each specific antibody to its specific antigen. However, the variability is not evenly distributed throughout the variable domain of the antibody. It is mainly concentrated in the three CDRs or highly variable regions in the VHH domain. The more highly conserved parts of the VHH domain are called the framework region (FR). The natural heavy chain VHH domain contains four FRs, which mostly adopt a β-sheet configuration, connected by three CDRs that form a loop and in some cases form part of the β-sheet structure. The CDRs in each antibody chain are held together in close proximity by the FRs to form the antigen-binding site of the antibody. The constant domain is not directly involved in the binding of the antibody to the antigen, but has different effects, such as the role of the antibody in antibody-dependent cellular toxicity.

"Complementarity determining region" (CDR) in the present disclosure refers to the hypervariable region of the VHH domain that can form a complementary surface with the three-dimensional surface of the antigen-binding domain. From the N-terminus to the C-terminus, each VHH of the antibody contains three CDRs (i.e., CDR-1, CDR-2, and CDR-3), which define the binding affinity and specificity of the VHH domain.

In the present disclosure, "fragment crystallizable region" or "Fc region" refers to the terminal region of an antibody that interacts with cell surface receptors called Fc receptors and/or certain proteins in the complement system. Structurally, the Fc region includes at least a hinge region (a short sequence of the heavy chain connecting the 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) from the N-terminus to the C-terminus. For example, the Fc domain of the IgG1 antibody can be obtained by decomposing the IgG1 antibody with papain.

The "Percentage (%) sequence identity" for amino acid sequences herein refers to the percentage of amino acid residues of the candidate sequence that are exactly the same as the amino acid residues of the reference sequence; when performing the above-mentioned comparison, the candidate sequence and the reference sequence can be placed side by side, and a gap can be introduced when necessary so that the two sequences form the highest sequence similarity; when calculating the similarity, conservatively substituted amino acid residues are regarded as different residues. There are many methods in the relevant field that can be used to perform the above-mentioned alignment, such as publicly available software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). When performing the alignment, a person with ordinary knowledge in the technical field to which the present invention belongs can select appropriate parameters and calculation methods to obtain the best arrangement. In this specification, the sequence comparison between two polypeptide sequences is performed using the protein-protein BLAST analysis database Blastp provided by the National Center for Biotechnology Information (NCBI). The amino acid sequence similarity between candidate sequence A and reference sequence B (also referred to as the amino acid sequence similarity between sequence A and sequence B with a specific percentage (%) in this specification) is calculated as follows: %, where X is the number of identical amino acid residues obtained by aligning sequences A and B using the BLAST sequence alignment program, and Y is the total number of amino acid residues in the shorter of the two sequences A and B.

The disclosed content and claimed inventive concepts also include minor variations in the amino acid sequence of the antibody, wherein the variation in the amino acid sequence maintains at least 85% sequence similarity, for example, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity. The properties of the antibody can be changed by specific modifications without affecting its physiological activity. For example, certain amino acids can be changed and/or deleted without affecting the physiological activity of the antibody of the present invention (i.e., the ability to bind to CEACAM6). In particular, conservative amino acid substitutions are also included. Conservative substitutions are mutual substitutions between amino acids with similar/related side chains. Generally speaking, the amino acids encoded by genes can be divided into four major categories: (1) Acidic amino acids, namely aspartate and glutamate; (2) basic amino acids, namely lysine, arginine, and histidine; (3) non-polar amino acids, namely alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) non-charged polar amino acids, namely glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. A better classification is that serine and threonine are aliphatic-hydroxy, aspartate and glutamine are amide-containing, alanine, valine, leucine, and isoleucine are aliphatic, and phenylalanine, tryptophan, and tyrosine are aromatic. For example, it is conceivable that substitution of isoleucine or valine for leucine, glutamine for aspartate, serine for threonine, or one structurally similar amino acid for another will not result in a significant change in molecular binding or protein properties, especially when the substitution is not in the framework region, and substitutions between amino acids will not affect the above properties. By detecting the specific activity of the antibody derivative, it can be understood whether the change of an amino acid can form a functional antibody. Antibody fragments or analogs can be prepared by methods known to those of ordinary skill in the art to which the present invention belongs. The amino and carboxyl termini of the fragments or analogs are preferably adjacent to the boundaries of the functional domain.

The term "subject" in this disclosure refers to mammals including humans that can be treated with the recombinant antibodies, pharmaceutical compositions and/or methods of the present invention. Unless otherwise specified, the term "subject" refers to both males and females.

II. Invention Details

The present disclosure relates to a recombinant antibody specific for CEACAM6 and the use of the recombinant antibody in treating cancer, particularly cancer having CEACAM6 overexpressed thereon and/or therein.

Therefore, the first aspect of the present disclosure is about a recombinant antibody or a fragment thereof (e.g., a VHH domain). First, refer to Figure 1, which is a schematic diagram drawn according to certain embodiments of the present disclosure, for illustrating the recombinant antibody of the present invention. Structurally, the recombinant antibody of the present invention comprises a pair of VHH domains, and an Fc region of an immunoglobulin connected to the pair of VHH domains. As known to those skilled in the art to which the present invention belongs, the Fc region comprises two identical protein fragments connected to each other by disulfide bonds, wherein each protein fragment comprises a hinge region, a CH2 domain, and a CH3 domain from the N-terminus to the C-terminus. The pair of VHH domains are connected to the N-terminus of the protein fragment. As shown in FIG. 1 , the recombinant antibody of the present invention is in the form of HCAb (i.e., a VHH-Fc homodimeric complex), which comprises two heavy chains connected by a disulfide bond in the hinge region, wherein each heavy chain comprises a VHH domain, a hinge region, a CH2 domain and a CH3 domain from the N-terminus to the C-terminus.

Alternatively, the pair of VHH domains may be linked to the N-terminus of the protein fragment via a linker. In this case, the recombinant antibody of the present invention is in the form of HCAb (i.e., VHH-linker-Fc homodimer complex), which comprises two heavy chains connected by a disulfide bond in the hinge region, wherein each heavy chain comprises a VHH domain, a linker, a hinge region, a CH2 domain and a CH3 domain from the N-terminus to the C-terminus.

It is known that the binding properties of an antibody (including binding affinity and specificity) depend on its variable domain (i.e., the VH and VL domains of a general antibody, or the VHH domain of an HCAb); more specifically, on the CDR sequence in the variable domain (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of a general antibody; or CDR-1, CDR-2, and CDR-3 of an HCAb). According to certain embodiments of the present disclosure, each VHH domain of a recombinant antibody comprises three CDRs (i.e., CDR-1, CDR-2, and CDR-3), wherein CDR-1 comprises the amino acid sequence of sequence number: 1, CDR-2 comprises the amino acid sequence of sequence number: 2, and CDR-3 comprises the amino acid sequence of sequence number: 3.

According to certain embodiments, each VHH domain of the recombinant antibody comprises an amino acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence similarity to SEQ ID NO: 4. It is contemplated that the sequence of the VHH domain (e.g., the framework sequence) may be varied (e.g., substituted with conservative or non-conservative amino acid residues) without affecting the binding affinity and/or specificity of the antibody of the invention. Preferably, the sequence of the VHH domain is conservatively replaced with one or more amino acid residues having similar properties; for example, leucine (another non-polar amino acid residue) is replaced with isoleucine, alanine, valine, proline, phenylalanine or tryptophan (a non-polar amino acid residue); aspartic acid (another acidic amino acid residue) is replaced with glutamine (an acidic amino acid residue); or lysine (another basic amino acid residue) is replaced with arginine or histidine (a basic amino acid residue). According to certain preferred embodiments, the VHH domain of the recombinant antibody comprises an amino acid sequence having at least 90% sequence similarity to SEQ ID NO:4. More preferably, the VHH domain of the recombinant antibody comprises an amino acid sequence having at least 95% sequence similarity to SEQ ID NO: 4. In one working embodiment, the VHH domain of the recombinant antibody has the amino acid sequence of SEQ ID NO: 4 (i.e., its amino acid sequence has 100% sequence similarity to SEQ ID NO: 4).

Depending on the desired purpose, the immunoglobulin can be human immunoglobulin G1 (IgG1), immunoglobulin G2 (IgG2), immunoglobulin G3 (IgG3), immunoglobulin G4 (IgG4) or immunoglobulin A (IgA). According to certain exemplary embodiments, the immunoglobulin is human IgG1. In a specific embodiment, the pair of VHH domains are linked to the N-terminus of the Fc region of human IgG1, wherein each recombinant chain of the recombinant antibody of the present invention comprises the amino acid sequence of SEQ ID NO: 5.

Alternatively, the immunoglobulin may be mouse IgG1, IgG2, IgG3 or IgA. According to certain embodiments, the immunoglobulin is mouse IgG2. In a specific embodiment, the pair of VHH domains are linked to the N-terminus of the Fc region of mouse IgG2, wherein each recombinant chain of the recombinant antibody of the present invention comprises the amino acid sequence of SEQ ID NO: 6.

According to certain embodiments of the present disclosure, recombinant antibodies are prepared using ribosome display and DNA cloning techniques.

Ribosome display is a cell-free system for screening proteins and peptides from a large protein library in vitro. Methods for preparing ribosome display systems are well known to those skilled in the art. Generally, a ribosome display system is established by immunizing a host animal (e.g., a camel, preferably an alpaca) with a peptide (e.g., a CEACAM6 polypeptide) according to a commonly used process; for example, the host animal is immunized with the peptide once every two to three weeks until the desired antibody titer is generated. After the last immunization injection, peripheral lymphocytes are collected from the immunized animal and total RNA is isolated therefrom. After synthesizing the corresponding cDNA and amplifying the cDNA fragment encoding the VHH domain, the PCR product is transferred to the expression vector to construct a cDNA gene library. The cDNA encoding VHH can be transcribed into mRNA and protein in the form of ribosome display (i.e., protein-ribosome-mRNA (PRM) complex). In the screening stage (also called "biopanning stage"), after adding the PRM complex to the immobilized ligand (e.g., immobilized CEACAM6 polypeptide), the complex with binding affinity to the immobilized ligand is washed out using salt concentration, chelating agent or mobile ligand (which can form a complex with the binding domain of the protein to separate the mRNA). The mRNA can then be reverse transcribed into cDNA for the next round of screening. The screening stage can be repeated several times (for example, 3-5 times) to obtain cDNA encoding a VHH domain that has binding specificity for a peptide (eg, CEACAM6 polypeptide).

According to certain embodiments of the present disclosure, the obtained cDNA is then transferred to an expression vector (e.g., an IgG1 vector) comprising the Fc region of a human immunoglobulin. After being introduced into appropriate host cells, the expression vector can express recombinant antibodies in the host cells; the produced recombinant antibodies structurally comprise an Fc region derived from humans and a VHH domain derived from camelids linked to the N-terminus of the Fc region. The host cell is preferably a mammalian cell; for example, a Chinese hamster ovary (CHO) cell, or an embryonic kidney 293 cell. According to a preferred embodiment, the host cell is an embryonic kidney 293 cell.

Alternatively, the recombinant antibodies of the present invention can be prepared directly by DNA cloning techniques. The DNA encoding the recombinant antibodies of the present invention can be isolated and sequenced by conventional procedures (e.g., using oligonucleotide probes that can specifically bind to the genes encoding the light chain and heavy chain of mAb). The isolated DNA is then placed into an expression vector, and the expression vector is transfected into the above-mentioned appropriate host cells to synthesize the desired antibody in the host cells.

The recombinant antibody can be purified according to standard procedures in the art to which the present invention pertains (e.g., cross-flow filtration, affinity column chromatography, and gel filtration, etc.).

Depending on the desired purpose, the recombinant antibody of the present disclosure can be conjugated to a drug (e.g., an anticancer agent) (i.e., in the form of an antibody-drug conjugate (ADC)) or to a therapeutic agent (e.g., a therapeutic peptide) to improve its therapeutic efficacy.

According to certain embodiments of the present disclosure, administration of recombinant antibodies can inhibit tumor growth. Therefore, the present disclosure also relates to a pharmaceutical composition or drug for treating cancer. The pharmaceutical composition or drug comprises an effective amount of a recombinant antibody or a fragment thereof of the present disclosure; and optionally, a pharmaceutically acceptable excipient.

Generally speaking, the weight of the recombinant antibody or antibody fragment of the present invention is about 0.1% to 99% of the total weight of the pharmaceutical composition or drug. In certain embodiments, the weight of the recombinant antibody or antibody fragment of the present invention is at least 1% of the total weight of the pharmaceutical composition or drug. In certain embodiments, the weight of the recombinant antibody or antibody fragment of the present invention is at least 5% of the total weight of the pharmaceutical composition or drug. In certain embodiments, the weight of the recombinant antibody or antibody fragment of the present invention is at least 10% of the total weight of the pharmaceutical composition or drug. In other embodiments, the weight of the recombinant antibody or antibody fragment of the present invention is at least 25% of the total weight of the pharmaceutical composition or drug.

The pharmaceutical compositions or medicaments of the present invention may be prepared according to acceptable pharmaceutical manufacturing methods, such as those described in Remington's Pharmaceutical Sciences, ^17th edition, ed. Alfonoso R. Gennaro, Mack Publishing Company, Easton, Pa (1985). Pharmaceutically acceptable formulations are those that are compatible with the other ingredients of the dosage form and are acceptable to the organism.

The pharmaceutical composition or drug of the present invention can be formulated into solid, semisolid or liquid forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories and injections. Accordingly, the recombinant antibody or antibody fragment of the present invention can be administered by different routes such as oral, buccal, rectal, parenteral, intravenous injection and intraperitoneal injection. In the form of a dosage form, the recombinant antibody or antibody fragment of the present invention can be administered alone or in combination with other pharmaceutically active agents known to treat cancer. A person with ordinary knowledge in the art to which the present invention belongs can select an appropriate dosage form according to the different administration routes. The optimal administration route varies depending on the nature or severity of the disease or condition.

Preferably, the pharmaceutical composition or medicament of the present disclosure is formulated in a liquid form in the form of a sterile solution or suspension, which can be injected into a subject intravenously, intraarterially, intramuscularly, subcutaneously, intraspinally, intraperitoneally or intratumorally. The pharmaceutical composition or medicament can be formulated as an oily or aqueous isotonic suspension, solution or emulsion and can contain prescription drugs, such as suspension, stabilization or dispersion agents. Alternatively, the pharmaceutical composition or medicament can be prepared in a dry form, such as a powder, crystal or freeze-dried solid, and accompanied by water or isotonic saline that is sterile and pyrogen-free before use. The composition can also be placed in a sterile ampoule or vial.

When the recombinant antibody or antibody fragment of the present invention is formulated into a dosage form such as intravenous, percutaneous or subcutaneous injection, the antibody or antibody fragment can be prepared into a non-pyrogenic non-orally acceptable liquid solution. Taking into account factors such as pH, isotonicity and stability, those with ordinary knowledge in the art to which the present invention belongs know how to prepare the non-orally acceptable liquid solution. In addition to the recombinant antibody or antibody fragment of the present invention, a preferred pharmaceutical composition or drug for intravenous, percutaneous or subcutaneous injection should include an isotonic formulation, such as sodium chloride injection, Ringer's injection, glucose injection, glucose and sodium chloride injection, lactated Ringer's injection or other known formulations. The pharmaceutical composition or drug of the present invention may also contain stabilizers, preservatives, buffers, antioxidants or other additives known to those of ordinary skill in the art to which the present invention belongs. The interval between intravenous administration of the pharmaceutical composition or drug of the present disclosure will vary with the severity of the disease and the condition and potential specific reactions of each individual. In the case of continuous intravenous administration, the interval between each administration of the monoclonal antibody of the present invention may be 12 to 24 hours. The final course of intravenous treatment can be determined by medical personnel.

Another aspect of the present disclosure is a method for treating cancer in an individual, comprising administering to the individual an effective amount of the recombinant antibody, antibody fragment, pharmaceutical composition or agent of the present invention.

The effective amount administered to an individual is about 0.01 to 1,000 mg per kilogram of 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, 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 1, 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; preferably, about 0.1 to 100 mg per kg of individual body weight. The dose may be administered in a single aliquot or in multiple aliquots. A skilled person or clinician may adjust the dose or course of treatment according to the patient's physiological condition or the severity of the disease.

According to certain embodiments of the present disclosure, administration of a recombinant antibody can inhibit the growth of a tumor in an individual by reducing epidermal growth factor receptor (EGFR) signaling and the ability of cancer/tumor cells to self-renew and invade.

Optionally, the method of the present invention further comprises administering to the subject an effective amount of an anticancer agent before, simultaneously with, or after administering the recombinant antibody or antibody fragment of the present invention. Exemplary anticancer agents include, but are not limited to: alkylating agents (agents that inhibit cell replication by damaging DNA; for example, altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifos famide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa, and trabectedin); antimetabolites (agents that interfere with DNA and RNA by replacing the normal building blocks of RNA and DNA; for example, 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 in the cell cycle that are involved in DNA replication; for example, daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, bleomycin, dactinomycin, mitomycin-C mitomycin-C and mitoxantrone); mitotic inhibitors (drugs derived from natural products that stop cells from dividing into new cells; for example, cabazitaxel, docetaxel, nab-paclitaxel, paclitaxel, vinblastine, vincristine, and vinorelbine); corticosteroids (natural hormones or hormone-like drugs; for example, prednisone, methylprednisolone, and dexamethasone); and antibodies (e.g., rituximab, trastuzumab, gemtuzumab, alemtuzumab, tositumomab, cetuximab, ibritumomab, bevacizumab, panitumumab, catumaxomab, ofatumumab, ipilimumab, and brentuximab). According to certain preferred embodiments, the anticancer agent is an anti-EGFR antibody, such as cetuximab, panitumumab, nimotuzumab, necitumumab, duligotuzumab, depatuxizumab, matuzumab or zalutumumab. According to a specific embodiment, the anticancer agent is cetuximab. In this embodiment, administration of the recombinant antibody and cetuximab can additively or synergistically inhibit tumor growth by reducing EGFR signaling and the ability of cancer/tumor cells to self-renew and invade.

The subject that can be treated by the method of the present invention is a mammal; for example, humans, mice, rats, guinea pigs, hamsters, monkeys, pigs, dogs, cats, horses, sheep, goats, cows and rabbits. Preferably, the subject is a human.

The recombinant antibodies or antibody fragments disclosed herein can be administered to an individual via an appropriate route, such as oral, enteral, nasal, topical, mucosal or parenteral administration. Depending on the purpose of use, parenteral administration can be intratumoral, intramuscular, intravenous or intraperitoneal injection.

The following are several experimental examples to illustrate certain aspects of the present invention, so as to facilitate those with ordinary knowledge in the technical field to which the present invention belongs to implement the present invention, and these experimental examples should not be regarded as limiting the scope of the present invention. It is believed that after reading the description provided here, the skilled person can fully utilize and practice the present invention without over-interpretation. All public documents cited here are regarded as part of this specification in their entirety.

Materials and Methods

Cell culture

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

Preparation of recombinant antibodies ( Hereinafter referred to as " HCAb " )

HCAbs were expressed and purified using EXPI293F™ and EXPICHO-S™ cells according to the operator manual. Briefly, HCAb constructs were transfected into EXPI293F™ cells using EXPIFECTAMINE™ 293 and EXPIFECTAMINE™ CHO reagents. After cell culture medium was collected, HCAbs were purified using a protein G column.

The prepared HCAb comprises a homodimer of a VHH domain from a camel family fused with an Fc region from a human. According to the analysis results, each VHH domain comprises an amino acid sequence of sequence number: 4, wherein CDR-1, CDR-2 and CDR-3 comprise amino acid sequences of sequence numbers: 1-3, respectively (Table 1); and the Fc region from a human comprises an amino acid sequence of sequence number: 5.

Table 1 Amino acid sequence of HCAb of the present invention Name Amino acid sequence (SEQ ID NO: 4) HCAb QVKLEESGGGLVQAGGSLRLSCAAS GGSFSSYMLA WFRQAPGKEREFVA NISPGGYTYYADSAQG RFTISRENAKNTMYLQMNSLNPEDTAVYYCAA DIRVLAAAQVASYDY WGQGTQVTVSS The CDR sequences in the VHH domain are marked in bold, and are CDR-L1, CDR-L2, and CDR-L3 from N-terminus to C-terminus.

Binding affinity

Surface plasmon resonance (SPR) was used to determine the binding affinity of recombinant HCAb to recombinant CEACAM6. Specifically, the binding kinetics of HCAb to CEACAM6 were analyzed using a BIACORE ^™ machine. Recombinant CEACAM6 protein was immobilized on a sensor chip according to the user manual. Five-fold serial dilutions of HCAb (20, 10, 5, 2.5, and 1 nM) in HBS-P buffer were injected at a rate of 10 μl per minute onto the chip containing 3000 resonance units (RU) of CEACAM6. The resulting sensorgrams were superimposed and the binding kinetics obtained from two independent experiments were averaged and analyzed using software.

Immunohistochemical staining (Immunohistochemistry, IHC)

Immunohistochemical staining was performed to determine the binding affinity and specificity of recombinant HCAbs for tumor and normal tissues. Specifically, in tissue microarrays, 5-μm tissue sections were dewaxed with xylene and rehydrated. Endogenous peroxidase was blocked with 3% hydrogen peroxide, after which HCAbs were added to the slides and incubated at 4°C until the next day. CEACAM6 expression was detected using streptococcal avidin-biotin-peroxidase complex (DAKO), and peroxidase activity was visualized with 3,3’-diaminobenzidine (DAB). Slides were counterstained with hematoxylin and mounted with mounting agent.

Immunoblotting (Immunoblotting)

Cells were scraped with cold mammalian protein extract buffer containing phosphatase and protease inhibitors to collect cell protein lysate. Protein concentration was determined. Approximately 20 μg of protein was separated with 10% sodium dodecyl sulfate polyacrylamide gel and transferred to polyvinylidene difluoride membrane. Primary antibody was diluted with Tris buffer containing 3% BSA, added to the membrane and reacted at 4°C until the next day. The spots were detected using a gel treatment system with horseradish peroxidase (HRP) substrate.

Intrusion and transfer test

Cell invasion and metastasis assays were performed using 24-well transwell polycarbonate filters (8 μm pore size) coated or not with matrigel. Briefly, cancer cells were placed in the upper chamber, and 1% FBS or epidermal growth factor (EGF) was added to the lower chamber. After 24 h of incubation, the number of invasive and metastatic cells was counted. Non-penetrated cells were removed from the upper surface of the filter using a cotton swab. Penetrated cells were fixed and stained using a staining kit according to the user manual. For quantitative analysis, all cells that invaded or metastasized to the lower surface were stained and counted using an optical microscope.

Animal testing

1×10 ⁷ HT-29 cells were mixed with 0.1 ml of Matrigel and injected subcutaneously into 8-week-old severe combined immunodeficient (SCID) male mice. Seven days after cell injection, the mice were divided into four groups and intravenously injected with phosphate buffered saline (PBS), cetuximab (10 mg/kg), HCAb (10 mg/kg), and cetuximab (10 mg/kg) + HCAb (10 mg/kg) once every 7 days for a total of 6 times. Tumor size was measured weekly with a caliper, and tumor volume was calculated using the following formula: (length × ^width2 × 0.45).

Statistical analysis

All statistical analyses were performed using software. Unpaired Student's' t test or one-way ANOVA test was used to analyze the results to compare the results of two or more independent groups. For each analysis result, all data were obtained from three independent experiments and expressed as mean ± SD. *P ＜ 0.05, **P ＜ 0.01, ***P ＜ 0.001, ns indicates that the 95% two-tailed confidence interval is not significantly different.

Embodiment 1 Confirm HCAb

According to the results of IHC staining and SPR analysis, the HCAb of the present invention can recognize glycosylated CEACAM6 protein, and the binding affinity (KD) of the HCAb of the present invention to recombinant CEACAM6 is lower than 1 nM (results not shown). IHC data confirmed that compared with normal tissues (including skin, breast, lymph node, stomach, gastric muscle layer, lung, liver, colon, testis, prostate, kidney, esophagus, cerebellum, brain, adrenal gland, placenta, heart, spleen, skeletal muscle, cauda, salivary gland, gall bladder, pancreas and tonsils) in which no obvious antibody signal was detected, the HCAb of the present invention has binding affinity and specificity for cancerous tissues (including rectal adenocarcinoma, descending colon adenocarcinoma, metastatic colorectal adenocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma and cholangiocarcinoma) (results not shown).

These data indicate that the HCAb of the present invention can specifically target cancer cells.

Embodiment 2 HCAb Effects on inhibiting tumor cells

According to the results of immunoblot and invasion assays, tumor cells overexpress CEACAM6 protein, which promotes tumor progression by interacting with EGFR (results not shown). Reducing the expression of CEACAM6 can significantly inhibit the invasion of tumor cells (results not shown). Compared with the control group, the administration of the HCAb of the present invention can significantly inhibit the phosphorylation level of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), that is, inhibit the activation of the ERK1/2 MAP kinase pathway (results not shown). The results of Figures 2A and 2B further demonstrate that the administration of the HCAb of the present invention can reduce the spheroid formation of tumor cells and the invasion ability of tumor cells. It is noteworthy that co-administration of HCAb of the present invention and cetuximab can produce additive inhibitory effects on tumor cells ( FIG. 2B ).

These data indicate that the HCAb of the present invention can be used as an anticancer agent, which can be administered alone or co-administered with other active agents (eg, cetuximab) to achieve therapeutic purposes.

Embodiment 3 In vivo test

This example will evaluate the inhibitory activity of the HCAb of the present invention on tumor growth in animals. The data in Figure 3A demonstrates that, compared with the PBS control group, administration of HCAb can significantly inhibit tumor growth in animal models.

Cetuximab is a chimeric monoclonal antibody used 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 after 3-12 months of treatment. According to the results of this study, the expression of CEACAM6 was increased in head and neck squamous cell carcinoma (HNSCC) cell lines resistant to cetuximab (results not shown). To evaluate whether the HCAb of the present invention can enhance the therapeutic efficacy of cetuximab for cancer, the HCAb of the present invention and cetuximab were administered to tumor-bearing mice according to the procedures described in Materials and Methods. The data in Figure 3B demonstrate that co-administration of the HCAb of the present invention and cetuximab can significantly inhibit tumor growth compared to the control group. Therefore, the HCAb of the present invention can be used sequentially or simultaneously with a first-line anticancer agent (e.g., cetuximab) to improve the therapeutic efficacy.

In summary, the present disclosure provides a novel anti-CEACAM6 antibody. According to the embodiments of the present disclosure, the anti-CEACAM6 antibody of the present invention has binding affinity and specificity for tumor cells, and can inhibit tumor growth by reducing the expression of CEACAM6 and the activation of the ERK1/2 MAP kinase pathway. Accordingly, the anti-CEACAM6 antibody of the present invention can be used as a potential antibody for treating cancer.

Although the above embodiments disclose specific embodiments of the present invention, they are not intended to limit the present invention. A person having ordinary knowledge in the technical field to which the present invention belongs may make various changes and modifications without departing from the principle and spirit of the present invention. Therefore, the scope of protection of the present invention shall be based on that defined by the scope of the attached patent application.

without

In order to make the above and other purposes, features, advantages and embodiments of the present invention more clearly understood, the attached drawings are described as follows:

FIG. 1 is a schematic diagram according to an embodiment of the present disclosure, which is related to the structure of a recombinant antibody;

Figures 2A and 2B are bar graphs according to Example 2 of the present disclosure, which respectively illustrate the effects of the recombinant antibodies of the present invention on tumor cell metastasis and invasion capabilities; N.S.: not statistically significant, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; and

Figures 3A and 3B are line graphs drawn according to Example 3 of the present disclosure, which respectively illustrate the anti-tumor efficacy of the recombinant antibody of the present invention in an animal model.

According to conventional operation methods, various features and components in the figure are not drawn according to scale, and the drawing method is to present the specific features and components related to the present invention in the best way. In addition, the same or similar component symbols are used to refer to similar components/parts between different figures.

without

TW202434649A_112107050_SEQL.xml

Claims (12)

A recombinant antibody or a fragment thereof, which can bind to carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) and comprises a variable heavy chain (VHH) domain and an immunoglobulin crystallizable fragment region (Fc region), wherein the Fc region is fused with the VHH domain, and the VHH domain comprises: a first complementarity determining region (CDR-1), which comprises the amino acid sequence of sequence number: 1; a second CDR (CDR-2), which comprises the amino acid sequence of sequence number: 2; and a third CDR (CDR-3), which comprises the amino acid sequence of sequence number: 3. A recombinant antibody or a fragment thereof as described in claim 1, wherein the VHH domain comprises an amino acid sequence having at least 85% sequence similarity to sequence number: 4. A recombinant antibody or a fragment thereof as described in claim 2, wherein the VHH domain comprises an amino acid sequence having 100% sequence similarity with sequence number: 4. The recombinant antibody or fragment thereof as described in claim 1, wherein the VHH domain is a heavy chain antibody derived from a camel family animal. The recombinant antibody or fragment thereof as described in claim 1, wherein the immunoglobulin is human immunoglobulin G (IgG) or immunoglobulin A (IgA). The recombinant antibody or fragment thereof as described in claim 5, wherein the immunoglobulin is IgG1. A pharmaceutical composition comprising a recombinant antibody or a fragment thereof as described in claim 1, and a pharmaceutically acceptable formulation. A use of a recombinant antibody or a fragment thereof as described in claim 1 in preparing a drug, wherein the drug is used to treat cancer in an individual. The use as described in 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 tumor, prostate cancer, liver cancer, melanoma, esophageal cancer, multiple myeloma or head and neck squamous cell carcinoma. The use as described in claim 8, wherein the drug comprises an anticancer agent. The use as described in claim 10, wherein the anticancer agent is cetuximab. The use as described in claim 8, wherein the individual is a human.