Immunoconjugate, pharmaceutical composition comprising the same, and uses thereof in treating cancers

Abstract

This document discloses an immune conjugate comprising a recombinant antibody, a therapeutic agent, and a linker for linking the therapeutic agent to the recombinant antibody. According to some embodiments disclosed herein, the recombinant antibody comprises a VHH domain, wherein CDR-1, CDR-2, and CDR-3 of the VHH domain comprise the amino acid sequences of SEQ ID NO: 1-3 and 7-9, respectively. This document also discloses pharmaceutical compositions comprising the immune conjugate and methods for treating cancer using the immune conjugate or pharmaceutical composition of the present invention.

Description

Technical Field

[0001] This disclosure generally pertains to the field of disease treatment. More specifically, this disclosure relates to immune conjugates, pharmaceutical compositions comprising such immune conjugates, and their use in the treatment of cancer. Prior Technology

[0002] Cancer is a group of diseases involving abnormal cell growth that can invade or metastasize to healthy organs or tissues in affected individuals. It is a leading cause of death worldwide. According to the World Health Organization (WHO), cancer caused nearly 10 million deaths in 2020, or about one-sixth of all deaths. The most common types of cancer include lung cancer, breast cancer, prostate cancer, colorectal cancer, stomach cancer, skin cancer, and liver cancer. Major treatments for cancer include surgery, chemotherapy, radiation therapy, hormone therapy, and targeted therapy. However, due to limitations such as drug resistance, low specificity, low efficacy, and / or adverse side effects such as pain, anemia, bleeding, lymphedema, diarrhea, constipation, fatigue, loss of appetite, infection, neuropathy, and memory problems, most of these treatments do not produce satisfactory results for cancer patients.

[0003] In light of the foregoing, there is ongoing interest in developing novel drugs and / or methods for treating cancer in a more efficient and safer manner. Summary of the Invention

[0004] The following is a brief overview of this disclosure to provide the reader with a basic understanding. This overview is not an exhaustive review of this disclosure, nor does it identify key / essential elements of the invention or define its scope. Its sole purpose is to present some of the concepts disclosed herein in a simplified form as an introduction to the more detailed description that will follow.

[0005] As embodied and broadly described herein, a first aspect of this disclosure relates to an immune conjugate comprising a recombinant antibody or a single-domain antibody (sdAb) and a therapeutic agent, wherein the therapeutic agent is linked to the recombinant antibody or the sdAb, as appropriate, via a linker. According to certain embodiments of this disclosure, the recombinant antibody comprises (i) a single variable heavy chain domain (VHH domain) or a pair of homodimeric or heterodimeric VHHs and crystallizable region fragments (Fc regions) of an immunoglobulin, the Fc region being fused to each of the VHH domains, as appropriate, via a peptide linker. In some embodiments, the therapeutic agent is linked to the Fc region of the recombinant antibody via the linker.

[0006] According to some embodiments disclosed herein, the VHH domain of the recombinant antibody includes a first chain complementarity-determining region (CDR-1), a second CDR (CDR-2), and a third CDR (CDR-3), which respectively contain an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO: 1 or 7, SEQ ID NO: 2 or 8, and SEQ ID NO: 3 or 9.

[0007] According to some preferred embodiments, the VHH domain contains an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4, 10, or 11. In one particular embodiment, the VHH domain contains an amino acid sequence that is 100% identical to SEQ ID NO: 4, 10, or 11.

[0008] According to some embodiments, the VHH domain is derived from camel heavy chain antibodies.

[0009] According to some embodiments, the antibody system is in a chimeric, humanized, or human form. According to another specific embodiment, the sdAb system is in a chimeric form.

[0010] According to some embodiments, the immunoglobulin is human immunoglobulin G (IgG) or immunoglobulin A (IgA). In one particular 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.

[0011] According to some embodiments, the VHH domain is fused to the N-terminus of the Fc region of an immunoglobulin via a peptide linker, wherein the peptide linker comprises short peptides of Gly and Ser.

[0012] Depending on the desired purpose, the therapeutic agent may be a cytotoxic drug, a radioactive nucleus, a cytokine, a hormonal drug, an immunostimulant, or an immunotherapeutic agent. Preferably, the therapeutic agent is a cytotoxic drug. According to some embodiments of this disclosure, the therapeutic agent is auristatin, irinotecan, exatecan, levofolinate, 5-fluorouracil, or a derivative thereof. In one exemplary embodiment, the therapeutic agent is monomethylauristatin E (MMAE). In another exemplary embodiment, the therapeutic agent is monomethylauristatin F (MMAF).

[0013] This article also discloses a pharmaceutical composition for the treatment of cancer. The pharmaceutical composition comprises the disclosed immune conjugate and, where appropriate, a medically acceptable carrier.

[0014] Another aspect of this disclosure relates to a method for treating cancer in an individual. This method involves administering to the individual an effective amount of the disclosed immune conjugate or pharmaceutical composition.

[0015] Examples of cancers that can be treated by the method of the present invention include (but are not limited to) stomach cancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer, kidney cancer, colorectal cancer, cervical cancer, ovarian cancer, brain tumor, prostate cancer, hepatocellular carcinoma, melanoma, esophageal cancer, multiple myeloma, head and neck squamous cell carcinoma, or bile duct cancer.

[0016] This system is for mammals; humans are preferred.

[0017] The numerous incidental features and advantages of this disclosure will be better understood by referring to the following detailed description taken in conjunction with the accompanying drawings. Simple Explanation of the Diagram

[0018] This specification will be better understood by reading the following detailed description with reference to the accompanying drawings, which provide a brief overview of the subject.

[0019] Figure 1A is a schematic diagram depicting the structure of the recombinant antibody in the antibody-drug conjugate (ADC) of the present invention according to an embodiment of the present disclosure.

[0020] Figure 1B is a schematic diagram depicting the structure of the ADC of the present invention according to an embodiment of the present disclosure. A: Payload, i.e., PEG3-VC-MMAE.

[0021] Figures 2A and 2B respectively depict the quality analysis results of the ADC of the present invention according to some embodiments of this disclosure. Figure 2A: Full form of the ADC; Figure 2B: Reduced form of the ADC.

[0022] Figure 3 depicts the results of flow cytometry analysis according to Example 1 of this disclosure.

[0023] Figure 4 is a photograph depicting the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Example 2 of this disclosure.

[0024] Figures 5A to 5C are line graphs depicting the effect of the ADC of the present invention on designated cells according to Example 2 of this disclosure.

[0025] Figures 6A and 6B are line graphs depicting the tumor volume (Figure 6A) and body weight (Figure 6B) of tumor-bearing mice treated with the ADC of the present invention or the specified treatment according to Example 3 of this disclosure. The arrows in Figure 6A indicate the ADC treatment days (a total of 6 administrations).

[0026] Figure 7 shows the antitumor effect of chimeric ADCs containing ethathecan.

[0027] Figure 8 shows the body weight of mice after administration of the chimeric ADC containing ethathecan. Figure 9 shows that the chimeric HCAb has low affinity for normal tissues.

[0028] Figure 10 shows that chimeric HCAb specifically recognizes colorectal cancer, lung cancer, bile duct cancer, and pancreatic cancer tissues.

[0029] Figure 11 shows that humanized ADCs significantly inhibited cancer cell growth.

[0030] Figure 12 shows that humanized HCAb-ADC inhibits tumor growth in an animal model in a dose-dependent manner.

[0031] Figure 13 shows that no significant weight loss was observed after humanized HCAb-ADC treatment.

[0032] Figure 14 shows that sdAb-ADC significantly inhibits cancer cell growth.

[0033] Figure 15 shows that administration of sdAb-ADC inhibited tumor growth in an animal model.

[0034] Figure 16 shows that sdAb-ADC did not significantly induce weight loss in the animal model.

[0035] By convention, the various features / elements described are not drawn to scale, but are instead drawn to best illustrate the specific features / elements relevant to the present invention. Furthermore, the element symbols and names in the various figures are used to indicate elements / components. Implementation

[0036] The following detailed description, provided in conjunction with the accompanying drawings, is intended to describe examples of the invention and is not intended to represent the only form in which the examples of the invention can be constructed or utilized. This specification sets forth the functions of the examples and the sequence of steps for constructing and operating the examples. However, the same or equivalent functions and sequences can be achieved by different examples.

[0037] I. Definition

[0038] For convenience, certain terms used in the specification, examples, and appended claims are collected herein. Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have the meanings commonly understood and used by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, it shall be understood that singular terms shall include their plural forms and plural terms shall include their singular forms. Specifically, unless the context otherwise clearly indicates, as used herein and in the claims, the singular forms “a(a)” and “an(an)” include the plural forms. Moreover, 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.

[0039] In this application, the phrase "and / or" is a grammatical conjunction and should be interpreted as meaning that one or more related circumstances may occur.

[0040] While the numerical ranges and parameters describing the broad scope of this invention are approximate, the numerical values ​​described in specific examples should be reported as accurately as possible. However, any numerical value inherently contains some error, which is necessarily caused by the standard deviation found in individual test measurements. Furthermore, as used herein, the term "about" generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, when considered, the term "about" means within an acceptable standard error of the average value, as generally understood by one skilled in the art. Except in operational / working examples, or unless otherwise expressly stated, all numerical ranges, quantities, values, and percentages, such as the quantities of material, durations, temperatures, operating conditions, ratios of quantities, and the like disclosed herein, should be understood to be modified by the term "about" in all cases. Therefore, unless indicated to the contrary, the numerical parameters described in this disclosure and the appended claims are approximate values ​​that may vary as needed. At a minimum, each numerical parameter should be understood at least according to the number of significant digits reported and by applying common rounding techniques.

[0041] The term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific or multivalent antibodies (e.g., bispecific antibodies), chimeric antibodies, heavy chain antibodies (HCAbs), and antibody fragments, as long as they exhibit the desired biological activity. The term "antibody fragment" refers to a portion of a full-length antibody, generally the antigen-binding domain or variable domain (i.e., the VH domain and / or VL domain) of the full-length antibody. Examples of antibody fragments include VHH domains (also known as single-domain antibodies (sdAbs) or nano-antibodies (Nb)), VHH dimers, antigen-binding fragments (Fab), Fab', F(ab')2, single-chain variable fragments (scFv), bifunctional antibodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0042] As used herein, the term "recombinant antibody" refers to an antibody expressed and isolated from a cell or cell line transfected with an expression vector containing the coding sequence of the antibody, wherein the coding sequence is not naturally associated with the cell.

[0043] As used herein, the term "heavy chain antibody" (HCAb) refers to an antibody consisting of two heavy chains and containing no light chains (see, for example, Hamers-Casterman et al., Nature. 1993; 363: 446-448). Specifically, an HCAb consists of two heavy chains linked by covalent disulfide bonds, each heavy chain having a variable domain at one end. This heavy chain variable domain of an HCAb is called the "VHH domain" to distinguish it from the heavy chain variable domain (VH domain) of "conventional" antibodies that have two heavy chains and two light chains. HCAbs, naturally occurring in camels and sharks, bind antigens via their VHH domains.

[0044] As used herein, the “VHH domain” of the term HCAb refers to the amino-terminal region of the antibody heavy chain. These regions are generally the most variable parts of the antibody and contain antigen-binding sites. The term “variable” refers to the fact that certain portions of the VHH domain vary significantly in sequence between antibodies and are used for the binding and specificity of each particular antibody to its specific antigen. However, variability is not uniformly distributed throughout the entire VHH domain of the antibody. It is concentrated in three segments within the VHH domain called complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the VHH domain are called frameworks (FRs). Each VHH domain of the native heavy chain contains four FR regions, which are primarily β-sheet configurations linked by three CDRs, thereby forming loops that link β-sheet structures and, in some cases, a portion of the β-sheet structure. The CDRs in each chain are tightly held together by the FR regions and contribute to the formation of the antibody's antigen-binding site. Constant regions do not directly participate in antibody-antigen binding but exhibit various effector functions, such as antibody-dependent cytotoxicity.

[0045] The term "complementarity-determining region" (CDR) used in this article refers to the hypervariable region of the VHH domain, which forms a surface complementary to the three-dimensional surface of the binding antigen. 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.

[0046] The term "chimeric antibody" broadly refers to an engineered antibody that incorporates one or more regions from one antibody and one or more regions from other antibodies. Specifically, chimeric antibodies typically include the constant heavy chain (CH) and constant light chain (CL) domains from human antibodies, and the variable heavy chain (VH) and variable light chain (VL) domains from antibodies derived from non-human species. Suitable non-human sources can include animals such as mice, rats, hamsters, rabbits, and other animals.

[0047] The term "humanized antibody" refers to an antibody that is primarily derived from non-human sources, particularly its heavy and light chains, and is designed to reduce or inhibit human immune responses. This involves modifying the antibody to replace specific amino acids in its functional regions. Typically, the constant domains of humanized antibodies consist mainly of the human CH and CL domains.

[0048] The term "sdAb" refers to an antibody whose complementarity-determining region is part of a single-domain polypeptide. This includes (but is not limited to) heavy chain antibodies, antibodies that are naturally lacking in the light chain, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain scaffolds not derived from antibodies. Single-domain antibodies may encompass any existing or future variants in the art.

[0049] The term "constant region" or "constant domain" describes the carboxyl-terminal segments of the antibody light and heavy chains. These regions do not directly participate in the binding of the antibody to its antigen, but play a role in various effector functions, such as interaction with Fc receptors. They refer to the portions of the immunoglobulin molecule that have a more conserved amino acid sequence and contain antigen-binding sites compared to the variable region. Constant regions can include the CH1, CH2, and CH3 segments of the heavy chain, and the CL segment of the light chain.

[0050] The term "framework" or "FR" refers to the variable region residues flanking the complementarity-determining region (CDR). FR residues are present in various antibody forms, including chimeric antibodies, humanized antibodies, human antibodies, domain antibodies, bifunctional antibodies, linear antibodies, and bispecific antibodies. These residues are residues within the variable domain that are not part of the hypervariable region or the CDR region.

[0051] The term "linker" refers to a chemical entity consisting of covalent bonds or chains of atoms used to covalently link polypeptides to drug moieties.

[0052] As used herein, the term "crystallizable region" or "Fc region" refers to the antibody tail region that interacts with some proteins of the cell surface receptor and / or complement system, known as the Fc receptor. Structurally, the Fc region comprises at least a hinge region (a short sequence in 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. The Fc region of an IgG1 antibody can be generated, for example, by digesting the IgG1 antibody with papain.

[0053] The "percentage of sequence identity (%)" for any amino acid sequence identified herein is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the specific reference sequence after aligning the candidate sequence with a specific reference sequence, and, where necessary, introducing vacancies to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of sequence identity. Alignments performed for the purpose of determining the percentage of sequence identity can be performed using various methods within the skill level of this technique, such as publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR). Those skilled in this technique can determine the appropriate parameters for measuring alignments, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. For the purposes of this document, the sequence comparison between two amino acid sequences was performed using the Blastp (protein-protein BLAST) computer program available online at the National Center for Biotechnology Information (NCBI). The percentage of sequence similarity between a given sequence A and a topic sequence B (which can be alternatively expressed as a given sequence A having a certain percentage of sequence similarity with a given sequence B) is calculated by the following formula: X represents the number of amino acid residues that are rated as a consistent match by the sequence alignment program BLAST when comparing A and B, and Y represents the total number of amino acid residues in the subject sequence B.

[0054] As discussed herein, minor changes in the amino acid sequence of the antibody are considered to be covered within the inventive concept disclosed and claimed herein, provided that the changes 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. The antibodies disclosed herein can be specifically modified to alter the characteristics of peptides unrelated to their physiological activity. For example, certain amino acids can be altered and / or deleted without affecting the physiological activity of the antibody in this study. Specifically, this covers conserved amino acid substitutions. Conserved substitutions are substitutions occurring within side-chain-related amino acid families. Genetically encoded amino acids are generally classified into the following families: (1) acidic = aspartic acid, glutamic acid; (2) basic = lysine, arginine, histamine; (3) nonpolar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) nonpolar = glycine, aspartic acid, glutamic acid, cysteine, serine, threonine, tyrosine. A more preferred family is: serine and threonine belong to the aliphatic-hydroxyl family; aspartic acid and glutamic acid belong to the acetylamine-containing family; alanine, valine, leucine, and isoleucine belong to the aliphatic family; and phenylalanine, tryptophan, and tyrosine belong to the aromatic family. For example, it is reasonable to expect that independent substitutions such as isoleucine or valine replacing leucine, glutamic acid replacing aspartic acid, serine replacing threonine, or similar substitutions of a single amino acid with a structurally related amino acid will not significantly affect the binding or properties of the resulting molecule, especially when the substitution does not involve an amino acid within the framework site. Whether an amino acid change produces a functional peptide can be readily determined by analyzing the specific activity of the peptide derivative. Antibody fragments or analogs can be readily prepared by those generally skilled in this technique. Preferred amino and carboxyl terms of the fragment or analog appear near the boundaries of the functional region.

[0055] As used herein, the terms “connection,” “coupling,” “linking,” and “linkage” are used interchangeably to mean any way in which two components are linked by direct or indirect bonding.

[0056] The phrase "medically acceptable" means a substance that has been approved by federal or state regulatory agencies or is included in the United States Pharmacopeia, the European Pharmacopeia or other widely recognized pharmacopoeias for use in animals, and especially for human use.

[0057] The term "excipient" refers to a pharmaceutically acceptable substance, composition, or medium, which may be a liquid or solid filler, diluent, solvent, or encapsulating agent. Examples of excipients include encapsulating materials or additives such as absorption enhancers, antioxidants, binders, buffers, carriers, coating agents, colorants, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, fragrances, preservatives, propellants, release agents, sterilizing agents, sweeteners, solubilizers, wetting agents, and combinations thereof. Additionally, "excipient" can also refer to diluents, adjuvants (such as Freund's adjuvant, whether complete or incomplete Freund's adjuvant), or mediums.

[0058] As used herein, the terms "treat," "treating," and "treatment" are interchangeable and encompass the partial or complete prevention, relief, reduction, and / or management of cancer-related symptoms, secondary conditions, or illnesses. As used herein, the term "treatment" refers to the administration or delivery of the immune conjugates or pharmaceutical compositions disclosed herein to an individual with cancer-related symptoms, secondary conditions, or illnesses, with the aim of partially or completely alleviating, relieving, reducing, or decreasing one or more of the cancer-related symptoms, secondary conditions, or features, delaying their onset, inhibiting their progression, reducing their severity, and / or reducing their incidence. Cancer-related symptoms, secondary conditions, and / or illnesses include (but are not limited to) nausea, vomiting, loss of appetite, bowel changes, constipation, fatigue, muscle weakness, fractures, swelling or lumps, bleeding, cough, fever, neurological problems (e.g., seizures, visual changes, hearing changes, or facial drooping), weight changes (i.e., weight gain or weight loss), coma, and pain. Treatment may be administered to individuals presenting only with early signs of such symptoms, conditions, and / or conditions in order to reduce the risk of developing cancer-related symptoms, secondary conditions, and / or conditions. Treatment is generally considered "effective" if one or more symptoms or clinical markers decrease, a term defined herein. Alternatively, treatment is considered "effective" if the progression of symptoms, conditions, or conditions slows or stops.

[0059] As mentioned herein, the term "effective amount" refers to the quantity of a component sufficient to produce the desired response. For therapeutic purposes, an effective amount is also the quantity in which the therapeutically beneficial effect of the component exceeds any of its toxic or harmful effects. An effective amount of medicine does not need to cure the disease or condition, but will provide treatment for the disease or condition, thereby delaying, inhibiting, or preventing the onset of the disease or condition, or alleviating the symptoms of the disease or condition. An effective amount may be divided into one, two, or more doses in a suitable form for administration once, twice, or more times over a specified period of time. The specific effective amount or sufficient amount will vary depending on factors such as: the specific condition being treated, the patient's physical condition (e.g., the patient's body mass, age, or sex), the type of mammal or animal being treated, the duration of treatment, the nature of any concurrent treatments and the specific formulations used, and the structure of the compound or its derivatives. An effective amount may be expressed, for example, in grams, milligrams, or micrograms, or in milligrams per kilogram of body weight (mg / kg). Alternatively, the effective amount can be expressed as the concentration of the active ingredient (such as the immunoconjugate disclosed herein), such as mole concentration, mass concentration, volume concentration, weight mole concentration, mole fraction, mass fraction, and mixing ratio. A person with ordinary skill can calculate the human equivalent dose (HED) of the agent (such as the immunoconjugate of this invention) based on dosages determined from animal models. For example, the maximum safe starting dose for human individuals can be estimated by following industry guidance published by the U.S. Food and Drug Administration (FDA) entitled "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers."

[0060] The term "individual" refers to an animal, including the human species, that can be treated by the immune conjugates, pharmaceutical compositions, and / or methods of the present invention. Unless specifically indicating a sex, the term "individual" is intended to refer to both male and female sexes.

[0061] II. Description of the Invention

[0062] (I) Immune conjugates (ADCs)

[0063] The first embodiment of this disclosure provides an immune conjugate comprising a recombinant antibody or a single-domain antibody (sdAb) and a therapeutic agent, wherein the therapeutic agent is linked to the recombinant antibody (i.e., in the form of an ADC (Figure 1B)) or the sdAb (the antibody in Figure 1B may be replaced by the sdAb), as appropriate, via a linker. In some embodiments, the recombinant antibody and the sdAb may be in a chimeric, humanized, or human form, and the sdAb is in a chimeric form.

[0064] According to embodiments disclosed herein, the recombinant antibody or sdAb is specific for carcinoembryonic antigen-associated cell adhesion molecule 6 (CEACAM6; also known as "CD66c"). CEACAM6 is a member of the carcinoembryonic antigen (CEA) family known to be overexpressed in various types of solid tumors, including colorectal tumors, gastric tumors, endometrial tumors, breast tumors, ovarian tumors, cervical tumors, pancreatic tumors, lung tumors, and head and neck squamous cell carcinoma. Therefore, the recombinant antibody or sdAb can be used as a targeting element that specifically directs the immune conjugate to cancer / tumor cells expressing CEACAM6, thereby inducing cytotoxicity in a cancer / tumor-specific manner.

[0065] (I)-1 antibody

[0066] Recombinant antibodies

[0067] Referring now to Figure 1A, which is a schematic diagram depicting a recombinant antibody according to certain embodiments of the present disclosure. Structurally, the recombinant antibody of the present invention comprises a pair of homodimeric or heterodimeric VHH domains and an Fc region of an immunoglobulin linked to the paired VHH domains. As known in the art, the Fc region comprises two identical protein fragments linked by disulfide bonds, wherein each protein fragment comprises a hinge region, a CH2 domain, and a CH3 domain from its N-terminus to its C-terminus. The paired VHH domains are linked to the N-terminus of the protein fragments. In one embodiment, as depicted in Figure 1A, the recombinant antibody system of the present invention is in the form of an HCAb (i.e., a homodimeric VHH-Fc complex), comprising two heavy chains linked by disulfide bonds in the hinge region, wherein each heavy chain comprises a VHH domain, a hinge region, a CH2 domain, and a CH3 domain from its N-terminus to its C-terminus. In another embodiment, the HCAb may be a heterodimeric VHH-Fc complex. In some embodiments, the recombinant antibody may be in a chimeric, humanized, or human form.

[0068] Alternatively, the paired VHH domains are linked to the N-terminus of a protein fragment via a linker. In one embodiment, the recombinant antibody system of the present invention is in the form of HCAb (i.e., a homodimeric VHH-linker-Fc complex) comprising two heavy chains linked by disulfide bonds in a hinge region, wherein each heavy chain comprises a VHH domain, a linker, a hinge region, a CH2 domain, and a CH3 domain from its N-terminus to its C-terminus. Any linker suitable for linking VHH to the N-terminus of a protein fragment may be used in this disclosure. In some embodiments, the linker comprises a short peptide of Gly and Ser. In some embodiments, the peptide linker has a (GlymSern)p motif or a variant thereof, wherein m and n are integers from 1 to 10 and p is an integer from 1 to 30. Variations of this motif may include amino acid substitutions, such as Gly replaced by Ser, or vice versa. Examples of peptide linkers include an amino acid sequence, including (but not limited to) GGGSG (SEQ ID NO: 12), GGGGS (SEQ ID NO: 15) or repeating sequences thereof (such as a second, third, fourth or fifth repeating sequence), AHHSEDP (SEQ ID NO: 16), EPKTPKPQPQPQPQPQPNPTTE (SEQ ID NO: 17) or STPPTPSPSTPP (SEQ ID NO: 18), or combinations thereof. In one embodiment, the linker comprises GGGSG.

[0069] In another variant, the recombinant antibody may contain a single VHH domain linked to the N-terminus of a protein fragment, via a linker, as appropriate. The linker between the VHH domain and the protein fragment is as described herein.

[0070] It is generally accepted that the binding properties of an antibody (including binding affinity and specificity) are determined by its variable domains (i.e., the VH and VL domains of conventional antibodies, or the VHH domain of HCAb); more specifically, by the CDR sequence of the variable domains (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of conventional antibodies, or CDR-1, CDR-2, and CDR-3 of HCAb). According to some embodiments disclosed herein, each VHH domain of the recombinant antibody contains three CDRs (i.e., CDR-1, CDR-2, and CDR-3), wherein CDR-1 contains the amino acid sequence of SEQ ID NO: 1, CDR-2 contains the amino acid sequence of SEQ ID NO: 2, and CDR-3 contains the amino acid sequence of SEQ ID NO: 3. According to some other embodiments, CDR-1 contains the amino acid sequence of SEQ ID NO: 7, CDR-2 contains the amino acid sequence of SEQ ID NO: 8, and CDR-3 contains the amino acid sequence of SEQ ID NO: 9. Alternatively, CDR1, CDR2, and CDR3 of the VHH domain may contain a combination of SEQ ID NO: 1 or 7, SEQ ID NO: 2 or 8, and SEQ ID NO: 3 or 9.

[0071] For example, the amino acid sequence of the VHH domain of the recombinant antibody is provided as described below in SEQ ID NO: 4, wherein the CDRs (i.e., from the N-terminus to the C-terminus, CDR-1, CDR-2 and CDR-3) are marked in bold in the sequence. QVKLEESGGGLVQAGGSLRLSCAASGGSFSSYMLAWFRQAPGKEREFVANISPGGYTYYADSAQGRFTISRENAKNTMYLQMNSLNPEDTAVYYCAADIRVLAAAQVASYDYWGQGTQVTV

[0072] In some embodiments, the VHH domain of the recombinant antibody may be humanized to form a humanized antibody. For example, a humanized VHH is prepared comprising the amino acid sequence shown in SEQ ID NO: 10 below. [E]VQ [L]VESGGGLVQ [P]GGSLRLSCAASG [RT]FS [E]YMLAWFRQAPGKEREFVANISPGGYTYYADSA [K]GRFTISR [D]NAKNTMYLQMNSL [R]PEDTAVYYCAAD [L]RVLAAAQVASYDYWGQGTQVTV (Bold text indicates CDR, while underlined text indicates amino acids with sequences different from SEQ ID NO: 4).

[0073] For recombinant antibodies containing a pair of VHH domains, the VHHs can be the same (homodimer) or different (heterodimer).

[0074] Various methods for humanizing antibody sequences have been long established in the art. A widely used method is CDR transfer, which involves transferring the complementarity-determining region (CDR) sequence from a donor antibody to a human antibody framework with different specificities. Another technique for humanization is surface remodeling, which maintains the non-surface exposed residues of non-human origin while replacing the surface residues with human equivalents. Additionally, a method called "guided selection" allows for the identification of mouse antibodies that can serve as a fully human antibody source, thereby preserving the antigenic determinants and binding properties of the original antibody. Furthermore, a humanization method based on molecular dynamics calculations is described in patent application WO2009 / 032661.

[0075] Since the binding affinity and specificity of an antibody are primarily determined by its CDR sequence, it is understood that the framework (FR) sequence of the VHH domain can be varied (e.g., substituted with conserved or non-conserved amino acid residues) without affecting the binding affinity and / or specificity of the antibody of the present invention. Preferably, the FR sequence is substituted with one or more suitable amino acid conserved residues with similar properties; for example, leucine (a nonpolar amino acid residue) is substituted with isoleucine, alanine, valine, proline, phenylalanine, or tryptophan (another nonpolar amino acid residue); aspartic acid (an acidic amino acid residue) is substituted with glutamic acid (another acidic amino acid residue); or lysine (a basic amino acid residue) is substituted with arginine or histamine (another basic amino acid residue). Based on the conserved substitutions, those skilled in the art can substitute one or more amino acid residues of the FR sequence of the VHH domain of the recombinant antibody without affecting its activity and / or function (i.e., binding to CEACAM6). Therefore, recombinant antibodies containing substituted amino acids in the FR sequence of the VHH domain are intended to be included within the scope of this disclosure. According to some embodiments, each VHH domain of the recombinant antibody contains an amino acid sequence that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 4; preferably, at least 90% identical to SEQ ID NO: 4; more preferably, at least 95% identical to SEQ ID NO: 4.

[0076] Depending on the desired purpose, the Fc region linked to the VHH domain is the Fc region of human immunoglobulin G1 (IgG1), immunoglobulin G2 (IgG2), immunoglobulin G3 (IgG3), immunoglobulin G4 (IgG4), or immunoglobulin A (IgA). According to some exemplary embodiments, the Fc region of the recombinant antibody is the Fc region of human IgG1. In one particular embodiment, the paired VHH domain is linked to the N-terminus of the human IgG1 Fc region, wherein each heavy chain of the recombinant antibody of the present invention contains the amino acid sequence of SEQ ID NO: 13. Additionally, a signal peptide may be fused to the N-terminus of the VHH of the recombinant antibody (see SEQ ID NO: 5) to transport the antibody out of the cell membrane. After transmembrane translocation of the protein, the signal peptide is cleaved by a signal peptidase.

[0077] Alternatively, the Fc region of the recombinant antibody may be the Fc region of mouse IgG1, IgG2, IgG3, or IgA. According to some embodiments, the Fc region of the recombinant antibody may be the Fc region of mouse IgG2. In one particular embodiment, the paired VHH domains are linked to the N-terminus of the mouse IgG2 Fc region, wherein each heavy chain of the recombinant antibody of the present invention contains the amino acid sequence of SEQ ID NO: 19. The signal peptide may be fused to the N-terminus of the VHH of the recombinant antibody (see SEQ ID NO: 6) to transport the antibody out of the cell membrane. After transmembrane translocation of the protein, the signal peptide is cleaved by a signal peptidase.

[0078] According to certain embodiments of this disclosure, recombinant antibody systems are generated via ribosome display and DNA selection. Ribosome display is a cell-free system for in vitro selection of proteins and peptides from a large collection library. Methods for generating ribosome display systems are known in this art. Generally, a ribosome display system is established according to a commonly used procedure by immunizing a host animal (e.g., a camel, preferably an alpaca) with a peptide (e.g., the CEACAM6 peptide); for example, immunizing the host animal with the peptide every two or three weeks until the desired antibody titer is achieved. After the last immunization, peripheral lymphocytes from the immunized animal are collected, and total RNA is isolated from the lymphocytes. The corresponding cDNA fragment is synthesized, and the cDNA fragment encoding the VHH domain is amplified accordingly. The PCR product is then conjugated into an expression vector to construct a cDNA collection library. The cDNA encoding the VHH domain can be used to transcribe mRNA and translate proteins in a ribosome-displayed form (i.e., a protein-ribosome-mRNA (PRM) complex). In the selection phase (also known as the "biopanning phase"), the PRM complex is added to a fixed ligand (e.g., a fixed CEACAM6 peptide), and then the complex exhibiting binding affinity to the fixed ligand is dissociated by salt concentration, chelating agents, or mobile ligands. These mobile ligands form a complex with the protein-binding motif, thereby allowing the dissociation of the mRNA. The mRNA can then be reverse-transcribed back into cDNA for the next round of selection. This selection phase can be repeated several times (e.g., 3-5 times) to obtain cDNA encoding a VHH domain specific to the peptide (e.g., the CEACAM6 peptide).

[0079] According to some embodiments of this disclosure, the resulting cDNA is then selected into an expression vector (i.e., an IgG1 vector) containing the Fc region of a human immunoglobulin. After introduction into a suitable host cell, the expression vector can be used to express a recombinant antibody in the host cell; the resulting recombinant antibody structurally comprises a human-derived Fc region and a camel-derived VHH domain linked to the N-terminus of that Fc region. The host cell is preferably a mammalian cell, such as a Chinese hamster ovary (CHO) cell or an embryonic kidney 293 cell. According to a preferred embodiment, the host cell line is an embryonic kidney 293 cell.

[0080] Alternatively, the recombinant antibodies of this invention can be directly generated through DNA selection. The DNA encoding the recombinant antibodies of this invention can be easily isolated and sequenced using conventional procedures, such as using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mAbs. Once isolated, the DNA can be placed in an expression vector and then transfected into suitable host cells as described above, so as to synthesize the desired antibodies in the host cells.

[0081] Recombinant antibodies can be purified according to standard procedures in this technology, such as cross-flow filtration, affinity column chromatography, gel filtration and similar procedures. [, Single-domain antibodies , ] [, , ]

[0082] In some embodiments, a single VHH domain may also be used in the immunoconjugates described herein, which are single-domain antibodies (sdAbs). Generally, sdAbs are thermostable and pH stable. According to some embodiments disclosed herein, each VHH domain of the recombinant antibody comprises three CDRs (i.e., CDR-1, CDR-2, and CDR-3), wherein CDR-1 comprises the amino acid sequence of SEQ ID NO: 1 or 7, CDR-2 comprises the amino acid sequence of SEQ ID NO: 2 or 8, and CDR-3 comprises the amino acid sequence of SEQ ID NO: 3 or 9.

[0083] In some embodiments, the sdAb comprises the amino acid sequence of SEQ ID NO: 4, 10, or 11. The sdAb having SEQ ID NO: 11 is a mutant of SEQ ID NO: 4. QVKLEESGGGLVQAGGSLRLSCAASGGSFSSYMLAWFRQAPGKEREFVANISPGGYTYYADSAQGRFTISRENAKNTMYLQMNSLNPEDTAVYYCAADIRVLAAAQVASYDYWGQGTQVTVCS (SEQ ID NO: 11)

[0084] Methods for producing sdAbs and their modifications are known in the art. For example, sdAbs can be produced from living host cells that have been genetically engineered to produce proteins. Methods for constructing the genes of the cells used to produce proteins are well known in the art. See, for example, Ausabel et al. (1990), Current Protocols in Molecular Biology (Wiley, New York). sdAbs derived from non-human animals and subsequently modified, for example, by recombinant DNA methods known in the art, such modifications include humanization, deimmunization, and chimerism. Humanized sdAbs can also be obtained, for example, using transgenic mice that express human heavy and light chain genes but not endogenous mouse immunoglobulin heavy and light chain genes. In some embodiments, humanized sdAbs (such as nano-antibody molecules) are optimized by introducing conserved substitutions, substitutions from shared sequences, substitutions from germlines, and / or reverse mutations using any methods known in the art.

[0085] (I)-2 therapeutic agent

[0086] Generally, the therapeutic agents of the immune conjugates of the present invention can be cytotoxic drugs, radioactive nuclei, intercytokines, hormonal drugs, immunostimulants, or immunotherapeutic drugs.

[0087] Cytotoxic agents can be any molecule known to inhibit the growth of cancer / tumor cells or to exhibit cytotoxic / killing effects on cancer / tumor cells. Examples of cytotoxic agents suitable for producing the immune conjugates of the present invention include (but are not limited to) alkylating agents (agents that prevent cell replication by damaging DNA; such as altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine). (e.g., ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa, or trabectedin) and antimetabolites (drugs that interfere with DNA and RNA by acting as replacements for normal structural units of RNA and DNA; e.g., azacitidine, 5-fluorouracil) (5-FU), 6-mercaptopurine (6-MP), capecitabine, cladribine, clofarabine, cytarabine (Ara-C), decitabine, fluxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, neurabine (larabine), pemetrexed, pentostatin, pralatrexate, or thioguanine), anthracyclines, or antibiotics (agents that interfere with enzymes involved in DNA replication during the cell cycle; such as daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, bleomycin, dactinomycin D, mitomycin-C, or mitoxantrone) or mitotic inhibitors (agents that stop cell division to form new cells);Examples include olistatin, cabazitaxel, docetaxel, nab-paclitaxel, paclitaxel, vinblastine, vincristine, vinorelbine, tubulysin, or mertansine (DM1). According to one exemplary example of this disclosure, the cytotoxic drug is olistatin or a derivative thereof, or a topoisomerase 1 inhibitor, such as MMAE, MMAF, ezetidine, and ezetidine derivatives.

[0088] A radioactive nuclide, also known as a radionuclide, radioisotope, or radioactive isotope, can be any of the following: yttrium-90 (90Y), indium-111 (111In), iodine-131 (131I), samarium-153 (153Sm), argon-177 (177Lu), astatine-211 (211At), bismuth-212 (212Bi), actinium-225 (225Ac), radium-223 (223R), or thorium-227 (227Th). Preferably, the radioactive nucleus is mismatched with a chelating agent, such as ethylenediaminetetramethylenephosphonic acid (EDTMP), 1,4,7,10-tetraazacyclododecanetetramethylenephosphonic acid (DOTMP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane-1,4-diacetic acid (NODA), or diethylenetriaminepentaacetic acid (DTPA).

[0089] Cytokines can be any cytokine known to regulate or stimulate an individual's immune response, such as interleukin (IL)-2, IL-10, IL-12, IL-15, IL-18, IL-21, IL-23, IL-24, IL-27, IL-35, tumor necrosis factor (TNF)-α, interferon (IFN)-α, IFN-γ, granulocyte-macrophage community-stimulating factor (GM-CSF), or combinations thereof.

[0090] Hormonal drugs are known to regulate (primarily downregulate) the levels of cancer-related hormones in cancer patients. Examples of hormone drugs suitable for producing the immune conjugate of this invention include (but are not limited to) aromatase inhibitors (e.g., anastrozole, exemestane, or letrozole), selective estrogen receptor modulators (SERMs; e.g., tamoxifen or raloxifene), estrogen receptor antagonists (e.g., fulvestrant or toremifene), and luteinizing hormone-releasing hormone (LHRH) agonists (e.g., goserelin, leuprolide). Leuprolide or triptorelin, antiandrogens (such as apalutamide, enzalutamide, darolutamide, bicalutamide, flutamide, or nilutamide), CYP17 inhibitors (such as abiraterone or ketoconazole), progestins (such as medroxyprogesterone acetate or megestrol acetate), and adrenolytics (such as mitotane).

[0091] As the name suggests, immunostimulants are small molecules that can be used to stimulate an individual's immune system to fight cancer / tumor cells. Examples of immunostimulating agents (also known as immunostimulants) suitable for binding with the recombinant antibodies of the present invention include, but are not limited to, acemannan, bropirimine, burdock, deoxycholic acid (DCA), echinacea, elapegademase, flavonoids (e.g., rutin, isoliquiritigenin, or liquiritigenin), glatiramer acetate, oprelvekin, pegademase bovine, plerixafor, prolactin, trilaciclib, terpenes (e.g., triterpenes), TLR7 / TLR8 agonists, TLR9 agonists, Sting agonists, and NLRP agonists.

[0092] Regarding immunotherapy drugs, they can be any small molecule commonly used in immunotherapy for cancer treatment; for example, immune checkpoint inhibitors (ICIs; such as inhibitors of cytotoxic T lymphocyte antigen-4 (CTLA-4), planned cell death 1 (PD-1), or PD-L1) or immunomodulatory drugs (IMiDs; such as thalidomide, pomalidomide, lenalidomide, or their analogues or derivatives).

[0093] Alternatively, the therapeutic agent may be a plant toxin (such as ricin, abrin, saporin, volkensin, modeccin, gelonin, and viscumin), an exotoxin (such as Pseudomonas aeruginosa exotoxin and botulinum toxin, such as botulinum toxin A, botulinum toxin B, botulinum toxin C, botulinum toxin D, botulinum toxin E, and botulinum toxin F), or an endotoxin (such as diphtheria toxin) to achieve tumor killing.

[0094] (I)-3 Connecting an antibody or sdAb to a therapeutic agent via a linker

[0095] According to embodiments disclosed herein, the linker is configured to link a therapeutic agent to the Fc region of an sdAb or recombinant antibody. Depending on the intended purpose, the linker of the immunoconjugate of the present invention may be a cleavable linker (i.e., a linker that can be cleaved / reduced by an enzyme or under specific physical conditions) or a non-cleavable linker (also referred to as a "stable linker" that is not hydrolyzed in vivo; i.e., resistant to cleavage caused by an enzyme or physical process in vivo).

[0096] Exemplary cleavable linkers suitable for binding the therapeutic agents of the present invention to recombinant antibodies include (but are not limited to) protease-sensitive linkers (e.g., valine-citrulline (VC) dipeptide, valine-alanine (VA) dipeptide, valine-lysine (VL) dipeptide, valine-arginine (VR) dipeptide, or glutamate-valine-citrulline (EVC) tripeptide), pH-sensitive linkers (e.g., SS disulfide bond, hydrazone linker, ester linker, or acetylamine linker), and glutathione-sensitive linkers (e.g., N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) or N-succinimidyl-4-(2-pyridyldithio)valerate (SPP)).

[0097] Non-limiting examples of non-cleavable linkers include maleic diaminohexyl (MC), maleic diaminomethylcyclohexane-1-carboxylate (MCC), and succinimido-4-[N-maleic diaminomethyl]cyclohexane-1-carboxylate (SMCC).

[0098] Alternatively, the linker may be a linker known in this art for linking two functional motifs in an immune conjugate (e.g., linking the antibody and payload of an ADC).

[0099] A skilled person can select a suitable linker for generating the immune conjugate of the present invention according to the intended purpose. According to one exemplary embodiment, the linker connecting the recombinant antibody and the therapeutic agent comprises a polyethylene glycol (PEG) chain and a protease-sensitive linker linked to the PEG chain; preferably, the PEG chain has 1 to 10 repeating EG units. According to one example of this disclosure, the linker comprises a PEG chain having three repeating EG units (i.e., PEG3) and a VC dipeptide linked to the PEG chain. In the embodiments, the generated immune conjugate is in the form of "recombinant antibody (Fc)-PEG chain-VC dipeptide-therapeutic agent".

[0100] According to some embodiments disclosed herein, the immunoconjugates of the present invention are generated using trimannosyl ADC technology, a platform for site-specific linking of drug payloads to target antibodies (see, for example, WO 2018 / 126092 A1). In these embodiments, the recombinant antibody is first modified to bind to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more) azide groups; then, the azide-modified antibody is linked to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more) linker-payloads (each comprising a linker, a DBCO group bound to one end of the linker, and a therapeutic agent bound to the other end of the linker) via a copper-free click reaction between the azide group and a dibenzocyclooctynyl group (DBCO). It is understood that the azide group or DBCO group may alternatively be substituted with different groups suitable for click chemistry, such as alkynes, tetrakis, or transcyclooctynyl (TCO) groups. Depending on the desired purpose, the immune conjugate of the present invention can be alternatively generated by other known ADC synthesis methods, such as cysteine ​​binding, lysine binding, disulfide re-bridging, etc. Methods for synthesizing ADCs are known in this art; therefore, for the sake of brevity, their detailed descriptions are omitted herein.

[0101] According to certain exemplary embodiments of this disclosure, the recombinant antibody has four azide groups, and therefore, four therapeutic agents (e.g., four MMAE molecules) are linked to the Fc region of the recombinant antibody. In these embodiments, the drug-to-antibody ratio (DAR) of the resulting immune conjugate is approximately 4.

[0102] Examples of (I)-4 immune conjugates

[0103] In this disclosure, antibodies are linked to therapeutic agents, with or without a linker. Examples of chimeric HCAbs, humanized HCAbs, and sdAbs, with or without a linker, include (but are not limited to) the following sequences:

[0104] 1. Chimeric HCAb Sequence: Signal peptide-VHH-Fc (human IgG1) Question VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 13)

[0105] 2. Humanized HCAb Sequence: Signal peptide-humanized VHH-Fc (human IgG1) EVQLVESGGGLVQPGGSLRLSCAASGRTFSEYMLAWFRQAPGKEREFVANISPGGYTYYADSAKGRFTISRDNAKNTMYLQMNSLRPEDTAVYYCAADLRVLAAAQVASYDYWGQGTQVTVSS(GGGSG as applicable)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14)

[0106] 3. sdAb A standalone VHH can be used as an sdAb to be linked to a therapeutic agent, with or without a linker, as shown in SEQ ID NO: 4, 10 or 11.

[0107] Any therapeutic agent can be linked to the antibody described herein. Examples of therapeutic agents include (but are not limited to) oliquistatin, irinotecan, ethanotecan, levofolate, 5-fluorouracil, and MMAE. Examples of the chimeric HCAb, humanized HCAb, and sdAb mentioned above can be linked to any therapeutic agent. (II) Pharmaceutical compositions comprising the immune conjugate of the present invention

[0108] The second aspect of this disclosure relates to a pharmaceutical composition for treating cancer. According to some embodiments, the pharmaceutical composition comprises the immune conjugate of the present invention and, where applicable, a medically acceptable carrier.

[0109] Generally, the immune conjugate of the present invention is present in an amount of about 0.1% to 99% by weight, based on the total weight of the pharmaceutical composition. In some embodiments, the immune conjugate of the present invention is present in an amount of at least 1% by weight, based on the total weight of the pharmaceutical composition. In some embodiments, the immune conjugate is present in an amount of at least 5% by weight, based on the total weight of the pharmaceutical composition. In still other embodiments, the immune conjugate is present in an amount of at least 10% by weight, based on the total weight of the pharmaceutical composition. In still other embodiments, the immune conjugate is present in an amount of at least 25% by weight, based on the total weight of the pharmaceutical composition.

[0110] Pharmaceutical compositions can be prepared according to acceptable pharmaceutical procedures, such as those described in Remington's Pharmaceutical Sciences, 17th edition, edited by Alfonoso R. Gennaro, Mack Publishing Company, Easton, Pa (1985). A pharmaceutically acceptable carrier is one that is compatible with the other components in the formulation and is biologically acceptable.

[0111] A pharmaceutically acceptable carrier can be any pharmaceutically acceptable material or medium, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, which can be used to transport or transfer an active agent (e.g., the immunoconjugate of the present invention) from one organ or part of the body to another organ or part of the body. The carrier must be "acceptable" in the sense of compatibility with other components in the formulation, and the chosen carrier should minimize any degradation of the active agent and any adverse side effects on the individual. Preferably, the pharmaceutical composition is formulated in a liquid form, such as a sterile solution or suspension, which can be administered by injection, for example, intravenous, intra-arterial, intramuscular, subcutaneous, intrathecal, intraperitoneal, or intratumoral administration. The pharmaceutical composition may be formulated as an isotonic suspension, solution or emulsion in an oily or aqueous medium (such as sodium chloride injection, Ringer's injection, dextran injection, dextran and lactated Ringer's injection), and may contain a formulation agent, such as a suspending agent, stabilizer or dispersant.

[0112] As appropriate, the pharmaceutical compositions disclosed herein may further comprise one or more pharmaceutically acceptable additives, including buffers, antioxidants, diluents, disinfectants and similar additives.

[0113] (III) Uses of the immune conjugates and pharmaceutical compositions of the present invention

[0114] The third aspect of this disclosure relates to a method for treating cancer in an individual by using the immune binder or pharmaceutical composition of the present invention. The method comprises administering to the individual an effective amount of the immune binder or pharmaceutical composition as described in portions (i) and (ii) of this disclosure, respectively, to inhibit cancer growth, migration, or progression, and / or reduce or alleviate cancer-related symptoms.

[0115] According to some embodiments of this disclosure, in a system of mice, the individual was administered an immunoconjugate of the present invention at doses ranging from about 0.01 mg / kg to 100 mg / kg, such as about 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, 50 mg / kg, 51 mg / kg, 52 mg / kg, 53 mg / kg, 54 mg / kg, 55 mg / kg, 56 mg / kg, 57 mg / kg, 58 mg / kg, 59 mg / kg, 60 mg / kg, 61 mg / kg, 62 mg / kg, 63 mg / kg, 64 mg / kg, 65 mg / kg, 66 mg / kg, 67 mg / kg, 68 mg / kg, 69 mg / kg, 70 mg / kg, 71 mg / kg, 72 mg / kg, 73 mg / kg, 74 mg / kg, 75 mg / kg, 76 mg / kg, 77 mg / kg, 78 mg / kg, 79 mg / kg, 80 mg / kg, 81 mg / kg, 82 mg / kg, 83 mg / kg, 84 mg / kg, 85 mg / kg, 86 mg / kg, 87 mg / kg, 88 mg / kg, 89 mg / kg, 90 mg / kg, 91 mg / kg, 92 mg / kg, 93 mg / kg, 94 mg / kg, 95 mg / kg, 96 mg / kg, 97 mg / kg, 98 mg / kg, 99 mg / kg or 100 mg / kg. Preferably, about 0.The immunoconjugate of the present invention is administered at concentrations ranging from 1 mg / kg to 50 mg / kg. More preferably, the immunoconjugate of the present invention is administered to the individual at concentrations ranging from 1 mg / kg to 10 mg / kg. According to one working example, the immunoconjugate of the present invention is administered to the individual at concentrations of approximately 5 mg / kg.

[0116] Skilled personnel can readily determine the human equivalent dose (HED) of the immune conjugate of the present invention based on the dosage determined by animal studies provided in the working examples of this application. Therefore, the effective amount of the immune conjugate of the present invention suitable for human individuals can be in the range of about 1 μg to 10 mg per kilogram of human body weight; such as about 1 μg / kg, 2 μg / kg, 3 μg / kg, 4 μg / kg, 5 μg / kg, 6 μg / kg, 7 μg / kg, 8 μg / kg, 9 μg / kg, 10 μg / kg, 20 μg / kg, 30 μg / kg, 40 μg / kg, 50 μg / kg, 60 μg / kg, 70 μg / kg, 80 μg / kg, 90 μg / kg, 100 μg / kg, 110 μg / kg, 120 μg / kg, 130 μg / kg, 140 μg / kg, 150 μg / kg, 160 μg / kg, 170 μg / kg, 180 μg / kg, 190 μg / kg, 200 μg / kg, 210 μg / kg, etc. μg / kg, 220 μg / kg, 230 μg / kg, 240 μg / kg, 250 μg / kg, 260 μg / kg, 270 μg / kg, 280 μg / kg, 290 μg / kg, 300 μg / kg, 310 μg / kg, 320 μg / kg, 330 μg / kg, 340 μg / kg, 350 μg / kg, 360 μg / kg, 370 μg / kg, 380 μg / kg, 390 μg / kg, 400 μg / kg, 410 μg / kg, 420 μg / kg, 430 μg / kg, 440 μg / kg, 450 μg / kg, 460 μg / kg, 470 μg / kg, 480 μg / kg, 490 μg / kg, 500 μg / kg, 510 μg / kg, 520 μg / kg, 530 μg / kg, 540 μg / kg, 550 μg / kg, 560 μg / kg, 570 μg / kg, 580 μg / kg, 590 μg / kg, 600 μg / kg, 610 μg / kg, 620 μg / kg, 630 μg / kg, 640 μg / kg, 650 μg / kg, 660 μg / kg, 670 μg / kg, 680 μg / kg, 690 μg / kg, 700 μg / kg, 710 μg / kg, 720 μg / kg, 730 μg / kg, 740 μg / kg, 750 μg / kg, 760 μg / kg, 770 μg / kg, 780 μg / kg, 790 μg / kg, 800 μg / kg, 810 μg / kg, 820 μg / kg, 830 μg / kgThe dosage is 840 μg / kg, 850 μg / kg, 860 μg / kg, 870 μg / kg, 880 μg / kg, 890 μg / kg, 900 μg / kg, 910 μg / kg, 920 μg / kg, 930 μg / kg, 940 μg / kg, 950 μg / kg, 960 μg / kg, 970 μg / kg, 980 μg / kg, 990 μg / kg, or approximately 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg. This dosage can be administered in a single aliquot or alternatively in more than one aliquot. Skilled technicians or clinicians may adjust the dosage or regimen according to the patient's condition or the severity of their disease.

[0117] Depending on the intended purpose, the immune conjugate or pharmaceutical composition of the present invention may be administered to an individual once every 1-7 weeks, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, or for longer periods. It is understood that the dosing regimen may vary over time. According to some exemplary embodiments of this disclosure, the immune conjugate of the present invention is administered to an individual once a week. According to some preferred embodiments, the immune conjugate of the present invention is administered to an individual weekly for at least 3 weeks, such as 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or for longer periods. In one exemplary embodiment of this disclosure, the immune conjugate of the present invention is administered to an individual weekly for 6 consecutive weeks.

[0118] The recombinant immune conjugates or pharmaceutical compositions disclosed herein can be administered to an individual via appropriate routes, such as intratumoral, intramuscular, intravenous, or intraperitoneal injection. According to some embodiments of this disclosure, the recombinant immune conjugates or pharmaceutical compositions are administered to an individual via intravenous injection.

[0119] It should be understood that the method of the present invention can be applied to an individual alone or in combination with other therapies that have some beneficial effects on the prevention or treatment of cancer, such as surgery, chemotherapy, and / or radiation therapy. Depending on the intended / treatment purpose, the method of the present invention can be applied to an individual before, during, or after the administration of other therapies.

[0120] Examples of cancers that can be treated using the method of the present invention include (but are not limited to) stomach cancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer, kidney cancer, colorectal cancer, cervical cancer, ovarian cancer, brain tumor, prostate cancer, hepatocellular carcinoma, melanoma, esophageal cancer, multiple myeloma, squamous cell carcinoma of the head and neck, bile duct cancer, or combinations thereof.

[0121] Basically, the methods of this invention can be used to treat various mammalian systems, such as humans, mice, rats, guinea pigs, hamsters, monkeys, pigs, dogs, cats, horses, sheep, goats, cattle, and rabbits. Preferably, the system is human.

[0122] The following examples are provided to illustrate certain aspects of the invention and to assist those skilled in the art in carrying out the invention. These examples should not be construed as limiting the scope of the invention in any way. Without further detailed description, it is believed that those skilled in the art can utilize the invention to the fullest extent based on the description herein. All publications cited herein are hereby incorporated by full reference.

[0123] The embodiments disclosed herein

[0124] Example 1: An immunoconjugate comprising: a recombinant antibody comprising (i) a single variable heavy chain domain (VHH domain) or a pair of homodimeric or heterodimeric VHHs, and a crystallizable region fragment (Fc region) of an immunoglobulin, the Fc region being fused to each of the (etc.) VHH domains, fused via a peptide linker as appropriate; or (ii) a single-domain antibody (sdAb) comprising a VHH domain, wherein the VHH domain comprises: a first chain complementarity-determining region (CDR-1) comprising an amino acid sequence of SEQ ID NO: 1 or 7, a second CDR (CDR-2) comprising an amino acid sequence of SEQ ID NO: 2 or 8, and a third CDR (CDR-3) comprising an amino acid sequence of SEQ ID NO: 3 or 9; and a therapeutic agent; wherein the therapeutic agent is linked to the Fc region of the recombinant antibody or the VHH of the sdAb, linked via a linker as appropriate. Alternatively, CDR1, CDR2 and CDR3 of the VHH domain may contain a combination of SEQ ID NO: 1 or 7, SEQ ID NO: 2 or 8 and SEQ ID NO: 3 or 9.

[0125] Example 2: An immunoconjugate comprising: a recombinant antibody comprising a single variable heavy chain domain (VHH domain) or a pair of homodimeric or heterodimeric VHHs, and a crystallizable region fragment (Fc region) of an immunoglobulin, the Fc region being fused to each of the VHH domains, optionally via a peptide linker, wherein the VHH domain comprises: a first chain complementarity-determining region (CDR-1) comprising an amino acid sequence of SEQ ID NO: 1 or 7, a second CDR (CDR-2) comprising an amino acid sequence of SEQ ID NO: 2 or 8, and a third CDR (CDR-3) comprising an amino acid sequence of SEQ ID NO: 3 or 9; a therapeutic agent; and a linker that links the therapeutic agent to the Fc region of the recombinant antibody. Alternatively, the CDR1, CDR2, and CDR3 of the VHH domain may comprise a combination of SEQ ID NO: 1 or 7, SEQ ID NO: 2 or 8, and SEQ ID NO: 3 or 9.

[0126] Example 3: An immune conjugate as described in any of Examples 1 to 2, wherein the VHH domain is fused to the N-terminus of the Fc region of the immunoglobulin via a peptide linker, wherein the peptide linker comprises short peptides of Gly and Ser.

[0127] Example 4: An immune conjugate as in Example 3, wherein the peptide linker has a (GlymSern)p motif or a variant thereof, wherein m and n are any integers from 1 to 10 and p is any integer from 1 to 30.

[0128] Example 5: An immune conjugate as in Example 4, wherein the peptide linker comprises an amino acid sequence having GGGSG (SEQ ID NO: 12), GGGGS (SEQ ID NO: 15) or a repeating sequence thereof (such as a second, third, fourth or fifth repeating sequence).

[0129] Example 6: An immune conjugate as described in any of Examples 1 to 2, wherein the peptide linker comprises an amino acid sequence having the following amino acid sequences: GGGSG (SEQ ID NO: 12), GGGGS (SEQ ID NO: 15) or a repeating sequence thereof (such as a second, third, fourth or fifth repeating sequence), AHHSEDP (SEQ ID NO: 16), EPKTPKPQPQPQPQPQPNPTTE (SEQ ID NO: 17) or STPPTPSPSTPP (SEQ ID NO: 18), or a combination thereof.

[0130] Example 7: An immune conjugate comprising: a single-domain antibody (sdAb) containing a VHH domain, wherein the VHH domain comprises: a first chain complementarity-determining region (CDR-1) containing an amino acid sequence of SEQ ID NO: 1 or 7, a second CDR (CDR-2) containing an amino acid sequence of SEQ ID NO: 2 or 8, and a third CDR (CDR-3) containing an amino acid sequence of SEQ ID NO: 3 or 9; and a therapeutic agent; wherein the therapeutic agent is linked to the Fc region of the recombinant antibody or the VHH of the sdAb, as appropriate, via a linker. Alternatively, CDR1, CDR2, and CDR3 of the VHH domain may comprise a combination of SEQ ID NO: 1 or 7, SEQ ID NO: 2 or 8, and SEQ ID NO: 3 or 9.

[0131] Example 8: An immune conjugate as described in any of Examples 1 to 6, wherein the recombinant antibody system is a chimeric antibody or a humanized antibody.

[0132] Example 9: An immune conjugate as in Example 1 or 7, wherein the sdAb is a humanized antibody.

[0133] Example 10: An immune conjugate as described in any one of Examples 1 to 9, wherein the VHH comprises: CDR-1 comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 1; CDR-2 comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 2; and CDR-3 comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 3.

[0134] Example 11: An immune conjugate as described in any one of Examples 1 to 10, wherein the VHH comprises: CDR-1 containing the amino acid sequence of SEQ ID NO: 1, CDR-2 containing the amino acid sequence of SEQ ID NO: 2, and CDR-3 containing the amino acid sequence of SEQ ID NO: 3.

[0135] Example 12: An immune conjugate as described in any one of Examples 1 to 9, wherein the VHH comprises: CDR-1 comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to that of SEQ ID NO: 7; CDR-2 comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to that of SEQ ID NO: 8; and CDR-3 comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to that of SEQ ID NO: 9.

[0136] Example 13: An immune conjugate as described in any of Examples 1 to 9 and 12, wherein the VHH comprises: CDR-1 containing the amino acid sequence of SEQ ID NO: 7, CDR-2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-3 containing the amino acid sequence of SEQ ID NO: 9.

[0137] Example 14: An immune conjugate as described in any of Examples 1 to 13, wherein the VHH domain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% similarity to SEQ ID NO: 4, 10, or 11.

[0138] Example 15: An immune conjugate as described in any of Examples 1 to 14, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 4, 10, or 11.

[0139] Example 16: An immune conjugate as described in any of Examples 1 to 15, wherein the recombinant antibody includes the VHH domain containing the amino acid sequence of SEQ ID NO: 4, 10 or 11.

[0140] Example 17: An immune conjugate as described in any of Examples 1 to 16, wherein the recombinant antibody includes the VHH domain containing the amino acid sequence of SEQ ID NO: 4 or 10.

[0141] Example 18: An immune conjugate as described in any of Examples 1 to 17, wherein the recombinant antibody comprises a single VHH domain containing an amino acid sequence of SEQ ID NO: 4, 10 or 11.

[0142] Example 19: An immune conjugate as described in any of Examples 1 to 17, wherein the recombinant antibody comprises a pair of homodimeric VHHs, wherein each of the VHH domains comprises an amino acid sequence of SEQ ID NO: 4, 10 or 11.

[0143] Example 20: An immune conjugate as described in any of Examples 1 to 17, wherein the recombinant antibody comprises a pair of heterodimeric VHHs, wherein the paired VHHs are different and each of the VHH domains comprises an amino acid sequence selected from SEQ ID NO: 4, 10 and 11.

[0144] Example 21: An immune conjugate as described in any of Examples 1 to 17, wherein the sdAb includes the VHH domain containing the amino acid sequence of SEQ ID NO: 4, 10 or 11.

[0145] Example 22: An immune conjugate as in Example 21, wherein the sdAb includes the VHH domain containing the amino acid sequence of SEQ ID NO: 10 or 11.

[0146] Example 23: An immune conjugate as described in any of Examples 1 to 22, wherein the VHH domain is derived from a camelid heavy chain antibody.

[0147] Example 24: An immune conjugate as described in any of Examples 1 to 17, wherein the recombinant antibody comprises the amino acid sequence of SEQ ID NO: 5, 6, 13, 14 or 19.

[0148] Example 25: An immune conjugate as described in any of Examples 1 to 24, wherein the therapeutic agent is a cytotoxic drug, a radioactive nucleus, a cytokine, a hormonal drug, an immunostimulant, or an immunotherapy drug.

[0149] Example 26: An immune conjugate as described in any of Examples 1 to 25, wherein the therapeutic agent is an alkylating agent (an agent that prevents cell replication by damaging DNA; such as hexamethylmelamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, metronidazole, melphalan, oxaliplatin, temozolomide, thiotepa, or trabectedine), an antimetabolite (an agent that interferes with DNA and RNA by acting as a replacement for normal structural units of RNA and DNA; such as azacitidine, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cladribine, clofarabine, cytarabine ( Ara-C), decitabine, fluorouridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, nerabine, pemetrexed, pentostatin, pralatrexate, or thioguanine), anthracyclines, or antibiotics (agents that interfere with enzymes involved in DNA replication during the cell cycle; such as donomycin, cranberry, epirubicin, adamycin, vararubicin, bleomycin, actinomycin D, mitomycin-C, or mitoxantrone) or mitotic inhibitors (agents that stop cell division to form new cells; such as olritatin, cabazitaxel, docetaxel, albumin-bound paclitaxel, paclitaxel, vinblastine, vincristine, vinorelbine, tobrexin, or maytansine (DM1)).

[0150] Example 27: An immune conjugate as described in any of Examples 1 to 26, wherein the therapeutic agent is olistatin or a derivative thereof, a topoisomerase 1 inhibitor, such as MMAE, MMAF, ethatec or an ethatec derivative.

[0151] Example 28: An immune conjugate as described in any of Examples 1 to 27, wherein the linker connecting the recombinant antibody or the sdAb to the therapeutic agent is a cleavable linker or a non-cleavable linker.

[0152] Example 29: An immune conjugate as in Example 28, wherein the cleavable linker is a protease-sensitive linker (e.g., valine-citrulline (VC) dipeptide, valine-alanine (VA) dipeptide, valine-lysine (VL) dipeptide, valine-arginine (VR) dipeptide, or glutamate-valine-citrulline (EVC) tripeptide), a pH-sensitive linker (e.g., SS disulfide linker, hydrazone linker, ester linker, or acetylamine linker), and a glutathione-sensitive linker (e.g., N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) or N-succinimidyl-4-(2-pyridyldithio)valerate (SPP)).

[0153] Example 30: An immune conjugate as in Example 28, wherein the non-cleavable linker is maleic diaminohexyl (MC), maleic diaminomethylcyclohexane-1-carboxylate (MCC), and succinimido-4-[N-maleic diaminomethyl]cyclohexane-1-carboxylate (SMCC).

[0154] Example 31: An immune conjugate as described in any of Examples 1 to 28, wherein the linker connecting the recombinant antibody or the sdAb to the therapeutic agent is a polyethylene glycol (PEG) chain or a protease-sensitive linker linked to the PEG chain.

[0155] Example 32: An immune conjugate as described in any of Example 31, wherein the PEG chain has three repeating EG units (i.e., PEG3).

[0156] Example 33: An immune conjugate as in Example 31, wherein the protease-sensitive linker linked to the PEG chain is a VC dipeptide linked to the PEG chain.

[0157] Example 34: An immune conjugate as described in any of Examples 1 to 33, wherein the immune conjugate is in the form of a recombinant antibody (Fc) or an sdAb-PEG chain-VC dipeptide-therapeutic agent. Example

[0158] Materials and Methods

[0159] Preparation of recombinant antibodies (hereinafter referred to as "HCAb")

[0160] Following the manufacturer's instructions, EXPI293F™ and EXPICHO-S™ cells were used to express and purify selected HCAbs via ribosomes. In short, EXPI293F™ cells were transfected with HCAb constructs using EXPIFECTAMINE™ 293 and EXPIFECTAMINE™ CHO reagents. Cell culture medium was collected, and HCAbs were purified using a Protein G column.

[0161] The resulting HCAb contains a homodimer (in the form of VHH-hIgG1) fused from a camel-derived VHH domain and a human-derived Fc region. According to the analysis, each VHH domain contains the amino acid sequence of SEQ ID NO: 4 or 10, with CDR-1, CDR-2, and CDR-3 containing the amino acid sequences of SEQ ID NO: 1-3 or SEQ ID NO: 7-9 respectively (Table 1), and the human-derived Fc region contains the amino acid sequence of SEQ ID NO: 5.

[0162] Table 1. Amino acid sequences of the VHH domain of HCAbs described in this paper. name Amino acid sequence (SEQ ID NO: 4) VHH domain of chimeric HCAb QVKLEESGGGLVQAGGSLRLSCAASGGSFSSYMLAWFRQAPGKEREFVANISPGGYTYYADSAQGRFTISRENAKNTMYLQMNSLNPEDTAVYYCAADIRVLAAAQVASYDYWGQGTQVTV name Amino acid sequence (SEQ ID NO: 10) VHH domain of humanized HCAb [E]VQ [L]VESGGGLVQ [P]GGSLRLSCAASG [RT]FS [E]YMLAWFRQAPGKEREFVANISPGGYTYYADSA [K]GRFTISR [D]NAKNTMYLQMNSL [R]PEDTAVYYCAAD [L]RVLAAAQVASYDYWGQGTQVTV The CDR sequences in the VHH domain are marked in bold and include CDR-L1, CDR-L2 and CDR-L3 sequentially from the N end to the C end.

[0163] Preparation of antibody-drug conjugates (ADCs) using trimannose-based ADC technology

[0164] According to the procedure described in WO 2018 / 126092 A1, a chimeric ADC is prepared using trimannosyl ADC technology, the entire contents of which are incorporated herein by reference. In short, HCAb (VHH-hIgG1) is first modified by β-galactosidase and N-acetylglucosamine synthase (NAGS). The resulting trimannosyl Ab is then bound to UDP-N-azidoacetylglucosamine (UDP-GlcNAz) using N-acetylglucosamine transferase I (GnT1) and N-acetylglucosamine transferase II (GnT2) to produce an HCAb with four azido (Az) groups at the terminal N-acetylglucosamine (i.e., trimannosyl HCAb-4Az). Next, trimannosyl HCAb-4Az is bound to the DBCO-PEG3-VC-PAB-MMAE molecule via a copper-free click reaction between the azide group and the DBCO group. Detailed steps for preparing the ADC of this invention are provided below.

[0165] (i) Preparation of trimannosyl Ab

[0166] 18.57 mg of HCAb (pH 6.08) was mixed with 1 mg of β-galactosidase from Akkermansia muciniphila (Akk) and 0.2 mg of NAGS. The mixture was diluted to a final concentration of 5 mg / mL with 25 mM sodium citrate (pH 6.08) and then incubated at 37 °C for 3 hours. The product was filtered through a 0.22 µm syringe filter.

[0167] (ii) Preparation of trimannosyl HCAb-4Az

[0168] The filtered sample was subjected to protein A column treatment. The column was then washed sequentially with 1× phosphate-buffered saline (PBS; pH 7.2) and 25 mM 3-(N-hydroxyl)propanesulfonic acid (MOPS; pH 7.5). 1.5 mg of GnT1, 0.5 mg of GnT2, and 10 mg of UDP-GlcNAz were mixed into a solution containing 25 mM MOPS and 10 mM MnCl2, and then added to the column and circulated at room temperature for 3 hours. After washing with 0.1 M sodium citrate (pH 6.1), the column was dissociated with 0.1 M sodium citrate (pH 3.0). The dissociated product was immediately neutralized with 1 M tris(hydroxymethyl)aminomethane (Tris buffer; pH 9.0) and then filtered through a 0.22 μm syringe filter.

[0169] According to quality analysis, the resulting antibody has four azide groups linked to its Fc region (data not shown).

[0170] (iii) Preparation of VHH-hIgG1-4(DBCO-PEG3-vc-PAB-MMAE)

[0171] Replace the buffer for trimannosyl HCAb-4Az (15.42 mg) with 25 mM 2-(N-hydroxyl) ethanesulfonic acid (MES) buffer (pH 6.5) and adjust the final concentration to 5 mg / mL. Slowly add a solution containing dimethyl sulfoxide (DMSO; 3.2 mL, 20% buffer) and DBCO-PEG3-VC-PAB-MMAE (10 mM DMSO stock solution, 20 equivalents of antibody, 1.6 mL) to the antibody solution. Incubate the reaction mixture at 37°C for 20 hours in a shaking incubator (150 rpm). Concentrate the ADC product and remove unbound PEG3-VC-PAB-MMAE using a centrifugal filter with a nominal molecular weight limit (NMWL) of 30 kDa and 25 mM MES (pH 6.5) (Settings: 4°C, 4000 rpm, 15 min; repeated four times). The ADC concentration was estimated by measuring the absorbance at 280 nm (using 1.36 mL mg⁻¹ cm⁻¹ as the absorption coefficient at 280 nm). The recovery yield was determined by the A280 value.

[0172] According to the results of liquid chromatography-mass spectrometry (LC-MS), the drug-to-antibody ratio (DAR) of the ADC was approximately 4 (Figures 2A and 2B), indicating that four effective loads (i.e., PEG3-VC-PAB-MMAE) were linked to the ADC. The resulting ADC is named "VHH-hIgG1-4 (DBCO-PEG3-VC-PAB-MMAE)," which has four MMAE molecules linked to the Fc region of hIgG1 via a linker composed of PEG3 and VC dipeptides. The concentration of the ADC was approximately 5.84 mg / mL, and the recovery yield was approximately 97.28%.

[0173] Preparation of antibody-drug conjugates (ADCs) via cysteine ​​binding.

[0174] ADC synthesis was performed using a cysteine ​​crosslinking method. In short, humanized HCAb and sdAb were treated with varying amounts of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) for 2 hours at 30°C in a binding buffer (100 mM sodium phosphate, 150 mM sodium chloride, and 2 mM EDTA, pH 7.5) to reduce disulfide bonds (SS reduced to -SH). The resulting free thiol group (-SH) facilitated the binding of the reagent linked to the maleic diiminohexyl group (MC). In the subsequent binding step, varying amounts of vc-MMAE (MC-Val-Cit-PAB-MMAE) were added to the solution and incubated at 30°C for 18 hours. The reaction was then quenched with N-acetycysteine ​​(NAC). The ADC product was purified by centrifugation at 4000 rpm using an Amicon® Ultra 0.5 mL centrifuge filter, and the buffer was replaced with PBS. The drug-to-antibody ratio (DAR) of the final product was determined by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) using a Tosoh TSKgel Butyl-NPR 4.6 mm ID × 10 cm column.

[0175] Flow cytometry analysis technology

[0176] The binding affinity of the HCAb of this invention for various types of cancer cell lines was determined by flow cytometry. In short, cancer cells were collected using enzyme-free cell dissociation buffer and then washed with ice-cold PBS containing 2% fetal bovine serum (FBS). Cells were stained with the HCAb of this invention (1:100 dilution) in PBS containing 2% FBS for 1 hour at 4°C. After washing the cells three times with PBS, they were incubated with a secondary antibody (Alexa Fluor™ 488-bound donkey anti-mouse IgG (H+L)) at 4°C for 30 minutes. The expression level of CEACAM6 on the cell surface was analyzed by flow cytometry. A gate control strategy was used to exclude debris and dead cells, and a positive gate was set based on fluorescence intensity under isotype control guidance. The percentage of positive cells was calculated using the following formula: (Number of events in the positive gate / Total number of events in the live cell gate) × 100.

[0177] In vitro killing analysis

[0178] One × 10⁴ cancer cells were seeded overnight in 96-well plates. Different concentrations of ADC (1 μM to 1 pM, serially diluted 10-fold) were added to each well, and the cells were incubated at 37°C for 48 hours. The culture medium was removed, and the cells were washed with PBS. A solution containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide (MTT; 5 mg / mL) was added to each well, and the cells were incubated for 90 minutes. Next, MTT crystals were dissolved in DMSO. Cell viability was measured using an enzyme-linked immunosorbent assay (ELISA) reader at 450 nm absorbance. Cell viability (%) was determined by dividing the OD value of the ADC-treated cells by the OD value of the untreated (control) cells.

[0179] Animal research

[0180] One × 10⁷ HT-29 cells were subcutaneously injected into SCID mice. One week after cancer cell inoculation, the ADC of this invention was administered weekly via intravenous tail injection at a dose of 5 mg / kg. Simultaneously, FOLFIRI + cetuximab (a clinical regimen for colorectal cancer, wherein the FOLFIRI regimen comprises 40 mg / kg irinotecan, 30 mg / kg leucovorin calcium, and 55 mg / kg 5-fluorouracil, administered intraperitoneally) and cetuximab (10 mg / kg) were administered intravenously. Tumor volume was measured weekly using a diameter and calculated using the following formula: (length × width 2 × 0.45).

[0181] Example 1: Characterization of chimeric HCAbs

[0182] According to the procedures described in the "Materials and Methods" section of this disclosure, the binding affinity of the chimeric HCAb of the present invention to cancer cells was determined by flow cytometry. As shown in Figure 3 and Table 2, the HCAb of the present invention recognizes multiple types of cancer cells expressing CEACAM6.

[0183] Table 2. Binding affinity of HCAb to cancer cells expressing CEACAM6 cell lines Percentage of positive cells LS1034 (Ras-mutant colorectal cancer) 90.8% HT29 (Ras wild-type colorectal cancer) 86.2% CACO2 (Ras wild-type colorectal cancer) 80.4% FER (cetuximab-resistant head and neck cancer) 78.1% PC9 (non-small cell lung cancer) 82.2% MCF-7 (breast cancer) 31.3% MDA-MB-231 (Triple-negative breast cancer) 5.12% MDA-MB-468 (Triple-negative breast cancer) 89.6% H1299 (Non-small cell lung cancer) 0.5% CEACAM6 transduction to H1299 (H1299 CEACAM6) 86.3% THP-1 (human acute monocytic leukemia cells) 33.7% PBMC (a healthy donor) 10%

[0184] Example 2: Characterization of Chiped ADCs

[0185] According to the procedure described in the "Materials and Methods" section of this disclosure, HCAb was conjugated with MMAE, the HCAb containing a pair of isodimeric VHHs, each of which has SEQ ID NO: 4. SDS-PAGE data shows the conjugation of MMAE and HCAb (VHH-hIgG1) (Figure 4). Based on the analytical results, the ADC (DAR) was approximately 4 (Figures 2A and 2B).

[0186] The cytotoxicity of the chimeric ADC was assessed by culturing it for 48 hours with H1299 CEACAM6 cells (cancer cells expressing CEACAM6) or cells not expressing CEACAM6 (including LX2 and H1299 CEACAM5; serving as the control group in this study), and cell viability was subsequently determined by MTT assay. As depicted in Figure 5A, the ADC of this invention exhibited cytotoxic activity against cancer cells expressing CEACAM6, while no significant cytotoxicity was detected in cells not expressing CEACAM6, indicating that the ADC of this invention can specifically target and destroy cancer cells expressing CEACAM6. The cytotoxic effect of the ADC of this invention is confirmed in Figure 5B, which shows that, compared to THP-1 cancer cells (a human monocytic cell line derived from patients with acute monocytic leukemia), the ADC of this invention exhibited lower cytotoxicity against normal peripheral blood mononuclear cells (PBMCs). The IC50 of the chimeric HCAb-ADC against different cell types is summarized in Figure 5C.

[0187] Example 3: In vivo efficacy of chimeric ADCs in animal models

[0188] This example examines the antitumor effect of the chimeric ADC containing MMAE or ethatecan in Example 2 in an animal model. Figure 6A shows that, compared to the control group (PBS treatment), weekly administration of 5 mg / kg of the chimeric ADC containing MMAE for six weeks significantly inhibited tumor growth in the animal model. Notably, the antitumor effect of the chimeric ADC containing MMAE was superior to that of the current RAS wild-type colorectal cancer treatment regimen (i.e., the FOLFIRI + cetuximab regimen) (Figure 6A). Treatment with the ADC of this invention did not alter the body weight of mice (Figure 6B), indicating good tolerability in mice. Furthermore, the antitumor effect of the chimeric ADC containing ethatecan was examined in the same animal model. When the tumor volume reached approximately 700 mm³, SCID mice were intravenously administered 1 mg / kg of the chimeric ADC containing ethatecan. Two weeks after treatment, the tumor volume decreased to approximately 300 mm³ (Figure 7), and there was no significant change in body weight (Figure 8). In summary, this indicates that chimeric HCAbs containing MMAE or ethathecan have strong anticancer activity and are well tolerated in animal models.

[0189] Chimeric HCAbs exhibit low affinity for normal tissues (Figure 9). Immunohistochemical (IHC) staining with chimeric HCAbs was used to assess the expression of CEACAM6 in normal human tissues. Detection of weak or no signal with chimeric HCAbs indicates low expression of CEACAM6 in normal tissues.

[0190] Anti-CEACAM6 HCAbs specifically recognize colorectal cancer, lung cancer, cholangiocarcinoma, and pancreatic cancer tissues (Figure 10). Immunohistochemical (IHC) staining with chimeric HCAbs was used to assess CEACAM6 expression in different cancer types. Strong signal detection by chimeric HCAbs indicates high expression of CEACAM6 in cancer tissues.

[0191] In summary, this disclosure provides a novel ADC. According to examples of this disclosure, the chimeric ADC exhibits binding affinity and specificity to tumor cells and effectively inhibits tumor growth in animal models. Therefore, the chimeric ADC can serve as a novel and potential agent for the treatment of cancer.

[0192] It should be understood that the above description of the embodiments is merely illustrative and various modifications can be made by those skilled in the art. The above specification, examples, and materials provide a complete description of the structure and use of exemplary embodiments of the present invention. Although various embodiments of the present invention have been described above with some degree of specificity or with reference to one or more individual embodiments, those skilled in the art can make many changes to the disclosed embodiments without departing from the spirit or scope of the present invention.

[0193] Example 4: Anticancer effects of humanized HCAb-ADC

[0194] Following the procedures described in the "Materials and Methods" section of this disclosure, a humanized HCAb-ADC was generated by linking MMAE to a pair of homodimeric VHHs via cysteine ​​binding. Each of the homodimeric VHHs has SEQ ID NO: 4. The cytotoxicity of the humanized ADC was assessed by co-culturing the ADC with PC-9 cells (cancer cells expressing CEACAM6) for 96 hours, followed by cell viability determination using MTT assay. The humanized ADC significantly inhibited cancer cell growth, with an IC50 (half-maximal inhibitory concentration) of 10.2 nM (Figure 11). The humanized HCAb-ADC also inhibited tumor growth in an animal model in a dose-dependent manner, demonstrating a strong and specific killing effect on CEACAM6-expressing tumors (Figure 12). No significant weight loss was observed after treatment with the humanized HCAb-ADC, indicating good tolerability in animals (Figure 13). mpk represents mg / kg.

[0195] Example 5: Anticancer effects of sdAb-ADC

[0196] Following the procedures described in the "Materials and Methods" section of this disclosure, sdAb-ADC was generated by linking MMAE to a VHH-containing sdAb via cysteine ​​binding. The VHH has SEQ ID NO: 11. The cytotoxicity of sdAb-ADC was assessed by co-culturing sdAb-ADC with a cancer cell line expressing CEACAM6 for 96 hours, followed by cell viability determination using MTT assay. Compared to normal IgG-ADC (IC50 > 100 nM), sdAb-ADC significantly inhibited cancer cell growth, with an IC50 of approximately 1 nM (Figure 14). Furthermore, administration of sdAb-ADC (4 mg / kg) inhibited tumor growth in an animal model, demonstrating its strong killing effect on tumors expressing CEACAM6 (Figure 15). sdAb-ADC did not significantly cause weight loss, indicating good tolerability in animals (Figure 16).

[0197] In summary, this disclosure provides novel ADCs. According to examples of this disclosure, the ADCs of this invention (chimeric ADCs, humanized ADCs, and sdAb-ADCs) exhibit binding affinity and specificity to tumor cells and effectively inhibit tumor growth in animal models. Therefore, the ADCs of this invention can serve as novel and potential agents for the treatment of cancer.

[0198] It should be understood that the above description of the embodiments is merely illustrative and various modifications can be made by those skilled in the art. The above specification, examples, and materials provide a complete description of the structure and use of exemplary embodiments of the present invention. Although various embodiments of the present invention have been described above with some degree of specificity or with reference to one or more individual embodiments, those skilled in the art can make many changes to the disclosed embodiments without departing from the spirit or scope of the present invention. sequence list name sequence SEQ ID NO CDR-1 (Matching) GGSFSSYMLA 1 CDR-1 (Humanization) G [RT]FS [E]YMLA 7 CDR-2 (Matching) NISPGGYTYYADSAQG 2 CDR-2 (Humanization) NISPGGYTYYADSA [K]G 8 CDR-3 (Matching) DIRVLAAAQVASYDY 3 CDR-3 (Humanization) D [L]RVLAAAQVASYDY 9 VHH domain (Chimera or sdAb) QVKLEESGGGLVQAGGSLRLSCAASGGSFSSYMLAWFRQAPGKEREFVANISPGGYTYYADSAQGRFTISRENAKNTMYLQMNSLNPEDTAVYYCAADIRVLAAAQVASYDYWGQGTQVTV 4 Heavy chain-1 with signal peptide (Chimeric HCAb) MEFGLSWVFLVAILKGVQCQVKLEESGGGLVQAGGSLRLSCAASGGSFSSYMLAWFRQAPGKEREFVANISPGGYTYYADSAQGRFTISRENAKNTMYLQMNSLNPEDTAVYYCAADIRVLAAAQVASYDYWGQGTQVTVVD(GGGSG as applicable)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 5 Heavy Chain-2 (Chimeric HCAb) QVKLEESGGGLVQAGGSLRLSCAASGGSFSSYMLAWFRQAPGKEREFVANISPGGYTYYADSAQGRFTISRENAKNTMYLQMNSLNPEDTAVYYCAADIRVLAAAQVASYDYWGQGTQVTVSSMTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK 6 VHH domain (humanized or sdAb) [E]VQ [L]VESGGGLVQ [P]GGSLRLSCAASG [RT]FS [E]YMLAWFRQAPGKEREFVANISPGGYTYYADSA [K]GRFTISR [D]NAKNTMYLQMNSL [R]PEDTAVYYCAAD [L]RVLAAAQVASYDYWGQGTQVTV 10 Mutant (S122C) VHH domain (chimeric or sdAb) QVKLEESGGGLVQAGGSLRLSCAASGGSFSSYMLAWFRQAPGKEREFVANISPGGYTYYADSAQGRFTISRENAKNTMYLQMNSLNPEDTAVYYCAADIRVLAAAQVASYDYWGQGTQVTVCS 11 Linker connecting the N-terminus of VHH to Fc GGGSG GGGGS AHHSEDP EPKTPKPQPQPQPQPQPQPNPTTE STPPTPSPSTPP 12 15 16 17 18 Chimeric HCAb Question VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 13 Humanized HCAb EVQLVESGGGLVQPGGSLRLSCAASGRTFSEYMLAWFRQAPGKEREFVANISPGGYTYYADSAKGRFTISRDNAKNTMYLQMNSLRPEDTAVYYCAADLRVLAAAQVASYDYWGQGTQVTVSS (GGGSG if applicable)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 14 Heavy chain-2 with signal peptide (Chimeric HCAb) MEFGLSWVFLVAILKGVQCQVKLEESGGGLVQAGGSLRLSCAASGGSFSSYMLAWFRQAPGKEREFVANISPGGYTYYADSAQGRFTISRENAKNTMYLQMNSLNPEDTAVYYCAADIRVLAAAQVASYDYWGQGTQVTVSSMTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK 19

Claims

1. An immune conjugate comprising: a recombinant anti-carcinoembryonic antigen-associated cell adhesion molecule 6 (CEACAM6) antibody comprising (i) a single variable heavy chain domain (VHH domain) or a pair of homodimeric or heterodimeric VHHs and crystallizable region fragments (Fc regions) of immunoglobulins, the Fc region being fused to each of the (etc.) VHH domains; or (ii) a single-domain antibody (sdAb) comprising the VHH domain, wherein the VHH domain comprises: a first chain complementarity-determining region (CDR-1) comprising an amino acid sequence of SEQ ID NO: 1 or 7; a second CDR (CDR-2) comprising an amino acid sequence of SEQ ID NO: 2 or 8; and a third CDR (CDR-3) comprising an amino acid sequence of SEQ ID NO: 3 or 9; and a therapeutic agent; The therapeutic agent is linked via a linker to the Fc region of the recombinant antibody or the VHH of the sdAb, wherein the therapeutic agent is a cytotoxic drug, a radioactive nucleus, a cytokine, a hormonal drug, an immunostimulant, or an immunotherapy drug.

2. The immune conjugate of claim 1, wherein the recombinant antibody is a chimeric antibody, a humanized antibody, or wherein the sdAb is a humanized antibody.

3. The immune conjugate of claim 1 or 2, wherein the VHH comprises: CDR-1 comprising the amino acid sequence of SEQ ID NO: 1; CDR-2 comprising the amino acid sequence of SEQ ID NO: 2; and CDR-3 comprising the amino acid sequence of SEQ ID NO:

3.

4. The immune conjugate of claim 1 or 2, wherein the VHH comprises: CDR-1 comprising the amino acid sequence of SEQ ID NO: 7; CDR-2 comprising the amino acid sequence of SEQ ID NO: 8; and CDR-3 comprising the amino acid sequence of SEQ ID NO:

9.

5. An immune conjugate as claimed in claim 1 or 2, wherein the VHH domain contains an amino acid sequence that is at least 85% identical to SEQ ID NO: 4, 10 or 11.

6. The immune conjugate of claim 1 or 2, wherein the VHH domain comprises an amino acid sequence that is 100% identical to SEQ ID NO: 4, 10 or 11.

7. An immune conjugate as requested in item 1 or 2, wherein the VHH domain is derived from a camelid heavy chain antibody.

8. The immunoconjugate of claim 1 or 2, wherein the recombinant antibody comprises a pair of homodimeric VHHs, wherein each of the VHH domains comprises an amino acid sequence of SEQ ID NO: 4, 10 or 11.

9. The immunoconjugate of claim 1 or 2, wherein the recombinant antibody comprises a pair of heterodimeric VHHs, wherein the paired VHHs are distinct and each of the VHH domains comprises an amino acid sequence selected from SEQ ID NO: 4, 10 and 11.

10. The immunoconjugate of claim 1 or 2, wherein the recombinant antibody comprises a single VHH containing the amino acid sequence of SEQ ID NO: 4, 10 or 11.

11. The immune conjugate of claim 1 or 2, wherein the immunoglobulin is human immunoglobulin G (IgG) or immunoglobulin A (IgA).

12. The immune conjugate of claim 11, wherein the immunoglobulin is IgG1.

13. The immune conjugate of claim 1 or 2, wherein the recombinant antibody comprises the amino acid sequence of SEQ ID NO: 5, 6, 13, 14 or 19.

14. The immune conjugate of claim 1, wherein the cytotoxic drug is a mitotic inhibitor or a topoisomerase 1 inhibitor.

15. The immune conjugate of claim 1, wherein the cytotoxic drug is monomethylolpropamine E (MMAE) or monomethylolpropamine F (MMAF).

16. An immune conjugate as claimed in claim 1 or 2, wherein the linker is a cleavable linker or a non-cleavable linker.

17. The immune conjugate of claim 16, wherein the cleavable linker is a protease-sensitive linker, a pH-sensitive linker, or a glutathione-sensitive linker.

18. The immune conjugate of claim 17, wherein the protease-sensitive linker is a valine-citrulline (VC) dipeptide, a valine-alanine (VA) dipeptide, a valine-lysine (VL) dipeptide, a valine-arginine (VR) dipeptide, or a glutamate-valine-citrulline (EVC) tripeptide.

19. The immune conjugate of claim 17, wherein the pH-sensitive linker is an SS disulfide linker, a hydrazone linker, an ester linker, or a amide linker.

20. The immune conjugate of claim 17, wherein the glutathione-sensitive linker is N-succinimido-4-(2-pyridyldithio)butyrate (SPDB) or N-succinimido-4-(2-pyridyldithio)valerate (SPP).

21. The immunoconjugate of claim 16, wherein the non-cleavable linker comprises maleic diaminohexyl (MC), maleic diaminomethylcyclohexane-1-carboxylate (MCC), and succinimido-4-[N-maleic diaminomethyl]cyclohexane-1-carboxylate (SMCC).

22. A pharmaceutical composition comprising an immune conjugate as claimed in any one of claims 1 to 21 and a pharmaceutically acceptable carrier.

23. A pharmaceutical composition for a method of treating cancer in an individual, wherein the composition comprises an effective amount of an immune conjugate as claimed in any one of claims 1 to 21.

24. The pharmaceutical composition used in the method as claimed in claim 23, 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, hepatocellular carcinoma, melanoma, esophageal cancer, multiple myeloma, head and neck squamous cell carcinoma, or bile duct cancer.

25. The pharmaceutical composition used in the method as claimed in claim 23, wherein the system is human.

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