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

Abstract

Disclosed herein is an immunoconjugate comprising a recombinant antibody, a therapeutic agent, and a linker connecting the therapeutic agent to the recombinant antibody. According to some embodiments of the present disclosure, the recombinant antibody comprises a VHH domain, in which the CDR-1, CDR-2 and CDR-3 of the VHH domain respectively comprise the amino acid sequences of SEQ ID NOs: 1-3 and 7-9. Also disclosed herein are a pharmaceutical composition comprising the immunoconjugate, and methods of treating cancers by using the present immunoconjugate or pharmaceutical composition.

Description

IMMUNOCONJUGATE, PHARMACEUTICAL COMPOSITION COMPRISING THE SAME, AND USES THEREOF IN TREATING CANCERSBACKGROUND OF THE INVENTION

[0001] 1. FIELD OF THE INVENTION

[0002] The present disclosure in general relates to the field of disease treatment. More particularly, the present disclosure relates to an immunoconjugate, a pharmaceutical composition comprising the immunoconjugate, and uses thereof in the treatment of cancers.

[0003] 2. DESCRIPTION OF RELATED ART

[0004] Cancer is a group of diseases involving abnormal cell growth with the ability to invade or migrate to the healthy organs or tissues of the affected subject. It is a leading cause of death globally. According to the report of World Health Organization (WHO), cancer accounts for nearly 10 million deaths in 2020, or nearly one in six deaths. The most common types of cancer include lung cancer, breast cancer, prostate cancer, colorectal cancer, gastric cancer, skin cancer, and liver cancer. The primary treatments for cancers include surgery, chemotherapy, radiation therapy, hormonal therapy and targeted therapy. However, most of these treatments cannot produce a satisfactory effect on cancer patients due to the limitations of, for example, the development of drug-resistance, low-specificity, low-efficiency, and / or adverse side-effects, such as pain, anemia, bleeding, lymphedema, diarrhea, constipation, fatigue, appetite loss, infection, neuropathy and memory problems.

[0005] In view of the foregoing, there is a continuing interest in developing a novel agent and / or method for treating cancers in a more efficient and safer manner.SUMMARY

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

[0007] As embodied and broadly described herein, the first aspect of the disclosure is directed to an immunoconjugate comprising a recombinant antibody or a single-domain antibody (sdAb) and a therapeutic agent, wherein the therapeutic agent links to the recombinant antibody or thesdAb, optionally via a linker. According to certain embodiments of the present disclosure, the recombinant antibody comprises (i) a single variable heavy chain domain (VHH domain) or a pair of homodimeric or heterodimeric VHHs and a fragment crystallizable region (Fc region) of an immunoglobulin fused to each of the VHH domain(s) optionally via a peptide linker. In some embodiments, the therapeutic agent is connected to the Fc region of the recombinant antibody via the linker.

[0008] According to some embodiments of the present disclosure, the VHH domain of the recombinant antibody comprises a first chain complementarity determining region (CDR-1), a second CDR (CDR-2) and a third CDR (CDR-3), which respectively comprise the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NOs: 1 or 7, 2 or 8 and 3 or 9.

[0009] According to some preferred embodiments, the VHH domain comprises an amino acid sequence 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 specific embodiment, the VHH domain comprises an amino acid sequence 100% identical to SEQ ID NO: 4, 10 or 11.

[0010] According to certain embodiments, the VHH domain is derived from a camelid heavychain antibody.

[0011] According to certain embodiments, the antibody is in chimeric form, humanized form or human form. According to another certain embodiment, the sdAb is in a chimeric form.

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

[0013] According to certain embodiments, the VHH domain fused to the N-termini of the Fc region of the immunoglobulin via a peptide linker, wherein the peptide linker is a short peptide comprising Gly and Ser.

[0014] Depending on desired purpose, the therapeutic agent may be a cytotoxic drug, a radioactive nuclide, a cytokine, a hormone drug, an immune stimulating agent, or an immunotherapeutic drug. Preferably, the therapeutic agent is the cytotoxic drug. According to some embodiments of the present disclosure, the therapeutic agent is auristatin, irinotecan, exatecan, levofolinate, 5 -fluorouracil or a derivative thereof. In one exemplary embodiment, the therapeutic agent is monomethyl auristatin E (MMAE). In another exemplary embodiment, the therapeutic agent is monomethyl auristatin F (MMAF).

[0015] Also disclosed herein is a pharmaceutical composition for treating cancers. The pharmaceutical composition comprises the immunoconjugate of the present disclosure, and optionally, a pharmaceutically acceptable carrier.

[0016] Another aspect of the present disclosure pertain to a method of treating a cancer in a subject. The method comprises administering to the subject an effective amount of the immunoconjugate or pharmaceutical composition of the present disclosure.

[0017] Examples of the cancer treatable with the present method include, but are not limited to, gastric cancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer, renal cancer, colorectal cancer, cervical cancer, ovarian cancer, brain tumor, prostate cancer, hepatocellular carcinoma, melanoma, esophageal carcinoma, multiple myeloma, and head and neck squamous cell carcinoma.

[0018] The subject is a mammal; preferably, a human.

[0019] Many of the attendant features and advantages of the present disclosure will becomes better understood with reference to the following detailed description considered in connection with the accompanying drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present description will be better understood from the following detailed description read in light of the accompanying drawings briefly discussed below.

[0021] Fig. 1A is a schematic diagram depicting the structure of the recombinant antibody of the present antibody-drug conjugate (ADC) according to embodiments of the present disclosure.

[0022] Fig. IB is a schematic diagram depicting the structure of the present ADC according to embodiments of the present disclosure. A: payload, i.e., PEG3-VC-MMAE.

[0023] Figs. 2A and 2B respectively depict the results of mass analysis of the present ADC according to some embodiments of the present disclosure. Fig. 2A: intact form of the ADC; Fig. 2B : reduced form of the ADC.

[0024] Fig. 3 depicts the results of flow cytometer according to Example 1 of the present disclosure.

[0025] Fig. 4 is a photogram depicting the result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Example 2 of the present disclosure.

[0026] Figs. 5 A and 5B are line charts respectively depicting the effect of the present ADC on specified cells according to Example 2 of the present disclosure.

[0027] Figs. 6A and 6B are line charts respectively depicting the tumor volume (Fig. 6A) andbody weight (Fig. 6B) of tumor-bearing mice administered with the present ADC or specified treatment according to Example 3 of the present disclosure. The arrows in Fig. 6A indicated the day of the ADC treatment (a total of 6 doses).

[0028] Fig. 7 shows the anti-tumor effect of the chimeric ADC carrying exatecan.

[0029] Fig. 8 shows the body weight of the mice after administering the chimeric ADC carrying exatecan.Fig. 9 shows that the chimeric HCAb has a low affinity for normal tissues.

[0030] Fig. 10 shows that the chimeric HCAb specifically recognizes colorectal, lung, bile duct and pancreatic cancer tissues.

[0031] Fig. 11 shows that the humanized ADC significantly inhibits cancer cell growth.

[0032] Fig. 12 shows that the humanized HCAb-ADC inhibits tumor growth in animal models in a dose-dependent manner.

[0033] Fig. 13 shows that no significant weight loss is observed after the humanized HCAb- ADC treatment.

[0034] Fig. 14 shows that the sdAb-ADC significantly inhibits cancer cell growth.

[0035] Fig. 15 shows that the administration of sdAb-ADC suppresses tumor growth in animal models.

[0036] Fig. 16 shows that the sdAb-ADC does not significantly induce weight loss in animal models.

[0037] In accordance with common practice, the various described features / elements are not drawn to scale but instead are drawn to best illustrate specific features / elements relevant to the present invention. Also, reference numerals and designations in the various drawings are used to indicate elements / parts.DETAILED DESCRIPTION OF THE INVENTION

[0038] The detailed description provided below in connection with the appended drawings is intended as a description of the present examples and is not intended to represent the only forms in which the present example may be constructed or utilized. The description sets forth the functions of the example and the sequence of steps for constructing and operating the example. However, the same or equivalent functions and sequences may be accomplished by different examples.

[0039] I. DEFINITION

[0040] For convenience, certain terms employed in the specification, examples and appendedclaims are collected here. Unless otherwise defined herein, scientific and technical terminologies employed in the present disclosure shall have the meanings that are commonly understood and used by one of ordinary skill in the art. Also, unless otherwise required by context, it will be understood that singular terms shall include plural forms of the same and plural terms shall include the singular. Specifically, as used herein and in the claims, the singular forms “a” and “an” include the plural reference unless the context clearly indicates otherwise. Also, as used herein and in the claims, the terms "at least one" and "one or more" have the same meaning and include one, two, three, or more.

[0041] In this application, the phrase "and / or" is a grammatical conjunction that should be interpreted to mean that one or more of the connected instances may take place.

[0042] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in the respective testing measurements. Also, as used herein, the term "about" generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term "about" means within an acceptable standard error of the mean when considered by one of ordinary skill in the art. Other than in the operating / working examples, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages such as those for quantities of materials, durations of times, temperatures, operating conditions, ratios of amounts, and the likes thereof disclosed herein should be understood as modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and attached claims are approximations that can vary as desired. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0043] The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multi-specific or multivalent antibodies (e.g, bi-specific antibodies), chimeric antibodies, heavy-chain antibodies (HCAbs) and antibody fragments so long as they exhibit the desired biological activity. The term "antibody fragment" refers to a portion of a full-length antibody, generally the antigen binding or variable domain (i.e., VH and / or VL domains) of a full-length antibody. Examples of the antibody fragment include VHH domain (also known as single domain antibody (sdAb) or nanobody (Nb)), VHH dimer, fragment antigen-binding (Fab), Fab', F(ab')2, single-chain variable fragment (scFv), diabody, linear antibody, single-chain antibody molecule, and multi-specific antibody formed fromantibody fragments.

[0044] As used herein, the term "recombinant antibody" refers to an antibody that is expressed and isolated from a cell or cell line transfected with an expression vector comprising the coding sequence of the antibody, where said coding sequence is not naturally associated with the cell.

[0045] As used herein, the term "heavy-chain antibody" (HCAb) refers to an antibody which consists of two heavy chains and is devoid of light chains (See, for example, Hamers-Casterman, et al. Nature. 1993; 363: 446-448). Specifically, the HCAbs are composed of two heavy chains linked by a covalent disulfide bond, in which each heavy chain in the HCAb has a variable domain at one end. The variable domain of the heavy chain of the HCAb is referred to as “VHH domain” in order to distinguish it from the variable domain (VH domain) of the heavy chain of “conventional” antibody that has two heavy chains and two light chains. HCAbs, which naturally occur in camelids and sharks, can bind antigens via the VHH domains.

[0046] As used herein, the term "VHH domain" of an HCAb refers to the amino-terminal regions of heavy chains of the antibody. These regions are generally the most variable parts of an antibody and contain the antigen-binding sites. The term "variable" refers to the fact that certain portions of the VHH domains differ extensively in sequence among antibodies, and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the VHH domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the VHH domains. The more highly conserved portions of VHH domains are called the framework (FR). Each VHH domain of native heavy chains comprises four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions, and contribute to the formation of the antigen-binding site of antibodies. The constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as the participation of the antibody in antibodydependent cellular toxicity.

[0047] The term "complementarity determining region" (CDR) used herein refers to the hypervariable region of the VHH domain that forms a surface complementary to the 3-dimensional surface of a bound antigen. Proceeding from N-terminus to C-terminus, each VHH of the antibody comprises three CDRs (i.e., CDR-1, CDR-2, and CDR-3), which define the binding affinity and specificity of the VHH domains.

[0048] 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,a chimeric antibody typically includes the constant heavy (CH) and constant light (CL) domains from a human antibody, along with the variable heavy (VH) and variable light (VL) domains from an antibody derived from a non-human species. Suitable non-human sources can include animals such as mice, rats, hamsters, rabbits, and others.

[0049] The term "humanized antibody" refers to an antibody that is primarily derived from non- human sources, particularly concerning its heavy and light chains, and is designed to reduce or prevent immune responses in humans. This involves modifying the antibody to substitute specific amino acids in its functional regions. Typically, the constant domains of a humanized antibody are predominantly composed of human CH and CL domains.

[0050] The term "single-domain antibody" or "sdAb" refers to an antibody in which the complementary determining regions are part of a single domain polypeptide. This includes, but is not limited to, heavy-chain antibodies, antibodies that naturally lack light chains, single-domain antibodies derived from traditional four-chain antibodies, engineered antibodies, and singledomain scaffolds that are not derived from antibodies. Single-domain antibodies can encompass any existing or future variants in the field.

[0051] The term "constant region" or "constant domain" describes the carboxy-terminal segment of both the light and heavy chains of an antibody. This region does not participate directly in the binding of the antibody to its antigen but plays a role in various effector functions, such as interacting with Fc receptors. It refers to the part of the immunoglobulin molecule that has a more conserved amino acid sequence compared to the variable region, which contains the antigenbinding site. The constant region may include the CHI, CH2, and CH3 segments of the heavy chain, as well as the CL segment of the light chain.

[0052] The term "framework" or "FR" pertains to the variable region residues that flank the complementarity-determining regions (CDRs). FR residues are found in various antibody formats, including chimeric, humanized, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies. These residues are those in the variable domain that are not part of the hypervariable or CDR regions.

[0053] The term "linker" refers to a chemical entity that consists of a covalent bond or a chain of atoms, which serves to covalently connect a polypeptide to a drug moiety.

[0054] The term "fragment crystallizable region" or "Fc region", as used herein, refers to the tail region of an antibody that interacts with cell surface receptors called Fc receptors and / or some proteins of the complement system. In structure, the Fc region comprises, from N-terminus to C- terminus, at least a hinge region (a short sequence of the heavy chain that links the CHI and CH2domains), a CH2 domain (the second constant domain of the heavy chain) and a CH3 domain (the third constant domain of the heavy chain). An Fc region of an IgGl antibody can, for example, be generated by digestion of an IgGl antibody with papain.

[0055] "Percentage (%) sequence identity" with respect to any amino acid sequence identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the specific reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining the percentage of sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, sequence comparison between two amino acid sequences was carried out by computer program Blastp (protein-protein BLAST) provided online by Nation Center for Biotechnology Information (NCBI). The percentage sequence identity of a given sequence A to a subject sequence B (which can alternatively be phrased as a given sequence A that has a certain % sequence identity to a given sequence B) is calculated by the formula as follows:where X is the number of amino acid residues scored as identical matches by the sequence alignment program BLAST in that program’s alignment of A and B, and where Y is the total number of amino acid residues in the subject sequence B.

[0056] As discussed herein, minor variations in the amino acid sequences of antibodies are contemplated as being encompassed by the presently disclosed and claimed inventive concept(s), providing that the variations in the amino acid sequence maintain at least 85% sequence identity, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity. Antibodies of the present disclosure may be modified specifically to alter a feature of the peptide unrelated to its physiological activity. For example, certain amino acids can be changed and / or deleted without affecting the physiological activity of the antibody in this study (i.e., the ability to bind to coronavirus). In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic = aspartate, glutamate; (2) basic = lysine, arginine,histidine; (3) nonpolar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More preferred families are: serine and threonine are aliphatic-hydroxy family; asparagine and glutamine are an amide-containing family; alanine, valine, leucine and isoleucine are an aliphatic family; and phenylalanine, tryptophan, and tyrosine are an aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the peptide derivative. Fragments or analogs of antibodies can be readily prepared by those of ordinary skill in the art. Preferred amino- and carboxyl-termini of fragments or analogs occur near the boundaries of functional regions.

[0057] As used herein, the terms "link", "couple", "conjugate" and "connect" are used interchangeably to refer to any means of connecting two components either via direct linkage or via indirect linkage between two components.

[0058] The phrase "pharmaceutically acceptable" refers to substances that have received approval from a federal or state regulatory agency, or that are included in the United States Pharmacopeia, European Pharmacopeia, or other widely recognized pharmacopeias for use in animals, particularly in humans.

[0059] The term "excipient" denotes a pharmaceutically acceptable substance, composition, or vehicle, 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 their combinations. Additionally, "excipient" may also refer to a diluent, adjuvant (such as Freund's adjuvant, whether complete or incomplete), or vehicle.

[0060] As used herein, the term "treat," "treating" and "treatment" are interchangeable, and encompasses partially or completely preventing, ameliorating, mitigating and / or managing a symptom, a secondary disorder or a condition associated with cancers The term "treating" as used herein refers to application or administration of the immunoconjugate or pharmaceutical composition of the present disclosure to a subject, who has a symptom, a secondary disorder or a condition associated with cancers, with the purpose to partially or completely alleviate, ameliorate,relieve, delay onset of, inhibit progression of, reduce severity of, and / or reduce incidence of one or more symptoms, secondary disorders or features associated with cancers. Symptoms, secondary disorders, and / or conditions associated with cancers include, but are not limited to, nausea, vomiting, loss of appetite, bowel changes, constipation, fatigue, muscle weakness, broken bones, swelling or lump, blooding, cough, fever, neurological problems (e.g., seizures, vision changes, hearing change or drooping of the face), body weight changes (i.e., weight gain or weight loss), coma and pain. Treatment may be administered to a subject who exhibits only early signs of such symptoms, disorder, and / or condition for the purpose of decreasing the risk of developing the symptoms, secondary disorders, and / or conditions associated with cancers. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced as that term is defined herein. Alternatively, a treatment is "effective" if the progression of a symptom, disorder or condition is reduced or halted.

[0061] The term "effective amount" as referred to herein designate the quantity of a component which is sufficient to yield a desired response. For therapeutic purposes, the effective amount is also one in which any toxic or detrimental effects of the component are outweighed by the therapeutically beneficial effects. An effective amount of an agent is not required to cure a disease or condition but will provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered or prevented, or the disease or condition symptoms are ameliorated. The effective amount may be divided into one, two, or more doses in a suitable form to be administered at one, two or more times throughout a designated time period. The specific effective or sufficient amount will vary with such factors as the particular condition being treated, the physical condition of the patient (e.g., the patient’s body mass, age, or gender), the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives. Effective amount may be expressed, for example, in grams, milligrams or micrograms or as milligrams per kilogram of body weight (mg / Kg). Alternatively, the effective amount can be expressed in the concentration of the active component (e.g., the immunoconjugate of the present disclosure), such as molar concentration, mass concentration, volume concentration, molality, mole fraction, mass fraction and mixing ratio. Persons having ordinary skills could calculate the human equivalent dose (HED) for the medicament (such as the present immunoconjugate) based on the doses determined from animal models. For example, one may follow the guidance for industry published by US Food and Drug Administration (FDA) entitled “Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers” in estimating a maximum safe dosage for use in human subjects.

[0062] The terms "subject" refers to an animal including the human species that is treatable by the immunoconjugate, pharmaceutical composition and / or method of the present invention. The term "subject" is intended to refer to both the male and female gender unless one gender is specifically indicated.

[0063] II DESCRIPTION OF THE INVENTION

[0064] (I) Immunoconjugate (ADC)

[0065] The first aspect of present disclosure provides an immunoconjugate comprising a recombinant antibody or a single domain antibody (sdAb) and a therapeutic agent, wherein the therapeutic agent links to the recombinant antibody, i.e., in the form of an ADC (Fig. IB) or a sdAb (the antibody in Fig. IB can be replaced by sdAb), optionally via a linker. In some embodiments, the recombinant antibody and the sdAb may be in chimeric form, humanized form or human form and the sdAb is in a chimeric form.

[0066] According to the embodiments of the present disclosure, the recombinant antibody or sdAb is specific to carcinoembryonic antigen -related cell adhesion molecule 6 (CEACAM6; also known as "CD66c"), a member of the carcinoembryonic antigen (CEA) family known to be overexpressed in various types of solid tumors, including colorectal, gastric, endometrial, breast, ovarian, cervical, pancreatic and lung tumors, and head and neck squamous cell carcinoma. Accordingly, the recombinant antibody or sdAb may serve as a targeting element that directs the immunoconjugate specifically towards the CEACAM6-expressing cancer / tumor cells, thereby eliciting a cytotoxic effect in a cancer / tumor-specific manner.

[0067] (I)-l Antibodies

[0068] Recombinant antibody

[0069] Reference is now made to Fig. 1A, which is a schematic diagram depicting a recombinant antibody according to certain embodiments of the present disclosure. In structure, the present recombinant antibody comprises a pair of VHH domains that is homodimeric or heterodimeric, and an Fc region of an immunoglobulin linked to the pair of VHH domains. As known in the art, the Fc region comprises two identical protein fragments linked by a disulfide bond, in which each protein fragment comprises, from N-terminus to C-terminus, a hinge region, a CH2 domain and a CH3 domain. The pair of VHH domains is linked to the N-termini of the protein fragments. In one embodiment, as depicted in Fig. 1A, the present recombinant antibody is in the form of an HCAb (z. e., a homodimeric VHH-Fc complex) that comprises two heavy chains linked via the disulfide bond in the hinge region, in which each heavy chain comprises, from N- terminus to C-terminus, a VHH domain, a hinge region, a CH2 domain, and a CH3 domain. Inanother embodiment, the HCAb can be a heterodimeric VHH-Fc complex. In some embodiments, the recombinant antibody can be in chimeric form, humanized form or human form.

[0070] Alternatively, the pair of VHH domains are linked to the N-termini of the protein fragments via a linker. In one embodiment, the present recombinant antibody is in the form of an HCAb (z.e., a homodimeric VHH-linker-Fc complex) that comprises two heavy chains linked via the disulfide bond in the hinge region, in which each heavy chain comprises, from N-terminus to C-terminus, a VHH domain, a linker, a hinge region, a CH2 domain, and a CH3 domain. Any linker suitable for connecting the VHH and the N-termini of the protein fragment can be used in the present disclosure. In some embodiment, the linker is a short peptide comprising Gly and Ser. In some embodiments, the peptide linker has a (GlymSern)Pmotif, wherein m and n is any of integer of 1 to 10 and p is any of integer of 1 to 30, or a variant thereof. Variants of this motif may include amino acid substitutions, such as replacing Gly with Ser or vice vers. Examples of the peptide linker comprises an amino acid sequence including, but not limited to, GGGSG (SEQ ID NO: 12), GGGGS (SEQ ID NO: 15) or its repeats sequence (such as second, three, four or five repeats sequence), AHHSEDP (SEQ ID NO: 16), EPKTPKPQPQPQPQPQPNPTTE (SEQ ID NO: 17) or STPPTPSPSTPP (SEQ ID NO: 18) or a combination thereof. In a certain embodiment, the linker comprises GGGSG.

[0071] In another aspect, the recombinant antibody can comprise a single VHH domain that linked to the N-termini of the protein fragments optionally via a linker. The linking between the VHH domain and the protein fragment is as those described herein.

[0072] It is well-accepted that the binding properties (including the binding affinity and specificity) of an antibody are determined by its variable domain(s) (z'.e., the VH and VL domains of a conventional antibody, or the VHH domain of an HCAb); more specifically, by the CDR sequences of the variable domain(s) (z.e., the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 of a conventional antibody, or the CDR-1, CDR-2 and CDR-3 of an HCAb). According to some embodiments of the present disclosure, each VHH domain of the recombinant antibody comprises three CDRs (z.e., CDR-1, CDR-2 and CDR-3), in which the CDR-1 comprises the amino acid sequence of SEQ ID NO: 1, the CDR-2 comprises the amino acid sequence of SEQ ID NO: 2, and the CDR-3 comprises the amino acid sequence of SEQ ID NO: 3. According to some another embodiments, the CDR-1 comprises the amino acid sequence of SEQ ID NO: 7, the CDR-2 comprises the amino acid sequence of SEQ ID NO: 8, and the CDR-3 comprises the amino acid sequence of SEQ ID NO: 9. Alternatively, the CDR1, CDR2, and CDR3 of the VHH domain may comprise a combination of either SEQ ID NOs: 1 or 7, either SEQ ID NOs: 2 or 8, and either SEQ ID NOs: 3 or 9.

[0073] As an example, the amino acid sequences of the VHH domain of the recombinant antibody is provided as SEQ ID NO: 4, described below, in which the CDRs (z.e., the CDR-1, CDR-2 and CDR-3, from N-terminus to C-terminus) are marked in bold letters, in sequence.QVKLEESGGGLVQAGGSLRLSCAASGGSFSSYMLAWFRQAPGKEREFVANISPGGYTY YADSAQGRFTISRENAKNTMYLQMNSLNPEDTAVYYCAADIRVLAAAQVASYDYWGQ GTQVTV

[0074] In some embodiments, the VHH domain of the recombinant antibody can be humanized to form humanized antibodies. As an example, a humanized VHH is prepared, which comprises an amino acid sequence as set forth in following SEQ ID NO: 10.EVQLVESGGGLVQPGGSLRLSCAASGRTFSEYMLAWFRQAPGKEREFVANISPGGYTY YADSAKGRFTISRDNAKNTMYLQMNSLRPEDTAVYYCAADLRVLAAAQVASYDYWG QGTQVTV (the bold text denotes the CDRs, while the underlined text indicates the amino acids that differ from the sequence of SEQ ID NO:4).

[0075] For a recombinant antibody comprising a pair of VHH domains, the VHH can be the same (homodimeric) or different (heterodimeric).

[0076] Various methods for humanizing antibody sequences are well-established in the field. A widely utilized approach is CDR grafting, which involves transferring the complementarity - determining region (CDR) sequences from a donor antibody onto a human antibody framework with different specificities. Another technique for humanization is resurfacing, which maintains non-surface exposed residues of non-human origin while substituting surface residues with human equivalents. Additionally, a method known as "guided selection" allows for the identification of mouse antibodies from which fully human antibodies can be derived, preserving the epitope and binding properties of the original antibody. Furthermore, a humanization approach based on molecular dynamics calculations is described in patent application W02009 / 032661.

[0077] Since the binding affinity and specificity of an antibody are mainly determined by the CDR sequences thereof, as could be appreciated, the framework (FR) sequences of the VHH domain may vary (e.g., being substituted by conserved or non-conserved amino acid residues) without affecting the binding affinity and / or specificity of the present antibody. Preferably, the FR sequence is conservatively substituted by one or more suitable amino acid(s) with similar properties; for example, the substitution of leucine (an nonpolar amino acid residue) by isoleucine, alanine, valine, proline, phenylalanine, or tryptophan (another nonpolar amino acid residue); the substitution of aspartate (an acidic amino acid residue) by glutamate (another acidic amino acid residue); or the substitution of lysine (an basic amino acid residue) by arginine or histidine (anotherbasic amino acid residue). Based on the conservative substitution, a skilled artisan may substitute the amino acid residue(s) of the FR sequences of the VHH domain of the recombinant antibody without affecting its activity and / or effect (i.e., binding to CEACAM6). Accordingly, the recombinant antibodies comprising substituted amino acid(s) in their FR sequences of VHH domain are intended to be included within the scope of the present disclosure. According to some embodiments, each VHH domain of the recombinant antibody comprises an amino acid sequence of at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 4; preferably, at least 90% identical to SEQ ID NO: 4; more preferably, at least 95% identical to SEQ ID NO: 4.

[0078] Depending on desired purposes, the Fc region linked to the VHH domain is an Fc region of human immunoglobulin G1 (IgGl), 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 an Fc region of human IgGl. In one specific embodiment, the pair of VHH domains is linked to the N-termini of the Fc region of human IgGl, in which each heavy chain of the present recombinant antibody comprises the amino acid sequence of SEQ ID NO: 13. In addition, a signal peptide can be fused to N-termini of the VHH of the recombinant antibody (see SEQ ID NO:5) so as to transport the antibody out of the cell membrane. After a protein is translocated across the membrane, the signal peptide is cut off by signal peptidase.

[0079] Alternatively, the Fc region of the recombinant antibody is an Fc region of mouse IgGl, IgG2, IgG3 or IgA. According to certain embodiments, the Fc region of the recombinant antibody is an Fc region of mouse IgG2. In one specific embodiment, the pair of VHH domains is linked to the N-termini of the Fc region of mouse IgG2, in which each heavy chain of the present recombinant antibody comprises the amino acid sequence of SEQ ID NO: 19. A signal peptide can be fused to N-termini of the VHH of the recombinant antibody (see SEQ ID NO:6) so as to transport the antibody out of the cell membrane. After a protein is translocated across the membrane, the signal peptide is cut off by signal peptidase.

[0080] According to certain embodiments of the present disclosure, the recombinant antibody is produced by ribosome display and DNA cloning. Ribosome display is a cell-free system for in vitro selection of proteins and peptides from large libraries. The methods of producing a ribosome display system are known in the art. In general, the ribosome display system is established by immunizing a host animal (e.g., a camelid, preferably a Lama Pacos (alpaca)) with a peptide (e.g., a CEACAM6 polypeptide) in accordance with commonly adopted procedures; for example, immunizing the host animal with the peptide every two or three weeks, until a desired antibodytiter is reached. After the final immunization, peripheral lymphocytes of the immunized animal are collected, and total RNAs are isolated therefrom. Corresponding cDNA fragments are synthesized, and a cDNA library is constructed accordingly via amplifying the cDNA fragment encoding the VHH domains followed by ligating the PCR products into an expression vector. The VHH-encoding cDNAs are useful in transcribing mRNAs and translating proteins in ribosome display format (z.e., protein -ribosome-mRNA (PRM) complex). In the selection stage (also known as the “biopanning stage”), the PRM complexes are added to an immobilized ligand (e.g., an immobilized CEACAM6 polypeptide), and the complexes exhibiting binding affinity to the immobilized ligand are then eluted by salt concentrations, chelating agents, or mobile ligands which complex with the binding motif of the protein allow dissociation of the mRNA. The mRNA can then be reverse transcribed back into cDNA for the next round of selection. The selection stage may be repeated several times (for example, 3-5 times) so as to obtain the cDNA encoding VHH domains specific to the peptide (e.g., the CEACAM6 polypeptide).

[0081] According to some embodiments of the present disclosure, the thus-obtained cDNA is then cloned into an expression vector (i.e., IgGl vector), which comprises the Fc region of a human immunoglobulin. After introducing a suitable host cell, the expression vector is useful in expressing the recombinant antibody in the host cell; the thus-produced recombinant antibody in structure comprises a human-derived Fc region, and camelid-derived VHH domains linked to the N-terminus of the Fc region. The host cell is preferably a mammalian cell, for example, Chinese hamster ovary (CHO) cells or embryonic kidney 293 cells. According to one preferred embodiment, the host cell is an embryonic kidney 293 cells.

[0082] Alternatively, the present recombinant antibody may be directly produced by DNA cloning. DNA encoding the present recombinant antibody may be easily isolated and sequenced by use of conventional procedures, such as using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the mAb. Once isolated, the DNA may be placed into an expression vector, which is then transfected into a suitable host cell as described above, so as to synthesize the desired antibody in the host cell.

[0083] The recombinant antibody may be purified according to standard procedures in the art, such as cross-flow filtration, affinity column chromatography, gel filtration and the like.Single domain antibody

[0084] In some embodiments, the VHH domain alone can also be used in the immunoconjugate described herein, which is a single domain antibody (sdAb). Generally, sdAbs are thermo- and pH-stable. According to some embodiments of the present disclosure, each VHH domain of the recombinant antibody comprises three CDRs (i.e., CDR-1, CDR-2 and CDR-3), in which theCDR-1 comprises the amino acid sequence of SEQ ID NO: 1 or 7, the CDR-2 comprises the amino acid sequence of SEQ ID NO: 2 or 8, and the CDR-3 comprises the amino acid sequence of SEQ ID NO: 3 or 9.

[0085] In some embodiments, the sdAb comprises the amino acid sequences of SEW ID NO: 4, 10 or 11. The sdAb having SEQ ID NO: 11 is a mutant of SEQ ID NO:4.QVKLEESGGGLVQAGGSLRLSCAASGGSFSSYMLAWFRQAPGKEREFVANISPGGYTY YADSAQGRFTISRENAKNTMYLQMNSLNPEDTAVYYCAADIRVLAAAQVASYDYWGQ GTQVTVCS (SEQ ID NO: 11)

[0086] Methods for producing a sdAb and its modification are known in the art. For example, a sdAb can be produced by living host cells that have been genetically engineered to produce protein. Methods for the genetic construction of cells for the production of proteins are well known in the art. See, for example, Ausabel et al., Eds. (1990), Current Protocols in Molecular Biology (Wiley, New York). A sdAb derived from a non-human animal and then modified, for example, humanized, de-immunized, chimeric, can be obtained using recombinant DNA methods known in the art. Humanized sdAb can also be obtained, for example, using transgenic mice that express human heavy and light chain genes but are not able to express endogenous mouse immunoglobulin heavy and light chain genes. In some embodiments, a humanized sdAb, such as a nanobody molecule, is optimized by introducing conservative substitutions, substitutions from consensus sequences, substitutions from the germline and / or reverse mutations via any methods known in the art.

[0087] (I)-2 Therapeutic agent

[0088] In general, the therapeutic agent of the present immunoconjugate may be a cytotoxic drug, a radioactive nuclide, a cytokine, a hormone drug, an immune stimulating agent, or an immunotherapeutic drug.

[0089] The cytotoxic drug may be any molecule known to inhibit cell growth or exhibit cytotoxic / killing effect on cancer / tumor cells. Examples of the cytotoxic drug suitable to produce the present immunoconjugate include, but are not limited to, alkylating agents (agents that keep cells from replication by damaging their DNA; e.g., altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa or trabectedin), antimetabolites (agents that interfere with DNA and RNA by acting as a substitute for the normal building blocks of RNA and DNA; e.g., azacitidine, 5-fluorouracil (5-FU), 6-mercaptopurine (6- MP), capecitabine, cladribine, clofarabine, cytarabine (Ara-C), decitabine, floxuridine, fludarabine,gemcitabine, hydroxyurea, methotrexate, nelarabine, pemetrexed, pentostatin, pralatrexate or thioguanine), anthracyclines or antibiotics (agents that interfere with enzymes involved in copying DNA during the cell cycle; e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, bleomycin, dactinomycin, mitomycin-C or mitoxantrone), or mitotic inhibitors (agents that stop cells from dividing to form new cells; e.g., auristatin, cabazitaxel, docetaxel, nab-paclitaxel, paclitaxel, vinblastine, vincristine, vinorelbine, tubulysin ormertansine (DM1)). According to one exemplary example of the present disclosure, the cytotoxic drug is auristatin or a derivative thereof or topoisomerase 1 inhibitors, e.g., MMAE, MMAF, exatecan, and exatecan-derivatives.

[0090] The radioactive nuclide, also known as radionuclide, radioisotope or radioactive isotope, may be any of yttrium-90 (90Y), indium-ill (mIn), iodine-131 (131I), samarium-153 (153Sm), lutetium-177 (177Lu), astatine-211 (2nAt), bismuth-212 (212Bi), actinium-225 (225Ac), radium-223 (223R) or thorium-227 (227Th). Preferably, the radioactive nuclide is complexed with a chelator, such as ethylenediaminetetramethylenephosphonic acid (EDTMP), 1,4,7,10- tetraazacy cl ododecanetetram ethyl enephosphonic acid (DOTMP), 1,4,7,10- tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA), l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), l,4,7-triazacyclononane-l,4-diacetic acid (NODA), or diethylenetriaminepentaacetic acid (DTPA).

[0091] The cytokine may by any cytokine known to modulate or stimulate the immune response of a subject, for example, 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)-a, interferon (IFN)-a, IFN-y, granulocytemacrophage colony-stimulating factor (GM-CSF), or a combination thereof.

[0092] Hormone drug is known to regulate (mainly down-regulate) the expression level of a cancer-related hormone in the cancer patients. Exemplary hormone drugs suitable to produce the present immunoconjugate include, but are not limited to, aromatase inhibitors (e.g., anastrozole, exemestane or letrozole), selective estrogen receptor modulators (SERM; e.g., tamoxifen or raloxifene), estrogen receptor antagonists (e.g., fulvestrant or toremifene), luteinizing hormone- releasing hormone (LHRH) agonists (e.g., goserelin, leuprolide or triptorelin), anti-androgens (e.g., apalutamide, enzalutamide, darolutamide, bicalutamide, flutamide or nilutamide), CYP17 inhibitors (e.g., abiraterone or ketoconazole), progestins (e.g., medroxyprogesterone acetate or megestrol acetate), and adrenolytics (e.g., mitotane).

[0093] As its name implies, the immune stimulating agent is a small-molecule useful in stimulating the immune system of a subject against cancer / tumor cells. Examples of the immune stimulating agent (also known as immunostimulant) suitable to be conjugated with the present recombinant antibody include, but are not limited to, acemannan, bropirimine, burdock,deoxy cholic acid (DCA), echinacea, elapegademase, flavonoids (e.g., rutin, isoliquiriti genin or liquiritigenin), glatiramer acetate, oprelvekin, pegademase bovine, plerixafor, prolactin, trilaciclib, terpenes (e.g., tri terpenoid), TLR7 / TLR8 agonist, TLR9 agonist, Sting agonist, andNLRP agonist.

[0094] Regarding the immunotherapeutic drug, it may be any small-molecules commonly used in immunotherapy for treating cancers; for example, immune checkpoint inhibitors (ICIs, e.g., an inhibitor of cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed cell death 1 (PD-1) or PD- Ll), or immunomodulatory drugs (IMiDs; e.g., thalidomide pomalidomide, lenalidomide, or the analogues or derivatives thereof).

[0095] Alternatively, the therapeutic agent may be a plant toxin (e.g, ricin, abrin, saporin, volkensin, modeccin, gelonin and vi scumin), an exotoxin (e.g., Pseudomonas 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 (e.g., diphtheria toxin) to achieve the tumor-killing effect.

[0096] (I)-3 Linker linking the antibody or sdAb to the therapeutic agent

[0097] According to the embodiments of the present disclosure, the linker is configured to link the therapeutic agent to the sdAb or the Fc region of the recombinant antibody. Depending on intended purpose, the linker of the present immunoconjugate may be a cleavable linker (i.e., a linker that is cleavable / reducible by an enzyme or under a specific physical condition), or a non- cleavable linker (also known as “stable linker” that is not hydrolyzed in vivo i.e., being resistant to cleavage by an enzymatic or physical process in vivo).

[0098] Exemplary cleavable linkers suitable for conjugating the present therapeutic agent to the recombinant antibody include, but are not limited to, a protease-sensitive linker (e.g., valinecitrulline (VC) dipeptide, valine-alanine (VA) dipeptide, valine-lysine (VL) dipeptide, valinearginine (VR) dipeptide or glutamate-valine-citrulline (EVC) tripeptide), a pH-sensitive linker (e.g., S-S disulfide linkage, hydrazone linker, ester linker or amide linker), and a glutathionesensitive linker (e.g., N-Succinimidyl 4-(2-pyridyldithio) butanoate (SPDB) or N-succinimidyl-4- (2 -pyridyldithio) pentanoate (SPP)).

[0099] Non-limiting examples of the non-cleavable linker include, maleimidocaproyl (MC), maleimidomethyl cyclohexane- 1 -carboxylate (MCC), and succinimidyl-4-[N-maleimidomethyl] cyclohexane- 1 -carboxylate (SMCC).

[0100] Alternatively, the linker may be a linker known in the art to connect two functional motifs in an immunoconjugate (e.g., connecting the antibody and the payload of an ADC).

[0101] A skilled artisan may select a suitable linker for producing the present immunoconjugatein accordance with an intended purpose. According to one exemplary embodiment, the linker connecting the recombinant antibody and therapeutic agent comprises a polyethylene glycol (PEG) chain and a protease-sensitive linker connected to the PEG chain; preferably, the PEG chain has 1 to 10 repeats of EG units. According to one example of the present disclosure, the linker comprises a PEG chain having three repeats of EG units (i.e., PEG3), and a VC dipeptide linked to the PEG chain. In the embodiment, the immunoconjugate is produced in the form of “recombinant antibody(Fc)-PEG chain- VC dipeptide-therapeutic agent”.

[0102] According to some embodiments of the present disclosure, the present immunoconjugate is produced by trimannosyl ADC technology, a platform for linking drug payload(s) to a target antibody in a site-specific manner (see, for example, WO 2018 / 126092 Al). In these embodiments, the recombinant antibody is first modified to conjugate with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more) azide groups; the azide-modified antibody is then capable of linking to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more) dibenzocyclooctyne group (DBCO)- conjugated linker-payloads (each of which comprises a linker, a DBCO group conjugated to one end of the linker, and a therapeutic agent conjugated to the other end of the linker) via a copper- free click reaction occurred between the azide and DBCO groups. As could be appreciated, the azide or DBCO group may be alternatively substituted by different groups suitable for click chemistry, for example, an alkyne, tetrazine, or trans-cyclooctyne (TCO) group. Depending on desired purpose, the present immunoconjugate may alternatively be produced by other methods known to synthesize ADCs, for example, cysteine conjugation, lysine conjugation, disulfide rebridging, etc. The methods for synthesizing ADCs are known in the art; hence, the detailed description is omitted herein for the sake of brevity.

[0103] According to certain exemplary embodiments of the present disclosure, the recombinant antibody has four azide groups, and accordingly, 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 thus-produced immunoconjugate is about 4.

[0104] (I)-4 Embodiments of Immunoconjugate

[0105] In the present disclosure, the antibody is connected to a therapeutic agent via or not via a linker. Examples of the chimeric-HCAb, humanized HCAb, sdAb in connection with or without a linker include, but are not limited to the following sequences:

[0106] 1. Chimeric-HCAbSequences: Signal peptide- VHH-Fc(Human IgGl)QVKLEESGGGLVQAGGSLRLSCAASGGSFSSYMLAWFRQAPGKEREFVANISPGGYTYYADSAQGRFTISRENAKNTMYLQMNSLNPEDTAVYYCAADIRVLAAAQVASYDYW GQGTQVTVVD(optionalGGGSG)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV I<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<AL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK (SEQ ID NO: 13)

[0107] 2. Humanized HCAbSequence: Signal peptide-humanized VHH-Fc(Human IgGl)EVQLVESGGGLVQPGGSLRLSCAASGRTFSEYMLAWFRQAPGKEREFVANISPGGYT YYADSAKGRFTISRDNAKNTMYLQMNSLRPEDTAVYYCAADLRVLAAAQVASYDY WGQGTQVTVSS(optionalGGGSG)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV I<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CT<VSNI<AL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK (SEQ ID NO: 14)

[0108] 3. sdAbThe VHH alone can be used as the sdAb to link to a therapeutic agent via or not via a linker, which is illustrated in SEQ ID NO: 4, 10 or 11.

[0109] Any therapeutic agent can be connected to the antibody described herein. Examples of the therapeutic agent include, but are not limited to, auristatin, irinotecan, exatecan, levofolinate, 5 -fluorouracil and MMAE. The above-mentioned embodiments of chimeric HCAb, humanized HCAb and sdAb can connect to any of the therpautic agent.(II) Pharmaceutical compositions comprising the present immunoconjugates

[0110] The second aspect of the present disclosure is directed to a pharmaceutical composition for the treatment of cancers. According to some embodiments, the pharmaceutical composition comprises the present immunoconjugate, and optionally, a pharmaceutically acceptable carrier.

[0111] Generally, the immunoconjugate of this invention is present at a level of about 0 1% to 99% by weight, based on the total weight of the pharmaceutical composition. In some embodiments, the immunoconjugate of this invention is present at a level of at least 1% by weight, based on the total weight of the pharmaceutical composition. In certain embodiments, the immunoconjugate is present at a level of at least 5% by weight, based on the total weight of thepharmaceutical composition. In still other embodiments, the immunoconjugate is present at a level of at least 10% by weight, based on the total weight of the pharmaceutical composition. In still yet other embodiments, the immunoconjugate is present at a level of at least 25% by weight, based on the total weight of the pharmaceutical composition.

[0112] The pharmaceutical composition may be prepared in accordance with acceptable pharmaceutical procedures, such as described in Remington’s Pharmaceutical Sciences, 17thedition, ed. Alfonoso R. Gennaro, Mack Publishing Company, Easton, Pa (1985). Pharmaceutically acceptable carriers are those that are compatible with other ingredients in the formulation and biologically acceptable.

[0113] The pharmaceutically acceptable carrier may be any pharmaceutically acceptable material or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, that is useful in carrying or transporting the active agents (e.g., the present immunoconjugate) from one organ, or portion of the body, to another organ, or portion of the body. The carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, and is selected to minimize any degradation of the active agent and to minimize any adverse side effects in the subject. Preferably, the pharmaceutical composition is formulated into liquid forms, e.g., sterile solutions or suspensions that can be administered by, for example, intravenous, intraarterial, intramuscular, subcutaneous, intrathecal, intraperitoneal or intratumoral injection. The pharmaceutical composition may be formulated as isotonic suspensions, solutions or emulsions in oily or aqueous vehicles (such as sodium chloride injection, Ringer’s injection, dextrose injection, dextrose and lactated Ringer’s injection), and may contain formulary agents such as suspending, stabilizing or dispersing agents.

[0114] Optionally, the pharmaceutical composition of the present disclosure may further comprise one or more pharmaceutically-acceptable additives, including buffering agent, antioxidant, diluent, antiseptic and the like.

[0115] (III) Uses of the present immunoconjugate and pharmaceutical composition

[0116] The third aspect of the present disclosure pertains to a method of treating a cancer in a subject by using the present immunoconjugate or pharmaceutical composition. The method comprises administering to the subject an effective amount of the immunoconjugate or pharmaceutical composition as respectively described in Sections (i) and (ii) of the present disclosure, so as to inhibit the growth, migration or progress of the cancer, and / or alleviate or ameliorate the symptoms associated with the cancer.

[0117] According to some embodiments of the present disclosure, the subject is a mouse, inwhich about 0.01 to 100 mg / Kg of the present immunoconjugate is administered to the subject, such as about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 mg / kg. Preferably, about 0.1 mg / kg to 50 mg / kg of the present immunoconjugate is administered to the subject. More preferably, about 1 mg / kg to 10 mg / kg of the present immunoconjugate is administered to the subject. According to one working example, about 5 mg / kg of the present immunoconjugate is administered to the subject.

[0118] A skilled artisan may readily determine the human equivalent dose (HED) of the present immunoconjugate, based on the doses determined from animal studies provided in working examples of this application. Accordingly, the effective amount of the present immunoconjugate suitable for use in a human subject may be in the range of about 1 pg / kg to 10 mg / kg body weight for human; such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320,330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520,530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720,730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920,930, 940, 950, 960, 970, 980, 990 pg / kg, or about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg / kg. The dose can be administered in a single aliquot, or alternatively in more than one aliquot. A skilled artisan or clinical practitioner may adjust the dosage or regime in accordance with the physical condition of the patient or the severity of the diseases.

[0119] Depending on intended purpose, the present immunoconjugate or pharmaceutical composition may be administered to the subject 1-7 times every week, 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 longer. As could be appreciated, the dosing regimen can vary over time. According to certain exemplary embodiments of the present disclosure, the present immunoconjugate is administered to the subject once every week (weekly). According to some preferred embodiments, the present immunoconjugate is administered to the subject weekly for at least 3 weeks, for example, 3, 4, 5, 6, 7, 8, 9 or 10 weeks, or longer. In one exemplary embodiment of the present disclosure, the present immunoconjugate is administered to the subject weekly for 6 consecutive weeks.

[0120] The recombinant immunoconjugate or pharmaceutical composition of the presentdisclosure may be administered to the subject by an appropriate route, such as intrautumoral, intramuscular, intravenous or intraperitoneal injection. According to some embodiments of the present disclosure, the recombinant immunoconjugate or pharmaceutical composition is administered to the subject via intravenous injection.

[0121] As would be appreciated, the present method can be applied to the subject, alone or in combination with additional therapies that have some beneficial effects on the prevention or treatment of cancers, for example, surgery, chemotherapy and / or radiation therapy. Depending on the intended / therapeutic purpose, the present method can be applied to the subject before, during, or after the administration of the additional therapies.

[0122] Examples of the cancer treatable with the present method include, but are not limited to, gastric cancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer, renal cancer, colorectal cancer, cervical cancer, ovarian cancer, brain tumor, prostate cancer, hepatocellular carcinoma, melanoma, esophageal carcinoma, multiple myeloma, head and neck squamous cell carcinoma, or a combination thereof.

[0123] Basically, the subject treatable with the present method is a mammal, for example, human, mouse, rat, guinea pig, hamster, monkey, swine, dog, cat, horse, sheep, goat, cow, and rabbit. Preferably, the subject is a human.

[0124] The following Examples are provided to elucidate certain aspects of the present invention and to aid those of skilled in the art in practicing this invention. These Examples are in no way to be considered to limit the scope of the invention in any manner. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety.

[0125] Embodiments of the present disclosure

[0126] Embodiment 1 : An immunoconjugate comprises: a recombinant antibody comprising (i) a single variable heavy chain domain (VHH domain) or a pair of homodimeric or heterodimeric VHHs and a fragment crystallizable region (Fc region) of an immunoglobulin fused to each of the VHH domain(s) optionally via a peptide linker, 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 the amino acid sequence of SEQ ID NO: 1 or 7, a second CDR (CDR- 2) comprising the amino acid sequence of SEQ ID NO: 2 or 8 and a third CDR (CDR-3) comprising the amino acid sequence of SEQ ID NO: 3 or 9; and a therapeutic agent; wherein the therapeutic agent links to the Fc region of the recombinant antibody or the VHH of the sdAb,optionally via a linker. Alternatively, the CDR1, CDR2, and CDR3 of the VHH domain may comprise a combination of either SEQ ID NOs: 1 or 7, either SEQ ID NOs: 2 or 8, and either SEQ ID NOs: 3 or 9.

[0127] Embodiment 2: An immunoconjugate comprises: a recombinant antibody comprising a single variable heavy chain domain (VHH domain) or a pair of homodimeric or heterodimeric VHHs and a fragment crystallizable region (Fc region) of an immunoglobulin fused to each of the VHH domain(s) optionally via a peptide linker, wherein the VHH domain comprises a first chain complementarity determining region (CDR-1) comprising the amino acid sequence of SEQ ID NO: 1 or 7, a second CDR (CDR-2) comprising the amino acid sequence of SEQ ID NO: 2 or 8 and a third CDR (CDR-3) comprising the amino acid sequence of SEQ ID NO: 3 or 9; a therapeutic agent; and a linker linking the therapeutic agent links to the Fc region of the recombinant antibody. Alternatively, the CDR1, CDR2, and CDR3 of the VHH domain may comprise a combination of either SEQ ID NOs: 1 or 7, either SEQ ID NOs: 2 or 8, and either SEQ ID NOs: 3 or 9.

[0128] Embodiment 3: The immunoconjugate of any one of Embodiments 1 to 2, wherein the VHH domain fused to the N-termini of the Fc region of the immunoglobulin via a peptide linker, wherein the peptide linker is a short peptide comprising Gly and Ser.

[0129] Embodiment 4: The immunoconjugate of Embodiment 3, wherein the peptide linker has a (GlymSern)Pmotif, wherein m and n is any of integer of 1 to 10 and p is any of integer of 1 to 30, or a variant thereof.

[0130] Embodiment 5: The immunoconjugate of Embodiment 4, wherein the peptide linker comprises an amino acid sequence having GGGSG (SEQ ID NO: 12), GGGGS (SEQ ID NO: 15) or its repeats sequence (such as second, three, four or five repeats sequence).

[0131] Embodiment 6: The immunoconjugate of any one of Embodiments 1 to 2, wherein the peptide linker comprises an amino acid sequence having GGGSG (SEQ ID NO: 12), GGGGS (SEQ ID NO: 15) or its repeats sequence (such as second, three, four or five repeats sequence), AHHSEDP (SEQ ID NO: 16), EPKTPKPQPQPQPQPQPNPTTE (SEQ ID NO: 17) or STPPTPSPSTPP (SEQ ID NO: 18) or a combination thereof.

[0132] Embodiment 7: An immunoconjugate comprises: a single domain antibody (sdAb) comprising a VHH domain, wherein the VHH domain comprises a first chain complementarity determining region (CDR-1) comprising the amino acid sequence of SEQ ID NO: 1 or 7, a second CDR (CDR-2) comprising the amino acid sequence of SEQ ID NO: 2 or 8 and a third CDR (CDR- 3) comprising the amino acid sequence of SEQ ID NO: 3 or 9; and a therapeutic agent; wherein the therapeutic agent links to the Fc region of the recombinant antibody or the VHH of the sdAb,optionally via a linker. Alternatively, the CDR1, CDR2, and CDR3 of the VHH domain may comprise a combination of either SEQ ID NOs: 1 or 7, either SEQ ID NOs: 2 or 8, and either SEQ ID NOs: 3 or 9.

[0133] Embodiment 8: The immunoconjugate of any one of Embodiments 1 to 6, wherein the recombinant antibody is a chimeric antibody, a humanized antibody.

[0134] Embodiment 9: The immunoconjugate of Embodiment 1 or 7, wherein the sdAb is a humanized antibody.

[0135] Embodiment 10: The immunoconjugate of any one of Embodiments 1 to 9, wherein the VHH comprises CDR- 1 comprising the 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 the 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 the 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.

[0136] Embodiment 11: The immunoconjugate of any one of Embodiments 1 to 10, 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.

[0137] Embodiment 12: The immunoconjugate of any one of Embodiments 1 to 9, wherein the VHH comprises CDR- 1 comprising the 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: 7, CDR-2 comprising the 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: 8 and CDR-3 comprising the 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: 9.

[0138] Embodiment 13: The immunoconjugate of any one of Embodiments 1 to 9 and 12, 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.

[0139] Embodiment 14: The immunoconjugate of any one of Embodiments 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% identical to SEQ ID NO: 4 or 10 or 11.

[0140] Embodiment 15: The immunoconjugate of any one of Embodiments 1 to 14, wherein the VHH domain comprises an amino acid sequence of SEQ ID NO: 4 or 10 or 11.

[0141] Embodiment 16: The immunoconjugate of any one of Embodiments 1 to 15, wherein the recombinant antibody comprises the VHH domain comprising an amino acid sequence of SEQ ID NO: 4, 10 or 11.

[0142] Embodiment 17: The immunoconjugate of any one of Embodiments 1 to 16, wherein the recombinant antibody comprises the VHH domain comprising an amino acid sequence of SEQ ID NO: 4 or 10.

[0143] Embodiment 18: The immunoconjugate of any one of Embodiments 1 to 17, wherein the recombinant antibody comprises a single VHH domain comprising an amino acid sequence of SEQ ID NO: 4, 10 or 11.

[0144] Embodiment 19: The immunoconjugate of any one of Embodiments 1 to 17, wherein the recombinant antibody comprises a pair of homedimeric VHHs, wherein each of the VHH domain comprising an amino acid sequence of SEQ ID NO: 4, 10 or 11.

[0145] Embodiment 20: The immunoconjugate of any one of Embodiments 1 to 17, wherein the recombinant antibody comprises a pair of heterodimeric VHHs, wherein the pair of the VHHs are different and each of the VHH domain comprising an amino acid sequence selected from SEQ ID NOs: 4, 10 and 11.

[0146] Embodiment 21: The immunoconjugate of any one of Embodiments 1 to 17, wherein the sdAb comprises the VHH domain comprising an amino acid sequence of SEQ ID NO: 4, 10 or 11.

[0147] Embodiment 22: The immunoconjugate of Embodiment 21, wherein the sdAb comprises the VHH domain comprising an amino acid sequence of SEQ ID NO: 10 or 11.

[0148] Embodiment 23: The immunoconjugate of any one of Embodiments 1 to 22, wherein the VHH domain is derived from a camelid heavy-chain antibody.

[0149] Embodiment 24: The immunoconjugate of any one of Embodiments 1 to 17, wherein the recombinant antibody comprises an amino acid sequence of SEQ ID NO: 5, 6, 13, 14 or 19.

[0150] Embodiment 25: The immunoconjugate of any one of Embodiments 1 to 24, wherein the therapeutic agent is a cytotoxic drug, a radioactive nuclide, a cytokine, a hormone drug, an immune stimulating agent, or an immunotherapeutic drug.

[0151] Embodiment 26: The immunoconjugate of any one of Embodiments 1 to 25, wherein the therapeutic agent is an alkylating agents (agents that keep cells from replication by damaging their DNA; e.g., altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil,cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa or trabectedin), antimetabolites (agents that interfere with DNA and RNA by acting as a substitute for the normal building blocks of RNA and DNA; e.g., azacitidine, 5 -fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cladribine, clofarabine, cytarabine (Ara-C), decitabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, nelarabine, pemetrexed, pentostatin, pralatrexate or thioguanine), anthracyclines or antibiotics (agents that interfere with enzymes involved in copying DNA during the cell cycle; e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, bleomycin, dactinomycin, mitomycin-C or mitoxantrone), or mitotic inhibitors (agents that stop cells from dividing to form new cells; e.g., auristatin, cabazitaxel, docetaxel, nab-paclitaxel, paclitaxel, vinblastine, vincristine, vinorelbine, tubulysin or mertansine (DM1)).

[0152] Embodiment 27: The immunoconjugate of any one of Embodiments 1 to 26, wherein the therapeutic agent is auristatin or a derivative thereof, topoisomerase 1 inhibitors, e.g., MMAE, MMAF, exatecan, or exatecan-derivatives.

[0153] Embodiment 28: The immunoconjugate of any one of Embodiments 1 to 27, wherein the linker linking the recombinant antibody or the sdAb to the therapeutic agent is a cleavable linker or a non-cleavable linker.

[0154] Embodiment 29: The immunoconjugate of Embodiment 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., S-S disulfide linkage, hydrazone linker, ester linker or amide linker), and a glutathione-sensitive linker (e.g., N-Succinimidyl 4-(2- pyridyldithio) butanoate (SPDB) or N-succinimidyl-4-(2 -pyridyldithio) pentanoate (SPP)).

[0155] Embodiment 30: The immunoconjugate of Embodiment 28, wherein the non-cleavable linker is a maleimidocaproyl (MC), maleimidomethyl cyclohexane-1 -carboxylate (MCC), and succinimidyl-4-[N-maleimidomethyl] cyclohexane-1 -carboxylate (SMCC).

[0156] Embodiment 31: The immunoconjugate of any one of Embodiments 1 to 28, wherein the linker linking the recombinant antibody or the sdAb to the therapeutic agent is a polyethylene glycol (PEG) chain or a protease-sensitive linker connected to the PEG chain.

[0157] Embodiment 32: The immunoconjugate of any one of Embodiment 31 , wherein the PEG chain having three repeats of EG units (i.e., PEG3).

[0158] Embodiment 33: The immunoconjugate of Embodiment 31, wherein the proteasesensitive linker connected to the PEG chain is a VC dipeptide linked to the PEG chain.

[0159] Embodiment 34: The immunoconjugate of any one of Embodiments 1 to 33, wherein the immunoconjugate is in the form of recombinant antibody (Fc) or sdAb-PEG chain- VC dipeptide-therapeutic agent.EXAMPLE

[0160] Materials and Methods

[0161] Preparation of recombinant antibody (hereinafter as “HCA b ”)

[0162] The HCAb selected by ribosome display was expressed using EXPI293F™ and EXPICHO-S™ cells and purified according to the manufacturer’s instructions. Briefly, EXPI293F™ cells were transfected with the HCAb construct using the EXPIFECTAMINE™ 293 and EXPIFECTAMINE™ CHO reagents. The cell culture medium was collected, and the HCAb was purified using a Protein G column.

[0163] The thus-produced HCAb comprised a homodimer of cam elid-derived VHH domain fused with a human-derived Fc region (in the form of VHH-hlgGl). According to the analytic results, each VHH domain comprised the amino acid sequence of SEQ ID NO: 4 or 10, in which the CDR-1, CDR-2 and CDR-3 respectively comprised the amino acid sequences of SEQ ID NOs: 1-3 or SEQ ID Nos: 7-9 (Table 1); and the human-derived Fc region comprised the amino acid sequence of SEQ ID NO: 5.

[0164] Table 1 Amino acid sequence of the VHH domain of the HCAb described hereinThe CDR sequences in VHH domain were marked in boldface, including CDR-L1, CDR-L2 and CDR-L3, from N- terminus to C-terminus, in sequence.

[0165] Preparation of antibody-drug conjugate (ADC) through trimannosyl ADC technology

[0166] The chimeric ADC was prepared by trimannosyl ADC technology in accordance with the procedures described in WO 2018 / 126092 Al, the entirety of which is incorporated herein by reference. In brief, the HCAb (VHH-hlgGl) was first modified by P-galactosidase and N- acetylglutamate synthase (NAGS). The thus-produced trimannosyl Ab was conjugated with UDP-N-azidoacetyl galactosamine (UDP-GlcNAz) by using N-acetylglucosaminyltransferase I (GnTl)and N-acetylglucosaminyltransferase II (GnT2) to produce a HCAb having 4 azide (Az) groups in the terminal N-acetylglucosamines (i.e., Trimannosyl HCAb-4Az). Next, the Trimannosyl HCAb- 4Az was conjugated with DBCO-PEG3-VC-PAB-MMAE molecules via a copper-free click reaction occurred between the azide and DBCO groups. The detailed steps for preparing the present ADC were provided below.

[0167] (i) Preparation of trimannosyl A b

[0168] 18.57 mg HCAb (pH 6.08) were mixed with 1 mg [3-galactosidase of Akkermansia muciniphila (Akk) and 0.2 mg NAGS. The mixture was diluted by 25 mM sodium citrate (pH 6.08) to a final concentration of 5 mg / mL, followed by incubating at 37°C for 3 hours. The product was filtered with a 0.22 pm syringe filter.

[0169] (ii) Preparation of Trimannosyl HCAb-4Az

[0170] The filtered sample was subj ected to a protein A column. The column was then washed with lx phosphate buffered saline (PBS; pH 7.2) and 25 mM 3-(N-morpholino) propanesulfonic acid (MOPS; pH 7.5), in sequence. 1.5 mg GnTl, 0.5 mg GnT2 and 10 mg UDP-GlcNAz were mixed in a solution containing 25 mM MOPS and 10 mM MnCL, followed by adding to the column and cycling at room temperature for 3 hours. After the washing step with 0.1 M sodium citrate (pH 6.1), the column was eluted with 0.1 M sodium citrate (pH 3.0). The eluted product was immediately neutralized by 1 M Tri s(hydroxymethyl)aminom ethane (Tris buffer; pH 9.0), and then filtered with a 0.22 pm syringe filter.

[0171] According to the mass analysis, the thus-produced antibody having 4 azide groups linked to its Fc region (data not shown).

[0172] (Hi) Preparation of VHH-hIgGl-4(DBCO-PEG3-vc-PAB-MMAE)

[0173] The buffer of trimannosyl HCAb-4Az (15.42 mg) was changed to 25 mM 2-(N- morpholino) ethanesulfonic acid (MES) buffer (pH 6.5), and the final concentration was adjusted to be 5 mg / mL. The solution containing dimethyl sulfoxide (DMSO; 3.2 mL, 20% of buffer) and DBCO-PEG3-VC-PAB-MMAE (10 mM stock of DMSO, 20 eq. for antibody, 1.6 mL) was slowly added to the antibody solution. The reaction mixture was incubated in shaking incubator (150 rpm) at 37°C for 20 hours. The centrifugal filter with 30 kDa of nominal molecular weight limit (NMWL) and 25 mM MES pH 6.5 was used for the concentration of the ADC product and the removal of unbound PEG3-VC-PAB-MMAE (set: 4°C, 4000 rpm, 15 minutes; repeat four times). The ADC concentration was estimated by measuring the absorbance at 280 nm (as absorption coefficient at 280 nm, 1.36 mLmg-lcm-1 was used). The recovery yield was determined by A280 value.

[0174] According to the results of liquid chromatography mass spectrometry (LC-MS), the drug-antibody ratio (DAR) of the ADC was about 4 (Figs. 2A and 2B), indicated that four payloads (i.e., PEG3-VC-PAB-MMAE) were linked to the ADC. The thus-produced ADC was designated as “VHH-hIgGl-4(DBCO-PEG3-VC-PAB-MMAE)” that had 4 MMAE molecules linked to the Fc region of hlgGl via the linker composed of PEG3 and VC dipeptide. The concentration of the ADC was about 5.84 mg / mL, and the recovery yield was about 97.28%.

[0175] Preparation of antibody-drug conjugate (ADC) through cysteine-conjugation

[0176] ADC synthesis was performed based on a cysteine cross-linking method. Briefly, the humanized HCAb and sdAb were treated with different equivalents of Tris(2- carboxyethyl)phosphine hydrochloride (TCEP) in a conjugation buffer (100 mM sodium phosphate, 150 mM sodium chloride, and 2 mM EDTA, pH 7.5) at 30°C for 2 hours to reduce the disulfide bonds (S-S to -SH). The resulting free thiol (-SH) groups facilitated conjugation with a maleimidocaproyl (MC)-linked agent. In the subsequent conjugation step, different equivalents 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-acetylcysteine (NAC). The ADC products were purified and buffer-exchanged to PBS using an Amicon® Ultra 0.5 mL centrifugal filter with 4000 rpm centrifugation. 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.

[0177] Flow cytometry

[0178] The binding affinity of the present HCAb towards various types of cancer cell lines was determined by flow cytometry. In brief, the cancer cells were harvested using enzyme-free cell dissociation buffer and then washed with ice-cold PBS containing 2% fetal bovine serum (FBS). The cells were respectively stained with the present HCAb (1 : 100 dilution) in PBS containing 2% FBS for 1 hour at 4°C. After washing the cells with PBS 3 times, the cells were incubated with the secondary antibody (Alexa Fluor™ 488-conjugated donkey anti-mouse IgG (H+L)) for 30 minutes at 4°C. The expression level of CEACAM6 of the cell surface was analyzed by flow cytometry. Gating strategies were used to exclude debris and dead cells and a positive gate was set based on fluorescence intensity, guided by isotype controls. The percentage of positive cells was calculated using the formula (events in positive gate / total events in live cell gate) x 100.

[0179] In vitro killing assay

[0180] DIO4cancer cells were seeded on a 96-well plate overnight. Different concentrations of ADC (1 pM to 1 pM, serial 10-fold diluted) were added into the wells and incubated at 37°Cfor 48 hours. The medium was removed and cells were washed by PBS. The solution containing 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 5 mg / mL) was added to each well and incubated for 90 minutes. The MTT crystal was then solved in DMSO. The cell viability was measured by an enzyme-linked immunosorbent assay (ELISA) reader at the absorbance at 450 nm. Cell viability (%) was determined by dividing the OD value of ADC treatment by the OD value of no treatment (control).

[0181] Animal study

[0182] 1 X 107HT-29 cells were subcutaneously inoculated into SCID mice. One week after cancer cell inoculation, the present ADC was administered via weekly intravenous tail injection at a dose of 5 mg / kg. Meanwhile, FOLFIRI+cetuximab (a clinical regimen for colorectal cancer, in which the FOLFIRI regimen included 40 mg / kg of Irinotecan, 30 mg / kg of levofolinate calcium, and 55 mg / kg of 5 -fluorouracil was administered via intraperitoneal injection) and cetuximab (10 mg / kg) was administered via intravenous injection. Tumor volumes were measured every week using calipers, and were calculated using the following equation: (length *width2x 0.45).

[0183] Example 1 Characterization of the chimeric HCAb

[0184] The binding affinity of the present chimeric HCAb towards cancer cells were determined by flow cytometry in accordance with the procedures described in “Materials and Methods” of the present disclosure. As the data depicted in Fig. 3 and Table 2, the present HCAb recognized various types of CEACAM6-expressing cancer cells.

[0185] Table 2 Binding affinity of the HCAb towards CEACAM6-expressing cancer cellsCell line Positive percentagesLS1034 (Ras -mutation type 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 transduced H1299 (H1299 CEACAM6) 86.3%THP-1( human acute monocytic leukemia cell) 33.7%PBMC (one healthy donor) 10%

[0186] Example 2 Characterization of the chimeric ADC

[0187] The HCAb, which comprises a pair of homedimeric VHHs, each having SEQ ID NO:4, was conjugated with MMAE in accordance with the procedures described in “Materials and Methods” of the present disclosure. The data of SDS-PAGE demonstrated the coupling of MMAE to the HCAb (VHH-hlgGl) (Fig. 4). According to the analytic results, the (DAR) of the ADC was about 4 (Figs. 2A and 2B).

[0188] The cytotoxicity capability of the chimeric ADC was evaluated by incubating the ADC with H1299 CEACAM6 cells (CEACAM6-expressing cancer cells) or CEACAM6-non- expressing cells (including LX2 and H1299 CEACAM5; serving as control group in the study) for 48 hours, and the cell viability was then determined by MTT assay. As the results depicted in Fig. 5 A, the present ADC exhibited cytotoxic activity to CEACAM6-expressing cancer cells, while no obvious cytotoxicity was detected in CEACAM6-non-expressing cells, indicated that the present ADC was capable of specifically targeting and destroying CEACAM6-expressing cancer cells. The cytotoxicity effect of the present ADC was confirmed by Fig. 5B, which demonstrated that as compared to THP- 1 cancer cells (a human monocytic cell line derived from an acute monocytic leukemia patient), the present ADC exhibited low cytotoxicity towards normal peripheral blood mononuclear cells (PBMCs). The IC50 summary of chimeric HCAb-ADC against different cells was shown in Figure 5C.

[0189] Example 3 In vivo efficacy of the chimeric ADC in animal model

[0190] The anti-tumor effect of the chimeric ADC of Example 2 carrying MMAE or exatecan in animal models was examined in this example. The data of Fig. 6A indicated that compared to the control group (PBS treatment), the administration of 5 mg / kg of the chimeric ADC carrying MMAE every week for six times greatly suppressed tumor growth in the animal model. Of note, the anti-tumor effect of the chimeric ADC carrying MMAE was better than that of current regimens of RAS-wild type colorectal cancer therapy (z.e., FOLFIRI+cetuximab regimen) (Fig. 6A). The treatment of the present ADC did not change the mouse body weight (Fig. 6B), suggesting it is well-tolerated in mice. In addition, the anti-tumor effect of the chimeric ADC carrying exatecan was examined in the same animal model. 1 mg / kg of the chimeric ADC carrying exatecan was intravenously administrated into SCID mice when tumor volume reached -700 mm3. Two weeks after the treatment, the tumor volume was reduced to -300 mm3(Fig. 7) and the body weight was not significantly altered (Fig. 8). Taken together, indicating the chimeric HCAbcarrying either MMAE or exatecan have great anti-cancer activity and were well-tolerated in animal models.

[0191] The chimeric HCAb has a low affinity for normal tissues (Fig. 9). Immunohistochemical (IHC) staining was performed to assess the expression of CEACAM6 in normal human tissues using the chimeric HCAb. The weak or no signals detected by the chimeric HCAb demonstrated CEACAM6 is low-expressed in normal tissues.

[0192] Anti-CEACAM6 HCAb specifically recognizes colorectal, lung, bile duct and pancreatic cancer tissues (Fig. 10). Immunohistochemical (IHC) staining was performed to assess the expression of CEACAM6 in different types of cancers using the chimeric HCAb. The strong signals detected by the chimeric HCAb demonstrated CEACAM6 is high-expressed in cancer tissues.

[0193] In conclusion, the present disclosure provides a novel ADC. According to the examples of the present disclosure, the chimeric ADC exhibited binding affinity and specificity to tumor cells, and efficiently suppressed tumor growth in the animal model. Accordingly, the chimeric ADC may serve as a novel and potential agent for treating cancers.

[0194] It will be understood that the above description of embodiments is given by way of example only and that various modifications may be made by those with ordinary skill in the art. The above specification, examples and data provide a complete description of the structure and use of exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those with ordinary skill in the art could make numerous alterations to the disclosed embodiments without departing from the spirit or scope of this invention.

[0195] Example 4 The anti-cancer effect of the humanized HCAb-ADC

[0196] The humanized HCAb-ADC was generated by linking MMAE to the humanized HCAb which comprises a pair of homedimeric VHHs, each having SEQ ID NO:4, through cysteine- conjugation, following the procedures described in “Materials and Methods” of the present disclosure. The cytotoxicity capability of the humanized ADC was evaluated by incubating the ADC with PC-9 cells (CEACAM6-expressing cancer cells) for 96 hours, and the cell viability was then determined by MTT assay. The humanized ADC significantly inhibited cancer cell growth with an IC50 ( Half Maximal Inhibitory Concentration ) 10.2 nM (Fig. 11). The humanized HCAb-ADC also inhibited tumor growth in animal models in a dose-dependent manner which demonstrates its strong and specific killing effect on CEACAM6-expressing tumors (Fig. 12). No significant weight loss was observed after the humanized HCAb-ADC treatment indicates it is well-tolerated in animals (Fig. 13). The mpk represents mg / kg.

[0197] Example 5 The anti-cancer effect of the sdAb-ADC

[0198] The sdAb-ADC was generated by linking MMAE to the sdAb comprising the VHH having SEQ ID NO: 11 through cysteine -conjugation, following the procedures described in "Materials and Methods" of the present disclosure. The cytotoxicity capability of the sdAb-ADC was evaluated by incubating the sdAb-ADC with CEACAM6-expressing cancer cell lines for 96 hours, and the cell viability was then determined by MTT assay. The sdAb-ADC significantly inhibited cancer cell growth with an IC50 ~1 nM compared to normal IgG-ADC (IC50>100nM) (Fig. 14). In addition, the administration of sdAb-ADC (4 mg / kg) suppressed tumor growth in animal models which demonstrates its strong killing effect on CEACAM6-expressing tumors (Fig. 15). The sdAb-ADC did not significantly induce weight loss which indicates it is well-tolerated in animals (Fig. 16).

[0199] In conclusion, the present disclosure provides novel ADCs. According to the examples of the present disclosure, the present ADC (chimeric-, humanized-, and sdAb-ADC) exhibited binding affinity and specificity to tumor cells, and efficiently suppressed tumor growth in the animal model. Accordingly, the present ADCs may serve as a novel and potential agent for treating cancers.

[0200] It will be understood that the above description of embodiments is given by way of example only and that various modifications may be made by those with ordinary skill in the art. The above specification, examples and data provide a complete description of the structure and use of exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those with ordinary skill in the art could make numerous alterations to the disclosed embodiments without departing from the spirit or scope of this invention.SEQUENCE LISTING

Claims

WHAT IS CLAIMED IS:

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 fragment crystallizable region (Fc region) of an immunoglobulin fused to each of the VHH domain(s) optionally via a peptide linker, 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 the amino acid sequence of SEQ ID NO: 1 or 7; a second CDR (CDR-2) comprising the amino acid sequence of SEQ ID NO: 2 or 8; and a third CDR (CDR-3) comprising the amino acid sequence of SEQ ID NO: 3 or 9; and a therapeutic agent; wherein the therapeutic agent links to the Fc region of the recombinant antibody or the VHH of the sdAb, optionally via a linker.

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

3. The immunoconjugate of claim 1 or 2, wherein the VHH comprises CDR-1 comprising the 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 the 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 the 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..

4. The immunoconjugate of claim 1 or 2, wherein the VHH comprises CDR-1 comprising the 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: 7, CDR-2 comprising the 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: 8 and CDR-3 comprising the 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: 9..

5. The immunoconjugate of claim 1 or 2, wherein the VHH domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: k4, 10 or 11.

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

7. The immunoconjugate of claim 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 homedimeric VHHs, wherein each of the VHH domain comprising 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 pair of the VHHs are different and each of the VHH domain comprising an amino acid sequence selected from SEQ ID NOs: 4, 10 and 11.

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

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

12. The immunoconjugate of claim 11, wherein the immunoglobulin is IgGl.

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

14. The immunoconjugate of claim 1 or 2, wherein the therapeutic agent is a cytotoxic drug, a radioactive nuclide, a cytokine, a hormone drug, an immune stimulating agent, or an immunotherapeutic drug.

15. The immunoconjugate of claim 14, wherein the cytotoxic drug is auristatin, exatecan, irinotecan, levofolinate, 5 -fluorouracil or a derivative thereof.

16. The immunoconjugate of claim 14, wherein the cytotoxic drug is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

17. The immunoconjugate of claim 1 or 2, wherein the linker is a cleavable linker or non-cleavable linker.

18. The immunoconjugate of claim 17, 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., S-S disulfide linkage, hydrazone linker, ester linker or amide linker), and a glutathionesensitive linker (e.g., N-Succinimidyl 4-(2-pyridyldithio) butanoate (SPDB) or N-succinimidyl-4- (2 -pyridyldithio) pentanoate (SPP)).

19. The immunoconjugate of claim 17, wherein the non-cleavable linker include, maleimidocaproyl (MC), maleimidomethyl cyclohexane- 1 -carboxylate (MCC), and succinimidyl- 4-[N-maleimidomethyl] cyclohexane-l-carboxylate (SMCC).

20. A pharmaceutical composition comprising the immunoconjugate any one of claims 1 to 19, and a pharmaceutically acceptable carrier.

21. A pharmaceutical composition for use in a method of treating a cancer in a subject, wherein the composition comprises an effective amount of the immunoconjugate of any one of claims 1 to 19.

22. The pharmaceutical composition for use in the method of claim 21, wherein the cancer is gastric cancer, lung cancer, bladder cancer, breast cancer, pancreatic cancer, renal cancer, colorectal cancer, cervical cancer, ovarian cancer, brain tumor, prostate cancer, hepatocellular carcinoma, melanoma, esophageal carcinoma, multiple myeloma, or head and neck squamous cell carcinoma.

23. The pharmaceutical composition for use in the method of claim 21, wherein the subject is a human.

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