Single-domain antibodies against gpa33, and derivatives and uses thereof

Abstract

The application belongs to the field of immunology and relates to a single-domain antibody against GPA33, a protein derived therefrom and application thereof. The single-domain antibody is composed of a heavy chain, the heavy chain comprising heavy chain CDR1 shown in any one of SEQ ID NO:41-SEQ ID NO:47, heavy chain CDR2 shown in any one of SEQ ID NO:48-SEQ ID NO:55 and heavy chain CDR3 shown in any one of SEQ ID NO:56-SEQ ID NO:68. Compared with the prior art, the application has the beneficial effect that the application uses biological genetic engineering technology to screen out single-domain antibodies specific to GPA33, the antibodies have obvious preliminary affinity, can block the release of cytokines by specific cells, have good binding activity through prokaryotic expression and have good drugability.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology or immunology, specifically to single-domain antibodies against GPA33, their derived proteins, and their applications. Background Technology

[0002] Colorectal cancer is one of the most common malignant tumors in Western countries and a leading cause of cancer death. Statistics from 2018 show that there were 18.1 million people worldwide suffering from cancer-related complications and 9.6 million deaths. The cure rate for colorectal cancer was 6.1%, and the mortality rate was 9.2%. Due to the high resistance to traditional therapies developed in microdisseminated colorectal cancer in recent years, the development of novel treatment methods and drugs is urgently needed. Our goals include generating less immunogenicity, constructing humanized or chimeric antibodies to reduce the patient's immune response, allowing for antibody reuse, and achieving more specific antigen recognition.

[0003] GPA33 (Cell surface A33 antigen, Swiss Prot database accession number: Q99795) is a type I single transmembrane protein with a molecular weight of 35.6 kDa, composed of 319 amino acids. Its glycosylation modification is located between 1 and 235 amino acids, and it is located outside the cell membrane. GPA33 is a cell surface differentiation antigen belonging to the immunoglobulin superfamily. The GPA33 allele is located on chromosome 1q24, containing 7 exons, with a total genomic DNA length of 37787 bp.

[0004] GPA33 exhibits tissue-specific expression. It is specifically expressed in gastrointestinal epithelial tissues, primarily distributed in mucosal epithelial cells, with lower levels in other tissues. Although the function of GPA33 is not fully understood, oncology studies have found high expression of GPA33 in over 95% of colorectal cancers. Due to the high intestinal tissue specificity of GPA33 expression, it not only provides an indicator for intestinal tumor detection but also plays a crucial role in studying the occurrence and progression of intestinal tumors.

[0005] As early as 2009, French researchers discovered that PPARγ ligands upregulate the mRNA and protein levels of GPA33 in a time- and concentration-dependent manner. Using DNA microarray technology, researchers found that the GPA33 gene is a target gene of PPARγ in HT29-Cl.16E cells; administration of the PPARγ agonist GW7845 to different human colon cancer cells (including HT29-Cl.16E, Caco2, and SW1116) showed a positive correlation between the two. The main mechanism involved is that PPARγ activation induces KLF4 expression; KLF4 binds to the promoter of GPA33, thereby increasing GPA33 expression.

[0006] GPA33 is also associated with osteoarthritis. Currently, clinical diagnosis of osteoarthritis mainly relies on imaging examinations. However, osteoarthritis often presents with few or no symptoms in its early stages, and patients only seek medical attention when secondary inflammation, pain, and impaired joint movement occur. By this time, joint damage has already occurred. Developing methods for early diagnosis of osteoarthritis is a pressing issue. A 2018 study reported significant differences in GPA33 gene expression between normal synovial tissue and osteoarthritic synovial tissue, suggesting that GPA33 could be used to develop products for diagnosing osteoarthritis. Researchers used quantitative real-time PCR to find that GPA33 gene expression was approximately 10 times higher in osteoarthritic synovial tissue compared to normal synovial tissue. Furthermore, similar results were obtained using immunoassay, in situ hybridization, microarray analysis, or high-throughput sequencing. Compared to traditional methods, gene diagnosis is more timely and sensitive, potentially reducing osteoarthritis mortality.

[0007] Immune dysregulation refers to the damage caused to one's own tissues and organs by an autoimmune response, resulting in symptoms. A 2021 study reported the expression pattern of GPA33 in human leukocyte subsets using mass spectrometry and flow cytometry. The results showed that GPA33 was expressed in B cells, dendritic cells, natural killer cells, and innate lymphocytes, with significant expression in CD4+ T cells. Primitive T cells and CXCR5+ regulatory T cells expressed high levels of GPA33, while primitive CD4+ T cells expressed moderate levels. The expression pattern of GPA33 highlights the functional heterogeneity of CD4+ central memory T cell populations. GPA33+CD4+ central memory T cells are completely undifferentiated; true central memory T cells lack immediate effector function, while GPA33+ central memory T cells exhibit rapid effector function. GPA33 expression in conventional CD4+ T cells suggests that it plays a role in localization and self-protection in the undifferentiated state. Meanwhile, research results also indicate that GPA33 can recognize human tTreg cells and provide a safer and more effective adoptive cell therapy strategy for isolating these cells. Treg cells can be generated during thymic development (tTreg cells) or derived from mature conventional CD4+ T cells (pTreg cells). Mouse studies have shown that tTreg cells are highly conserved, while pTreg cells can be reverted to conventional CD4+ T cells. Proteomics and transcriptomics studies have shown that GPA33 is expressed on a subset of human Treg cells. GPA33 is acquired late in tTreg cell development and is not expressed in TGF-β-induced Treg cells. GPA33 can recognize Treg cells in human blood that lack the ability to produce effector cytokines (IL-2, IFN-γ, IL-17). Treg cells with high GPA33 expression generally preferentially express the transcription factor Helios, which marks tTreg cells, and therefore can be robustly and stably expanded in vitro even in the absence of rapamycin. The expanded GPA33-overexpressing Treg cells were inhibitory and unable to produce pro-inflammatory cytokines.

[0008] Currently, there are few targeted clinical drugs for GPA33. Servier and Macro Genics have developed a bispecific antibody (DART) that recruits T cells through a dual-parental and highly targeted approach. DARTs are bispecific antibodies composed of the VH chain of one antibody and the VL chain of another antibody linked together. The VH of the second antibody is linked to the VL of the first antibody, forming a DART-like antibody, which is stabilized by an additional disulfide bond. MGD007 is a DART protein designed to guide T cells to target GPA33-expressing colorectal cancer cells. It entered Phase I clinical trials in July 2014 (code name NCT02248805). MGD007 has an Fc region fused to its structure, resulting in a longer serum half-life. Related xenograft studies have shown that tumor growth is inhibited at levels as low as 4 μg / kg. CD8+ and CD4+ T cells mediate the lysis of GPA33-expressing tumor cells, with increased activity, granzyme, and perforin. Notably, the suppressed T cell population can also be used to mediate the lysis of GPA33-expressing tumor cells. Along with CTL activity, T cell activation and expansion were observed in a GPA33-dependent manner. In cynomolgus monkeys, a 4-week course at a dose of 100 μg / kg was well-tolerated, consistent with the pharmacokinetics of Fc-containing molecules.

[0009] Several other laboratories are also continuously validating their independently developed antibodies and related technologies. A 2020 paper published by the Swiss Federal Institute of Technology (ETH Zurich) mentions a GPA33 antibody—A2—generated using phage display technology. A2 recognizes the "V" domain of GPA33 and can uniformly stain poorly, moderately, and well-differentiated colon adenocarcinoma samples. The A2 antibody is a recombinant mouse IgG2a subtype that preferentially targets GPA33-transfected CT26 mouse colon adenocarcinoma cells in immunized mice, distributing uniformly within the tumor mass, while other antibodies show patchy staining in tumor lesions. In vitro, A2 effectively induces the killing of GPA33-expressing cells through antibody-dependent cytotoxicity and inhibits the growth of GPA33-positive mouse CT26 and C51 lung metastases in vivo. On the other hand, antibody-based contrast agents are also under development as an auxiliary diagnostic tool for solid tumors. Researchers at Sichuan University constructed an anti-GPA33 GPA33scFv-Fc antibody by fusing GPA33-scFv with the Fc fragment of a human IgG1 antibody. GPA33-scFv-Fc specifically binds to GPA33-positive colorectal cancer cells and tumor tissue. After intravenous injection of GPA33-scFv-Fc labeled with the near-infrared fluorescent probe CF750 into GPA33-positive LS174T tumor graft mice, high-contrast images of the tumor grafts were dynamically recorded over 24 hours using an optical imaging system. Other researchers isolated exosomes from GPA33-positive LIM1215 cells and loaded them with doxorubicin; simultaneously, they coated carboxyl-containing superparamagnetic iron oxide nanoparticles (US) with a GPA33 antibody (GPA33Ab-US), hoping that the GPA33 antibody on the surface of these nanoparticles would bind to GPA33-positive exosomes and form a complex targeting GPA33-positive colon cancer cells. The results showed that GPA33Ab-US-Exo / Dox had good affinity and anti-proliferative activity in LIM1215 cells. Other in vivo studies have shown that GPA33Ab-US-Exo / Dox has good tumor targeting ability, can inhibit tumor growth, prolong the survival of mice, and reduce cardiotoxicity.

[0010] In summary, there is still a need in the field for antibodies that can bind with high affinity to GPA33, especially those specific to GPA33 and CD3.

[0011] Nanobodies are a rising star in the antibody field. Due to their small molecular weight, bivalent, trivalent, or bispecific antibodies can be obtained through simple molecular cloning techniques. Because of their small molecular size, nanobodies can achieve high yields in both prokaryotic expression systems (E. coli) and eukaryotic expression systems (CHO cells, 293 cells, etc.). The rapid development of nanobodies represents a powerful force with immense potential in antibody drug development, signifying an important direction for future antibody drug development.

[0012] Nanobodies exhibit high affinity and excellent penetration ability for their target binding sites, making them easier to bind to receptors and penetrate into tissues with low blood vessels. Furthermore, given the low immunogenicity of nanobodies, repeated administration to mice did not induce any humoral or cellular immunity. In addition, nanobodies can be used to construct various molecular structures, enabling molecular adjuvant therapy. Summary of the Invention

[0013] The purpose of this invention is to provide a single-domain antibody against GPA33 and its derived protein and its applications. The aforementioned single-domain antibody and its derived protein have a strong binding ability to GPA33 protein and can mediate cell internalization or ADCC.

[0014] Single-domain antibodies, as a type of monoclonal antibody, generally have higher affinity and better stability than traditional monoclonal antibodies, and they also possess the advantages of traditional monoclonal antibodies. Therefore, this invention utilizes the above advantages to obtain a single-domain antibody targeting GPA33. After prokaryotic expression, it exhibits a strong binding ability to the GPA33 protein. After eukaryotic expression, it was found that it can mediate cell internalization or ADCC activity, demonstrating certain drug-like properties.

[0015] In a first aspect, the present invention provides a single-domain antibody against GPA33, said single-domain antibody being composed of heavy chains, the heavy chains including heavy chain CDR1 shown in any one of SEQ ID NO:41-SEQ ID NO:47, heavy chain CDR2 shown in any one of SEQ ID NO:48-SEQ ID NO:55, and heavy chain CDR3 shown in any one of SEQ ID NO:56-SEQ ID NO:68.

[0016] Specifically, the heavy chain includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3; the amino acid sequences of said heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are one of the following (1)-(20):

[0017] (1) CDR1 shown in SEQ ID NO:47, CDR2 shown in SEQ ID NO:50, and CDR3 shown in SEQ ID NO:62;

[0018] (2) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:50, and CDR3 shown in SEQ ID NO:63;

[0019] (3) CDR1 shown in SEQ ID NO:47, CDR2 shown in SEQ ID NO:50, and CDR3 shown in SEQ ID NO:63;

[0020] (4) CDR1 shown in SEQ ID NO:45, CDR2 shown in SEQ ID NO:50, and CDR3 shown in SEQ ID NO:63;

[0021] (5) CDR1 shown in SEQ ID NO:47, CDR2 shown in SEQ ID NO:52, and CDR3 shown in SEQ ID NO:63;

[0022] (6) CDR1 shown in SEQ ID NO:47, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:63;

[0023] (7) CDR1 shown in SEQ ID NO:47, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:61;

[0024] (8) CDR1 shown in SEQ ID NO:42, CDR2 shown in SEQ ID NO:53, and CDR3 shown in SEQ ID NO:66;

[0025] (9) CDR1 shown in SEQ ID NO:42, CDR2 shown in SEQ ID NO:53, and CDR3 shown in SEQ ID NO:67;

[0026] (10) CDR1 shown in SEQ ID NO:42, CDR2 shown in SEQ ID NO:53, and CDR3 shown in SEQ ID NO:65;

[0027] (11) CDR1 shown in SEQ ID NO:44, CDR2 shown in SEQ ID NO:53, and CDR3 shown in SEQ ID NO:64;

[0028] (12) CDR1 shown in SEQ ID NO:43, CDR2 shown in SEQ ID NO:54, and CDR3 shown in SEQ ID NO:59;

[0029] (13) CDR1 shown in SEQ ID NO:41, CDR2 shown in SEQ ID NO:48, and CDR3 shown in SEQ ID NO:68;

[0030] (14) CDR1 shown in SEQ ID NO:47, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:60;

[0031] (15) CDR1 shown in SEQ ID NO:47, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:57;

[0032] (16) CDR1 shown in SEQ ID NO:47, CDR2 shown in SEQ ID NO:51, and CDR3 shown in SEQ ID NO:56;

[0033] (17) CDR1 shown in SEQ ID NO:47, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:56;

[0034] (18) CDR1 shown in SEQ ID NO:47, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:58;

[0035] (19) CDR1 shown in SEQ ID NO:47, CDR2 shown in SEQ ID NO:55, and CDR3 shown in SEQ ID NO:58;

[0036] (20) CDR1 shown in SEQ ID NO:45, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:58.

[0037] The CDR combinations (1)-(20) above correspond to SEQ ID NO.1-20 respectively.

[0038] All of the above sequences can be replaced with sequences that have "at least 80% homology" or sequences that replace only one or a few amino acids; preferably "at least 85% homology", more preferably "at least 90% homology", even more preferably "at least 95% homology", and most preferably "at least 98% homology".

[0039] In a preferred embodiment, the sequence of the single-domain antibody further includes a frame region FR; the frame region FR includes the amino acid sequences of FR1, FR2, FR3 and FR4;

[0040] The amino acid sequences of the framework region FR are as follows:

[0041] The FR1 or variants of FR1 shown in SEQ ID NO:69-72, wherein the variants of FR1 contain substitutions of up to 3 amino acids in the FR1;

[0042] The FR2 or variants of FR2 shown in SEQ ID NO:73-77, wherein the variants of FR2 contain substitutions of up to 3 amino acids;

[0043] The FR3 or variants of FR3 shown in SEQ ID NO:78-89, wherein the variants of FR3 contain substitutions of up to 3 amino acids;

[0044] The FR4 or variants of FR4 shown in SEQ ID NO:90-91, wherein the variants of FR4 contain substitutions of up to 3 amino acids.

[0045] In one embodiment, the single-domain antibody against GPA33 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence homology with an amino acid sequence selected from SEQ ID NO: 1-20, and is capable of specifically binding to the GPA33 protein.

[0046] In another preferred embodiment, the single-domain antibody against GPA33 has at least 95% sequence homology with the amino acid sequences selected from SEQ ID NO: 1-20 and is capable of specifically binding to the GPA33 protein.

[0047] A second aspect of the present invention is to provide a single-domain antibody against GPA33, wherein the amino acid sequences of the single-domain antibody are as shown in SEQ ID NO.1-20, or the single-domain antibody has at least 95% sequence homology with the amino acid sequences of SEQ ID NO.1-20.

[0048] In one embodiment, the nucleic acid molecule encoding the single-domain antibody against GPA33 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence homology with nucleotide sequences selected from SEQ ID NO: 21-40, and the single-domain antibody against GPA33 encoded therein is capable of specifically binding to the GPA33 protein.

[0049] Preferably, the coding sequences of the single-domain antibodies are as shown in SEQ ID NO.21-40, or have at least 95% sequence homology with SEQ ID NO.21-40.

[0050] A third aspect of the present invention is to provide an Fc fusion antibody or humanized antibody of the aforementioned single-domain antibody against GPA33.

[0051] A fourth aspect of the present invention is to provide nucleotide molecules encoding the aforementioned single-domain antibody against GPA33, the nucleotide sequences of which are shown in SEQ ID NO: 21-40, or have at least 95% sequence homology with SEQ ID NO: 21-40.

[0052] A fifth aspect of the present invention is to provide an expression vector comprising a nucleotide molecule encoding the aforementioned single-domain antibody or the aforementioned Fc fusion antibody.

[0053] A sixth aspect of the invention is to provide a host cell or non-human object that can express the aforementioned single-domain antibody against GPA33, or an expression vector comprising the aforementioned.

[0054] The present invention also provides a method for generating a single-domain antibody against GPA33 or an Fc fusion antibody thereto, comprising the steps of: (a) culturing the aforementioned host cells under conditions suitable for generating the single-domain antibody or an Fc fusion antibody thereto, thereby obtaining a culture containing the single-domain antibody against GPA33 or an Fc fusion antibody thereto; (b) isolating or recovering the single-domain antibody against GPA33 or an Fc fusion antibody thereto from the culture; and (c) optionally, purifying and / or modifying the single-domain antibody against GPA33 or an Fc fusion antibody thereto obtained in step (b).

[0055] A seventh aspect of the present invention is to provide a pharmaceutical composition comprising: (i) a single-domain antibody against GPA33 as described above, or an Fc fusion antibody of a single-domain antibody against GPA33 as described above; and (ii) one or more pharmaceutically acceptable excipients.

[0056] This invention also provides the use of the aforementioned single-domain antibody against GPA33 in the preparation of drugs that inhibit GPA33 gene expression, antitumor drugs, or antiarthritis drugs. Drugs that inhibit GPA33 gene expression are applicable to any condition with high GPA33 gene expression. Preferably, the tumor includes, but is not limited to, colorectal cancer. The arthritis may be osteoarthritis.

[0057] This invention also provides the use of single-domain antibodies against GPA33 in the preparation of drugs that mediate cell internalization or ADCC.

[0058] The present invention also provides the use of the aforementioned single-domain antibody against GPA33, or the Fc fusion antibody of the aforementioned single-domain antibody against GPA33, for the preparation of reagents, detection plates or kits (e.g., ELISA kits); wherein the reagents, detection plates or kits are used to detect the presence and / or content of GPA33 protein in a sample.

[0059] The single-domain antibody is VHH, which contains only the antibody heavy chain and not the antibody light chain.

[0060] Compared to existing technologies, the beneficial effects of this invention are as follows: This invention uses bioengineering technology to screen for single-domain antibodies specifically targeting GPA33. These antibodies exhibit significant initial affinity and excellent effects in blocking the release of cytokines from specific cells. They also demonstrate good binding activity upon prokaryotic expression, possessing good drug-like properties, and have the following advantages:

[0061] (1) The single-domain antibody obtained by the present invention has a flexible expression system. It can be expressed in a prokaryotic system or in a eukaryotic system of yeast cells or mammalian cells. Moreover, its expression cost in the prokaryotic expression system is low, which can reduce the production cost in the later stage.

[0062] (2) The single-domain antibody obtained by the present invention has simple multi-combination form modification. It can be obtained by simple tandem through genetic engineering to obtain multivalent and multispecific antibodies. Moreover, its immune heterogeneity is very low and it will not produce a strong immune response without humanization modification.

[0063] (3) The present invention provides a single-domain antibody with a wider affinity range. Before affinity maturation, its affinity range can be from nM to pM, providing multiple options for antibodies for different purposes in the later stage. Attached Figure Description

[0064] Figure 1 SDS-PAGE analysis of recombinant human GPA33 protein;

[0065] Figure 2 VHH sequence insertion rate analysis;

[0066] Figure 3 Enrichment of the library selected targeting GPA33;

[0067] Figure 4 GPA33 target partial prokaryotic expression antibody SDS-PAGE;

[0068] Figure 5 GPA33 target partial eukaryotic expression antibody SDS-PAGE;

[0069] Figure 6 GPA33 target antibody antigen binding activity;

[0070] Figure 7 GPA33 target tool antibody antigen binding activity;

[0071] Figure 8 GPA33 target antibody species cross-reactive antigen binding activity;

[0072] Figure 9 ADCC activity of GPA33 target nanobodies. Detailed Implementation

[0073] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0074] Single-domain antibodies (sdAbs, also referred to as nanobodies or VHHs by the developer Ablynx) are well known to those skilled in the art. A single-domain antibody is an antibody whose complementarity-determining region is part of a single-domain polypeptide. Therefore, a single-domain antibody contains a single complementarity-determining region (a single CDR1, a single CDR2, and a single CDR3). Examples of single-domain antibodies include antibodies containing only the heavy chain (which naturally does not contain a light chain), single-domain antibodies derived from conventional antibodies, and engineered antibodies.

[0075] Single-domain antibodies can be derived from any species, including mice, humans, camels, llamas, goats, rabbits, and cattle. For example, naturally occurring VHH molecules can be derived from antibodies provided by camelid species such as camels, dromedaries, llamas, and guanacos. Like complete antibodies, single-domain antibodies can selectively bind to specific antigens. Single-domain antibodies may contain only variable domains of the immunoglobulin chain, which have CDR1, CDR2, and CDR3, as well as a frame region.

[0076] As used herein, the term "sequence homology" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is typically expressed as a percentage. Preferably, homology is determined over the overall length of the sequences being compared. Thus, two copies having completely identical sequences have 100% homology.

[0077] In this invention, sequences with high homology to the CDR1-3 sequences disclosed in this invention can also be used to obtain nanobodies targeting GPA33.

[0078] In some embodiments, sequences having “at least 80% homology” with the sequences in (1)-(20), or sequences having “at least 85% homology”, “at least 90% homology”, “at least 95% homology”, or “at least 98% homology” can achieve the purpose of the invention (i.e., derived proteins).

[0079] In some embodiments, the inventive objective can also be achieved by replacing only one or a few amino acids compared to the sequences in SEQ ID NO: 1-20, for example, by including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions. In practice, when determining the degree of sequence homology between two amino acid sequences or when determining the combination of CDR1, CDR2, and CDR3 in a single-domain antibody, those skilled in the art can consider so-called “conserved” amino acid substitutions, in which case the substitution will preferably be a conserved amino acid substitution, which can generally be described as an amino acid residue being replaced by another amino acid residue having a similar chemical structure, and the substitution having little or no effect on the function, activity, or other biological properties of the polypeptide. Such conserved amino acid substitutions are common in the art; for example, a conserved amino acid substitution is one or a few amino acids from the following groups (a)-(d) being replaced by another or a few amino acids from the same group: (a) polar negatively charged residues and their uncharged amides: Asp, Asn, Glu, Gln;

[0080] (b) Polar positively charged residues: His, Arg, Lys; (c) Aromatic residues: Phe, Trp, Tyr; (d) Aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. Particularly preferred conserved amino acid substitutions are as follows:

[0081] Asp is replaced by Glu; Asn is replaced by Gln or His; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Asn or Gln; Arg is replaced by Lys; Lys is replaced by Arg or Gln; Phe is replaced by Met, Leu, or Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe or Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr, or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser. Furthermore, those skilled in the art will understand that the inventiveness of the single-domain antibody lies in the CDR1-3 regions, while the frame region sequences FR1-4 are not immutable, and the sequences of FR1-4 can adopt conserved sequence variants of the sequences disclosed in this invention.

[0082] The preferred host cell of the present invention is a bacterial cell, a fungal cell, or a mammalian cell.

[0083] This patent involves preparing a target protein and a truncated form of the target protein using genetic engineering technology. The obtained antigen protein is then used to immunize Bactrian camels in Alashan, Inner Mongolia. After multiple immunizations, peripheral blood lymphocytes or spleen cells of the camels are obtained. Through genetic engineering, the variable region coding sequence of the camel-derived antibody is recombined into a phage display vector. Specific antibodies against the antigen protein are screened using phage display technology, and their ability to bind to the antigen and their application in the treatment of autoimmune diseases are further tested.

[0084] The above technical solution will now be broken down and explained in detail, and described with specific embodiments:

[0085] Example 1: Preparation of recombinant human GPA33 extracellular domain protein:

[0086] The human recombinant extracellular domain protein used in this patent was expressed and purified by the company itself. The specific design scheme of the expression vector for the human recombinant GPA33 protein is as follows:

[0087] (1) The coding sequence of GPA33 was obtained by searching in NCBI. Its accession number is NM_005814.2. The accession number of the amino acid sequence encoded by this sequence is NP_005805.1, and the Uniprot ID is Q99795.

[0088] (2) The amino acid sequence corresponding to NP_005805.1 was analyzed for transmembrane region and extracellular terminus using TMHMM and SMART websites, respectively.

[0089] (3) The analysis results show that the extracellular terminus of GPA33 protein consists of amino acids 1-235, of which 1-21 are the signal peptide of the protein.

[0090] (4) The nucleotide sequence encoding the GPA33 protein from amino acid position 22 to 235 was cloned into the vector pcDNA3.4 using gene synthesis.

[0091] (5) The constructed vector was subjected to Sanger sequencing. After comparing with the original sequence and confirming that there were no errors, the recombinant plasmid was extracted in batches, endotoxin was removed, and it was transfected into suspension 293F for expression and purification of the target protein. The SDS-PAGE analysis results of the purified GPA33 recombinant protein are as follows: Figure 1 As shown, the purified protein has a purity of up to 90%, which meets the needs of animal immunity.

[0092] Example 2: Construction of a single-domain antibody library targeting the GPA33 protein:

[0093] One mg of the purified human recombinant GPA33 protein obtained in Example 1 was mixed with an equal volume of Freund's complete adjuvant and used to immunize a Bactrian camel from Alashan, Inner Mongolia. The camel was immunized once a week for a total of 7 weeks. Except for the first immunization, the remaining six immunizations were performed by mixing one mg of GPA33 protein with an equal volume of Freund's incomplete adjuvant. This immunization process was intended to concentrate the stimulation of the camel to produce antibodies against GPA33 protein.

[0094] After animal immunization, 150 mL of peripheral blood lymphocytes were collected from camels, and RNA was extracted from the cells. cDNA was synthesized using the extracted total RNA, and VHH (antibody heavy chain variable region) was amplified using nested PCR with the cDNA as a template.

[0095] Then, the pMECS vector and VHH fragment were digested with restriction endonucleases, and the digested fragments were ligated to the vector. The ligated fragments were electroporated into competent TG1 cells to construct a phage display library of the GPA33 protein, and the library size was determined to be approximately 1 × 10⁻⁶. 9 Simultaneously, the correct insertion rate of the target fragment in the library was detected by colony PCR identification, and the results are as follows: Figure 2 As shown.

[0096] The results showed that after PCR amplification of 30 colonies randomly selected from the library, 28 clones were able to amplify a band of 600 bp (predicted size), while 2 clones amplified an incorrect band. Therefore, the correct insertion rate was 28 ÷ 30 × 100% ≈ 93.3%.

[0097] Example 3: Screening for single-domain antibodies against GPA33 protein:

[0098] Take 200 μL of the recombinant TG1 cells from Example 2 and culture them in 2×TY medium. During the culture, add 40 μL of helper phage VCSM13 to infect the TG1 cells and culture them overnight to amplify the phage. The next day, precipitate the phage with PEG / NaCl and collect the amplified phage by centrifugation.

[0099] 500 μg of GPA33 protein diluted in 100 mM pH 8.3 NaHCO3 was coupled onto an ELISA plate and incubated overnight at 4°C. A negative control well (culture medium control) was also included. The next day, 200 μL of 3% skim milk was added, and the plate was blocked at room temperature for 2 hours. After blocking, 100 μL of the amplified phage library (approximately 2 × 10⁻⁶) was added. 11 (1 phage particle), incubate at room temperature for 1 hour; after 1 hour, wash 15 times with PBS + 0.05% Tween-20 to remove unbound phage.

[0100] Phages specifically bound to GPA33 protein were dissociated using trypsin at a final concentration of 25 mg / mL and then used to infect *E. coli* TG1 cells in logarithmic growth phase. The cells were cultured at 37°C for 1 hour, and the resulting phages were collected for the next round of screening. This screening process was repeated once to gradually enrich the cells. When the enrichment factor reached 10-fold or greater, the enrichment effect was as follows: Figure 3 As shown.

[0101] Figure 3 In this context, P / N = the number of monoclonal bacteria grown from phages eluted from positive wells in the biopanning process after infecting TG1 bacteria / the number of monoclonal bacteria grown from phages eluted from negative wells after infecting TG1 bacteria. This parameter gradually increases after enrichment occurs. I / E = the total number of phages added to positive wells in each round of the biopanning process / the total number of phages eluted from positive wells in each round of the biopanning process. This parameter gradually approaches 1 after enrichment occurs.

[0102] Example 4: Screening for GPA33-specific positive clones using phage enzyme-linked immunosorbent assay (ELISA):

[0103] According to the screening method in Example 3 above, three rounds of screening were performed on single-domain antibodies against GPA33 protein. The phage enrichment factor against GPA33 protein reached more than 10. After screening, 384 single colonies were selected from the positive clones and inoculated into 96-well plates of 2×TY medium containing 100 μg / mL ampicillin. A blank control was set up. After incubation at 37°C to the logarithmic phase, IPTG was added to a final concentration of 1 mM and incubated overnight at 28°C.

[0104] Crude antibody was obtained using the osmotic burst method. GPA33 recombinant protein was released into 100 mM NaHCO3 (pH 8.3), and 100 μg of protein was coated overnight at 4°C in an ELISA plate. 100 μL of the obtained crude antibody extract was transferred to an ELISA plate containing the antigen and incubated at room temperature for 1 h. Unbound antibody was washed away with PBST, and 100 μL of Mouse Anti-HA tag Antibody (HRP) (Mouse anti-HA horseradish peroxidase labeled antibody, ThermoFisher) diluted 1:2000 was added. The plate was incubated at room temperature for 1 h. Unbound antibody was washed away with PBST, and horseradish peroxidase chromogenic solution was added. The reaction was carried out at 37°C for 15 min, and then stop solution was added. The absorbance was read at 450 nm using an ELISA reader.

[0105] When the OD value of the sample well is more than 5 times that of the control well, it is determined to be a positive clone well. The bacteria in the positive clone well are transferred to LB medium containing 100 μg / mL ampicillin for plasmid extraction and sequencing.

[0106] Gene sequences of each clone were analyzed using the sequence alignment software VectorNTI. Clones with identical CDR1, CDR2, and CDR3 sequences were considered the same clone, while clones with different sequences were considered different clones. Ultimately, single-domain antibodies specifically targeting the GPA33 protein (SEQ ID NO. 1-20 and other single-domain antibodies with sequences not shown, such as 1G9, 1H5, 1H7, 2A10, 2A3, 3C6, 3F7, 3H10, 4B3, 4B5, 2D5, 2G5, 3B6, etc.) were obtained.

[0107] The amino acid sequence of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, constituting the entire VHH. The obtained single-domain antibody recombinant plasmid can be expressed in a prokaryotic system to ultimately obtain the single-domain antibody protein.

[0108] The CDR and FR sequences of 20 single-domain antibodies are shown in Table 1-6.

[0109] Table 1. CDR1 sequences of 20 single-domain antibodies Table 2. CDR2 sequences of 20 single-domain antibodies

[0110]

[0111] Table 3. CDR3 sequences of 20 single-domain antibodies

[0112]

[0113] Table 4. FR1 sequences of 20 single-domain antibodies

[0114]

[0115] Table 5. FR2 sequences of 20 single-domain antibodies

[0116]

[0117] Table 6. FR3 sequences of 20 single-domain antibodies

[0118]

[0119] The amino acid sequences SEQ ID NO.1-20 of the single-domain antibodies correspond one-to-one with the single-domain antibodies 1A2, 2H6, 4F11, 1E10, 3C4, 1A9, 1D7, 1B12, 4E2, 4C4, 2B2, 2E2, 4F7, 3G9, 2C2, 1A6, 2A4, 1A3, 2A12, and 4B9.

[0120] The FR4 sequence of the 4F7 single-domain antibody is SEQ ID NO: 90, while the FR4 sequence of the other 19 single-domain antibodies is SEQ ID NO: 91.

[0121] Example 5: Purification and expression of a specific single-domain antibody against GPA33 protein in the host bacterium *Escherichia coli*.

[0122] The plasmids (pMECS-VHH) of different clones obtained from sequencing analysis in Example 4 were electroporated into Escherichia coli HB2151 and plated on LB+amp+glucose culture plates containing ampicillin and glucose, and incubated overnight at 37°C. Single colonies were selected and inoculated into 5 mL of LB culture medium containing ampicillin and incubated overnight on a shaker at 37°C.

[0123] Inoculate 1 mL of overnight cultured bacteria into 330 mL of TB culture medium and incubate at 37°C in a shaker. When the OD600nm value reaches 0.6-0.9, add 1 M IPTG and incubate overnight at 28°C in a shaker. Centrifuge to collect E. coli and obtain crude antibody extract using the osmotic rupture method.

[0124] The antibody was purified by nickel column affinity chromatography. The purified single-domain antibody, such as... Figure 4 As shown, it includes VHH1 to 18.

[0125] VHH1-18 corresponds to the single-domain antibody of amino acid sequence SEQ ID NO.1-18, that is, VHH1-18 corresponds to: 1A2, 2H6, 4F11, 1E10, 3C4, 1A9, 1D7, 1B12, 4E2, 4C4, 2B2, 2E2, 4F7, 3G9, 2C2, 1A6, 2A4, 1A3.

[0126] Example 6: Construction of a eukaryotic expression vector for an Fc fusion antibody of a specific single-domain antibody against GPA33 protein

[0127] (1) Subcloning the target sequence obtained in Example 4 into a eukaryotic expression vector: The antibody screened in Example 4 was obtained by Sanger sequencing to obtain its nucleotide sequence;

[0128] (2) The codon-optimized nucleotide sequence (SEQ ID NO:21-40) was synthesized into the vector RJK-V4-hFC designed and modified by our company by sequence synthesis to obtain a recombinant eukaryotic expression vector. The modification method of the vector is as described in Example 10.

[0129] (3) Transform the recombinant eukaryotic expression vector constructed in step (2) into DH5α Escherichia coli, culture it for plasmid extraction, and remove endotoxins;

[0130] (4) The extracted plasmids were then sequenced and identified.

[0131] (5) After confirming the recombinant vector, prepare it for subsequent eukaryotic cell transfection and expression. After expressing the Fc protein of VHH by the method of Example 7 or 8, purify the above antibody by the method of Example 9.

[0132] Example 7: Expression of Fc fusion antibody of GPA33 protein-specific single-domain antibody in suspension ExpiCHO-S cells

[0133] (1) Three days before transfection, use 2.5×10 5 / mL cell passage and expansion culture ExpiCHO-S TM Cells, the calculated desired cell volume, were transferred to 120 mL (final volume) of fresh, preheated ExpiCHO. TM In a 500 mL shake flask of expression medium, the cell concentration was increased to approximately 4 × 10⁻⁶ cells / mL. 6 -6×10 6 live cells / mL;

[0134] (2) One day before transfection, ExpiCHO-S TM Cells were diluted to a concentration of 3.5 × 10⁻⁶. 6 Live cells / mL, incubate cells overnight;

[0135] (3) On the day of transfection, measure cell density and percentage of viable cells. Cell density should reach approximately 7 × 10⁻⁶ cells / day before transfection. 6 -10×10 6 live cells / mL;

[0136] (4) Use fresh ExpiCHO preheated to 37°C TM The expression medium was used to dilute the cells to 6 × 10⁶. 6 viable cells / mL. The calculated desired cell volume was transferred to 100 mL (final volume) of fresh, preheated ExpiCHO. TM In a 500 mL shake flask of expression medium;

[0137] (5) Gently invert to mix ExpiFectamine TM CHO reagent, using 3.7 mL OptiPRO TM Culture medium for diluting ExpiFectamine TM CHO reagent, vortex or mix well;

[0138] (6) Use 4 mL of refrigerated OptiPRO TMThe plasmid DNA was diluted with culture medium and swirled to mix. The plasmid DNA was a eukaryotic expression vector for an Fc fusion antibody of a specific single-domain antibody against GPA33 protein, prepared in Example 6.

[0139] (7) Incubate the ExpiFectamine CHO / plasmid DNA complex at room temperature for 1-5 minutes, then gently add it to the prepared cell suspension while gently shaking the flask during the addition process;

[0140] (8) The cells were cultured with shaking in humidified air at 37°C and 8% CO2.

[0141] (9) Add 600 μl of ExpiFectamine on the first day after transfection (18-22 hours later). TM CHO Enhancer and 24mLExpiCHO feed.

[0142] (10) Collect the supernatant about 8 days after transfection (when cell viability is less than 70%).

[0143] Example 8: Expression of Fc fusion antibody of GPA33 protein-specific single-domain antibody in suspension 293F cells

[0144] Recombinant single-domain antibody expression experimental procedure (taking a 500mL shake flask as an example):

[0145] (1) Three days before transfection, use 2.5×10 5 After passage and expansion of 293F cells at / mL, the calculated desired cell volume was transferred to a 500mL shake flask containing 120mL (final volume) of fresh, preheated OPM-293 CD05 Medium to achieve a cell concentration of approximately 2×10⁻⁶. 6 -3×10 6 Live cells / mL.

[0146] (2) On the day of transfection, measure cell density and percentage of viable cells. Cell density should reach approximately 2 × 10⁻⁶ cells / day before transfection. 6 -3×10 6 Live cells / mL.

[0147] (3) Dilute the cells to 1×10⁻⁵ using preheated OPM-293 CD05 Medium. 6 1 live cells / mL. Calculate the required cell volume and transfer it to a 500 mL shake flask containing 100 mL (final volume) of fresh, preheated culture medium.

[0148] (4) Dilute PEI (1 mg / mL) reagent with 4 mL Opti-MEM medium, swirl or pipette to mix; dilute plasmid DNA with 4 mL Opt-MEM medium, swirl to mix, and filter through a 0.22 μm filter. Incubate at room temperature for 5 min.

[0149] (5) Add the diluted PEI reagent to the diluted DNA and mix by inverting. Incubate the PEI / plasmid DNA complex at room temperature for 15-20 minutes, then gently add it to the prepared cell suspension while gently shaking the flask during the addition process.

[0150] (6) Culture the cells at 37°C, 5% CO2, and 120 rpm with shaking.

[0151] (7) Add 5 mL of OPM-CHO PFF05 feed at 24 h and 72 h after transfection.

[0152] (8) Collect the protein expression supernatant about 7 days after transfection (when cell viability is less than 70%).

[0153] Example 9: Purification of human Fc recombinant single-domain antibody

[0154] (1) The protein expression supernatant obtained in Example 7 or 8 was filtered with a 0.45 μm disposable filter to remove insoluble impurities;

[0155] (2) The above filtrate was purified by affinity chromatography using a protein purifier. The agarose packing material coupled with Protein A was used to purify the filtrate by utilizing the ability of human Fc to bind to Protein A.

[0156] (3) Pass the filtrate through a pre-packed Protein A column at a flow rate of 1 mL / min. In this step, the target protein in the filtrate will bind to the packing material.

[0157] (4) Wash the impurity proteins bound to the column with low-salt and high-salt buffers;

[0158] (5) Use a low pH buffer to systemically bind the target protein on the column;

[0159] (6) Quickly add the eluent to a Tris-HCl solution with a pH of 9.0 to neutralize it;

[0160] (7) After dialyzing the neutralized protein solution, perform SDS-PAGE analysis to confirm that the protein purity is above 95% and the concentration is above 0.5 mg / mL. Then, store it at low temperature for later use. The SDS-PAGE results of some antibodies are shown below. Figure 5 As shown. Figure 5 In the diagram, 1-7 are single-domain antibodies 1A2, 1A3, 1A6, 1E10, 2A4, 2A12, and 2B2, respectively.

[0161] Example 10: Construction of the eukaryotic expression vector RJK-V4-hFc for nanobodies

[0162] The target vector RJK-V4-hFC used in this invention is a modified version of the Invitrogen commercial vector pCDNA3.4 (vector data link: https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / pcdna3_4_topo_ta_cloning_kit_man.pdf) by incorporating the Fc region of the heavy chain coding sequence of human IgG (NCBI Accession No.: AB776838.1). In other words, this vector contains the CH2 and CH3 hinge regions of the IgG heavy chain. The specific modification scheme is as follows:

[0163] (1) Select the restriction enzyme sites XbaI and AgeI on pcDNA3.4;

[0164] (2) Multiple cloning sites (MCS) and 6×His tags were introduced at the 5' and 3' ends of the Fc fragment coding sequence, respectively, by overlapping PCR.

[0165] (3) The above fragment was amplified by PCR using a pair of primers with XbaI and AgeI restriction sites respectively;

[0166] (4) The recombinant DNA fragments in pcDNA3.4 and (3) were digested with restriction endonucleases XbaI and AgeI, respectively;

[0167] (5) The digested vector and the insert fragment were ligated with T4 ligase, and then the ligation product was transformed into E. coli, amplified, sequenced and verified to obtain the recombinant plasmid.

[0168] Example 11: Determination of the binding dose-response curve of a specific single-domain antibody (prokaryotic expression) to GPA33 protein.

[0169] (1) Coat 50 μL of 1 μg / mL GPA33 at 4℃ overnight.

[0170] (2) Wash the plate; add 200 μL of 5% milk and seal at 37°C for 1 hour.

[0171] (3) VHH was diluted to 2 μg / mL, and then the antibody was serially diluted 5-fold to a total of 8 concentration gradients. The VHH is a specific single-domain antibody against the prokaryotically expressed GPA33 protein obtained in Example 5;

[0172] (4) Wash the plate; add 50 μL of the single-domain antibody obtained by dilution in step (3), double replicates, and incubate at 37°C for 1 h.

[0173] (5) Wash the plate; add 50 μL of mouse anti-HA tag-HRP secondary antibody and incubate at 37℃ for 30 min.

[0174] (6) Wash the plate (wash several times); add 50 μL of TMB that has been brought back to room temperature, and react at room temperature in the dark for 15 min.

[0175] (7) Add 50 μL of stop solution (1N HCl) and save the microplate reader reading.

[0176] (8) Plot the curve and calculate EC50, as follows: Figure 6 As shown, all 20 single-domain antibodies targeting GPA33 protein exhibit excellent binding efficacy and specificity to GPA33 protein.

[0177] It should be noted that, Figure 6 In the diagram, for example, 4B9-1 and 2B5-2 represent the first expression of the 4B9 single-domain antibody and the second expression of the 2B5 single-domain antibody, respectively. The "-1" and "-2" symbols are only used to distinguish the number of expression times (the sequences of 4B9-1 and 2B5-2 are the same as those of 4B9 and 2B5, respectively). All other "-1" and "-2" symbols in the diagram serve the same purpose.

[0178] Example 12: Expression and purification of a tool antibody (Tab) targeting human GPA33

[0179] The Tab(MGD007) sequence was obtained from the International Immunogenetics Database (IMGT).

[0180] The searched sequences were commissioned to General Biosystems (Anhui) Co., Ltd. for codon optimization in mammalian cell expression systems and cloned into the pcDNA3.1 vector.

[0181] After resistance screening, plasmid-positive bacteria were selected for amplification, and plasmids were extracted using a plasmid extraction kit (Macherey Nagel, Cat#740412.50).

[0182] Add 100 μg of plasmid (40 μg heavy chain + 60 μg light chain) per 100 mL of cells and transiently transpose in 293F cells (culture medium: FreeStyle 293Expression medium, Thermo, Cat#12338026+F-68, Thermo, Cat#24040032) using PEI;

[0183] 6–24 h after transfection, add 5% volume of 10% Peptone (Sigma, Cat#P0521-100G) and incubate with 8% CO2 at 130 rpm for about 7–8 days.

[0184] When cell viability dropped to 50%, the expression supernatant was collected and purified using a Protein A (GE, Cat#17-5438-02) gravity column.

[0185] After PBS dialysis, the concentration was determined using Nanodrop, the purity was identified by SEC, and the binding capacity was verified by indirect ELISA.

[0186] The Tab obtained by this method has a concentration of not less than 2 mg / ml and a purity greater than 94%. Its EC50 for binding with GPA33 (ACRO, Cat#FO1-H52H1) is approximately 0.16 nM. The results are as follows: Figure 7 As shown.

[0187] Example 13: Determination of species cross-binding dose-response curve of Fc fusion antibody for GPA33 protein-specific single-domain antibody.

[0188] (1) Coat 50 μL of human, cynomolgus monkey and mouse GPA33 at 1 μg / mL and incubate overnight at 4℃.

[0189] (2) Wash the plate; add 200 μL of 5% milk and seal at 37°C for 1 hour.

[0190] (3) Dilute VHH-hFc to 2 μg / mL, and then perform a 5-fold serial dilution of the antibody to a total of 8 concentration gradients. Here, VHH-hFc is the Fc fusion antibody of the GPA33 protein-specific single-domain antibody from Example 8, which was purified in Example 9.

[0191] (4) Wash the plate; add 50 μL of the single-domain antibody obtained by dilution in step (3), double replicates, and incubate at 37°C for 1 h.

[0192] (5) Wash the plate; add 50 μL of goat anti-human IgG-HRP secondary antibody and incubate at 37°C for 30 min.

[0193] (6) Wash the plate (wash several times); add 50 μL of TMB that has been brought back to room temperature, and react at room temperature in the dark for 15 min.

[0194] (7) Add 50 μL of stop solution (1N HCl) and save the microplate reader reading.

[0195] (8) Plot the curve and calculate EC50, as follows: Figure 8As shown; hIgG refers to the isotype control, an immunoglobulin molecule that does not bind to any target and is obtained through commercial purchase; Tab was prepared in Example 12; it can be seen that the 20 single-domain antibodies against GPA33 of the present invention have excellent species cross-binding efficacy against GPA33 protein.

[0196] Example 14: Human-specific single-domain antibody against GPA33 and tool antibody-induced ADCC (antibody-dependent cell-mediated cytotoxicity):

[0197] After being revived and passaged for 3-4 generations, Colo205 cells were collected and seeded into 96-well plates at a rate of 20,000 cells per well.

[0198] The Tab, hIgG, and VHH-hFc samples were prepared into solutions with a maximum concentration of 10 μg / mL and then serially diluted 10-fold to obtain seven concentrations. Among them, hIgG refers to the isotype control, an immunoglobulin molecule that does not bind to any target and is obtained through commercial purchase. The Tab was prepared in Example 12. Here, VHH-hFc is the Fc fusion antibody of the specific single-domain antibody of GPA33 protein in Example 8, which was purified in Example 9.

[0199] Add the serially diluted antibody solution to the cell culture wells in equal volumes to the cell suspension;

[0200] For the sample wells and E / T wells (antibody concentration of 0), Jurkat-NFAT-luc-FcγRIIIa cells were collected and added to the cell culture wells at a rate of 20,000 cells per well.

[0201] After 6 hours of incubation, cell killing was detected using the One-Glo kit, and luminescence was read.

[0202] Calculate the fold of induction: (sample - BG) / (E / T - BG)

[0203] Based on the target cell killing rate and concentration, a four-parameter fitting was performed to calculate the EC50 concentration of each antibody-mediated ADCC effect. The results are as follows: Figure 9 As shown, all 20 single-domain antibodies of this invention can effectively mediate ADCC.

[0204] The fluorescence value of the mixture from the previous step was read using a flow cytometer.

[0205] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A single-domain antibody against GPA33, characterized in that: The single-domain antibody is composed of heavy chains, including heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3; The amino acid sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are as follows (a) or (b): (a) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:50, and CDR3 shown in SEQ ID NO:63; (b) CDR1 shown in SEQ ID NO:47, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:

63.

2. The single-domain antibody against GPA33 according to claim 1, characterized in that: The antibody sequence further includes a frame region FR; the frame region FR includes the amino acid sequences of FR1, FR2, FR3, and FR4; the amino acid sequences of the frame region FR are as follows: The FR1 or a variant thereof shown in SEQ ID NO:72, wherein the variant thereof contains substitutions of up to 3 amino acids; The FR2 or a variant thereof shown in SEQ ID NO:75, wherein the variant thereof contains substitutions of up to 3 amino acids; The FR3 or a variant thereof shown in SEQ ID NO:78 or 81, wherein the variant thereof contains substitutions of up to 3 amino acids in the FR3; The FR4 or a variant thereof shown in SEQ ID NO:91, wherein the variant thereof contains substitutions of up to 3 amino acids.

3. A single-domain antibody targeting GPA33, characterized in that: The amino acid sequences of the single-domain antibodies are shown in SEQ ID NO: 2 or 6, respectively.

4. The Fc fusion antibody or humanized antibody against the single-domain antibody against GPA33 as described in any one of claims 1-3.

5. A polynucleotide molecule encoding the single-domain antibody against GPA33 as described in any one of claims 1-3, characterized in that: Their nucleotide sequences are shown in SEQ ID NO: 22 or 26, respectively.

6. An expression carrier, characterized in that, It comprises a polynucleotide molecule encoding a single-domain antibody as described in any one of claims 1-3, an Fc fusion antibody as described in claim 4, or a polynucleotide molecule as described in claim 5.

7. A host cell, characterized in that, It can express the single-domain antibody against GPA33 as described in any one of claims 1-3, or the expression vector as described in claim 6.

8. The use of the single-domain antibody against GPA33 according to any one of claims 1-3 in the preparation of antitumor drugs or antiarthritis drugs, characterized in that, The tumor is colorectal cancer, and the arthritis is osteoarthritis.

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