Single-domain antibodies targeting human serum albumin and uses thereof

Abstract

The application provides a single-domain antibody targeting human serum albumin and an application thereof, and belongs to the field of biological medicines. The single-domain antibody is obtained by immunizing a camel with human serum albumin, and has high affinity, good species cross characteristics, better thermal stability and storage stability, and less aggregates, so that the half-life of a biological medicine can be effectively prolonged, and the beneficial effect of the biological medicine in treatment is facilitated.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a single-domain antibody targeting human serum albumin and its application. Background Technology

[0002] Human serum albumin (HSA) is the most abundant and chemically stable protein in blood. It is a single-chain, non-glycosylated polypeptide composed of 585 amino acids, with a molecular weight of 66.5 kDa and an isoelectric point between 4.7 and 4.9. Mature HSA has a heart-shaped molecular structure composed of three structurally similar α-helical domains (I, II, and III), each containing two subdomains (A and B). Domains IIA and IIIA can bind to endogenous and exogenous substances. The complete amino acid sequence contains 18 amino acids, with aspartic acid at the N-terminus and leucine at the C-terminus. It contains 35 cysteine ​​residues, forming 17 pairs of cross-linked disulfide bonds within the single-chain molecule, thus strengthening the molecule's stability and improving its resistance to acid and heat. The large number of polar amino acids within the molecule also contributes to HSA's high solubility.

[0003] HSA is an important component of the blood system, synthesized solely by hepatocytes, accounting for 40-60% of total plasma protein. Secreted into the circulatory system, it plays crucial physiological functions, including maintaining plasma colloid osmotic pressure, binding and transporting drugs and small molecules, and scavenging free radicals for antioxidant effects. Furthermore, HSA is the smallest molecular weight protein in plasma, is hydrophilic, and can freely pass through capillary walls, dynamically exchanging between the bloodstream and extracellular fluid, thus maintaining the balance of colloid osmotic pressure between blood and extracellular fluid.

[0004] Human serum albumin (HAS), due to its long half-life, good stability, and lack of immunogenicity, has been used to construct fusion proteins to achieve long-acting protein drugs. Single-domain antibodies and fusion proteins conjugated to HAS utilize the equilibrium between their free and bound forms in plasma to achieve sustained release and long-acting effects. Their application in increasing the half-life of therapeutically related proteins and peptides has been confirmed by numerous studies and clinical trials (e.g., WO04 / 041862, WO2006 / 122786, WO2008 / 020079, WO2008 / 142164, WO2009 / 068627, and WO2009 / 147248). Although HAS single-domain antibodies developed by Ebrinckx have been used in the development of long-acting protein drugs, the differentiated development of such antibodies, including the acquisition of antibodies targeting different binding epitopes or with different affinities, will provide more options for the development of long-acting platform systems based on them. Furthermore, due to different development protocols, single-domain antibodies with improved properties may be obtained through different immunized animals and optimized screening methods, thus better achieving the intended function. For example, patents CN201811314177.3 and CN201811314193.2 disclose different anti-human serum albumin single-domain antibodies, which have good specificity, high affinity and stability, can specifically recognize human serum albumin, and prolong the half-life of biopharmaceuticals.

[0005] Although there are existing binding proteins or single-domain antibodies against human serum albumin, the types are limited. Therefore, there is an urgent need to develop human serum albumin single-domain antibodies with better species cross-species characteristics, stability, and fewer aggregates. Summary of the Invention

[0006] To address the aforementioned shortcomings, this invention provides a single-domain antibody targeting human serum albumin and its application. This invention uses human serum albumin to immunize camels, and through screening, obtains a series of single-domain antibodies against human serum albumin. These single-domain antibodies exhibit high affinity, good cross-species characteristics, better thermal and storage stability, and fewer aggregates, effectively prolonging the half-life of biological drugs and contributing to their beneficial therapeutic effects.

[0007] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0008] On one hand, the present invention provides a single-domain antibody targeting human serum albumin, wherein the single-domain antibody comprises the following complementarity-determining regions:

[0009] (1) Contains a complementarity-determining region 1CDR1 containing any of the amino acid sequences or variant sequences shown in any one of SEQ ID NO:1-SEQ ID NO:2;

[0010] (2) Contains a complementarity-determining region 2CDR2 of any of the amino acid sequences or variant sequences shown in any one of SEQ ID NO:3-SEQ ID NO:4;

[0011] (3) Contains a complementarity-determining region 3CDR3 of any of the amino acid sequences or variant sequences shown in any one of SEQ ID NO:5-SEQ ID NO:6;

[0012] Preferably, the variant sequence is a CDR sequence having one or more amino acid substitutions, deletions, or additions compared to its source CDR; the substitutions are conservative substitutions.

[0013] Specifically, the single-domain antibody comprises:

[0014] (1) CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:3, CDR3 as shown in SEQ ID NO:5; and / or

[0015] (2) CDR1 as shown in SEQ ID NO:2, CDR2 as shown in SEQ ID NO:4, and CDR3 as shown in SEQ ID NO:6.

[0016] More specifically, the single-domain antibody comprises:

[0017] (1) The amino acid sequence as shown in SEQ ID NO:7; and / or

[0018] (2) The amino acid sequence as shown in SEQ ID NO:8; and / or

[0019] (3) The amino acid sequence as shown in SEQ ID NO:9; and / or

[0020] (4) The amino acid sequence as shown in SEQ ID NO:10.

[0021] On the other hand, the present invention provides a fusion protein, which is prepared by fusing any of the single-domain antibodies described above with one or more polypeptide molecules having therapeutic functions.

[0022] Specifically, the fusion protein is prepared by directly fusing the single-domain antibody with the therapeutic polypeptide molecule; or by connecting the single-domain antibody to a fusion protein containing one or more of the therapeutic polypeptide molecules via a linker peptide.

[0023] In another aspect, a multispecific molecule is provided, said multispecific molecule comprising any of the single-domain antibodies described above.

[0024] In another aspect, the present invention provides a conjugate comprising the single-domain antibody portion and the conjugation portion.

[0025] Specifically, the single-domain antibody portion and the conjugation portion are conjugated via a linker fragment.

[0026] In another aspect, the present invention provides a series of nucleic acid molecules encoding the single-domain antibodies.

[0027] Specifically, the nucleic acid molecules comprise one or more codon-optimized nucleic acid molecules.

[0028] More specifically, the nucleic acid molecule is:

[0029] (1) A nucleotide sequence as shown in SEQ ID NO:12; and / or

[0030] (2) A nucleotide sequence as shown in SEQ ID NO:13; and / or

[0031] (3) The nucleotide sequence shown in SEQ ID NO:14; and / or

[0032] (4) The nucleotide sequence shown in SEQ ID NO:15.

[0033] In another aspect, the present invention provides a series of carriers, wherein the carriers comprise one or more nucleic acid molecules described in this application.

[0034] Specifically, the vectors include, but are not limited to, plasmids, viruses, and bacteriophages.

[0035] In another aspect, the present invention provides a protein or polypeptide, which is obtained by expressing the above-mentioned vector through an expression system; the expression system is a bacterial, yeast, filamentous fungus, eukaryotic mammalian cell, insect cell, plant cell, or cell-free expression system.

[0036] In another aspect, the present invention provides a series of host cells comprising the above-mentioned nucleic acid molecules or the above-mentioned vectors.

[0037] Specifically, the host cells include, but are not limited to, microbial, plant, or animal cells. The vector described in this invention can be introduced into the host cells by methods known to those skilled in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0038] Specifically, the host cell is capable of expressing the aforementioned single-domain antibody, nucleic acid molecule, fusion protein, multispecific molecule, carrier, protein, or polypeptide.

[0039] In another aspect, the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the above-mentioned single-domain antibody, fusion protein, multispecific molecule, nucleic acid molecule, carrier, host cell, protein or polypeptide and / or conjugate.

[0040] Specifically, the pharmaceutical composition further comprises, optionally, a pharmaceutically acceptable carrier.

[0041] More specifically, the pharmaceutically acceptable carriers include, but are not limited to, diluents, excipients, fillers, wetting agents, disintegrants, flavoring agents, and binders.

[0042] Specifically, the pharmaceutical composition further comprises a combination therapy agent, which includes, but is not limited to, chemotherapeutic agents, radiotherapy agents, immunosuppressants, and cytotoxic drugs.

[0043] In another aspect, the present invention provides the use of the aforementioned single-domain antibodies, fusion proteins, multispecific molecules, nucleic acid molecules, vectors, host cells, proteins or peptides and / or conjugates in the preparation of pharmaceutical compositions.

[0044] Compared with the prior art, the positive and beneficial effects of the present invention are as follows:

[0045] This invention uses human serum albumin to immunize camels, and screens to obtain a series of single-domain antibodies against human serum albumin. The single-domain antibodies have high affinity, good species cross-linking characteristics, better thermal stability and storage stability, and fewer aggregates, which can effectively prolong the half-life of biological drugs and is beneficial to the therapeutic effect of biological drugs. Attached Figure Description

[0046] Figure 1 The images show the SDS-PAGE gel electrophoresis results. Lane M: marker; Lane 1: RYPE182; Lane 2: RYPE182-13; Lane 3: RYPE216; Lane 4: RYPE216-24; Lane 5: RYPE238; Lane 6: RYPE351; Lane 7: RYPE182; Lane 8: RYPE182-13; Lane 9: RYPE216; Lane 10: RYPE216-24; Lane 11: RYPE238; Lane 12: RYPE351. Lanes 1-6 represent reducing electrophoresis; Lanes 7-12 represent non-reducing electrophoresis.

[0047] Figure 2 The image shows the HPLC SEC detection results for RYPE182.

[0048] Figure 3The image shows the HPLC SEC detection results for RYPE182-13.

[0049] Figure 4 The image shows the HPLC SEC detection results for RYPE351. Detailed Implementation

[0050] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0051] Terminology Definition

[0052] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0053] The amino acid three-letter codes and single-letter codes used in this invention are as described in J. biol. chem, 243, p3558 (1968, IUPAC-IUB Committee).

[0054] The "single-domain antibody" described in this invention refers to the heavy chain of an antibody. The sequence of approximately 110 amino acids near the N-terminus of the antibody heavy chain varies considerably and is called the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is called the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDR). Each heavy chain variable region (VH or HCVR) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0055] The term "complementarity-determining region" (CDR) refers to one of the hypervariable regions within the variable domain of an antibody that primarily facilitates antigen binding. Typically, three CDRs (HCDR1, HCDR2, HCDR3) exist within each heavy chain variable domain. CDRs can be determined according to various numbering systems known in the art, such as Kabat, Chothia, or AbM or IMGT numbering systems. In this invention, the "Kabat numbering rule" (see Kabat et al. (1991)) is used to determine the amino acid sequence boundaries of the CDRs.

[0056] The term "framework region" or "FR" residues refers to the amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0057] The terms "specific binding" and "selective binding" refer to non-random binding reactions between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed by the dissociation equilibrium constant (KD) of the interaction. In this paper, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the dissociation equilibrium constant, the stronger the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. Typically, antibodies bind at a rate of approximately less than 10. -8 M, for example, approximately less than 10 -9 M, 10 -10 M, 10 -11 M or lower affinity (KD) binding.

[0058] The term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0059] The term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements that control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, vectors may also contain replication initiation sites.

[0060] The term "host cell" generally refers to an individual cell, cell line, or cell culture that may contain or already contains plasmids or vectors including the nucleic acid molecules described in this application, or that is capable of expressing the antibodies or antigen-binding fragments described in this application. The cells may include progeny of a single host cell. Due to natural, accidental, or intentional mutations, progeny cells may not necessarily be morphologically or genomically identical to the original parent cell, but they must be capable of expressing the antibodies or antigen-binding fragments described in this application. The cells can be obtained by in vitro transfection of cells using the vectors described in this application. The cells may be prokaryotic cells (e.g., *Escherichia coli*) or eukaryotic cells (e.g., yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells, or myeloma cells). In some cases, the cells may be mammalian cells. For example, the mammalian cells may be CHOK1 cells.

[0061] The term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, is well known in the art (see, for example, Remington's Pharmaceutical Sciences, Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delay agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and their analogues. Absorption delay agents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, p-hydroxybenzoate, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, which stabilize the desired activity of the active ingredient in a pharmaceutical product, including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0062] The term "pharmaceutical composition" generally refers to a formulation that exists in a form that allows for the effective biological activity of the active ingredient and does not contain any additional ingredients that would have unacceptable toxicity to the subject to which the composition will be administered. The composition is sterile.

[0063] Example 1. Construction of an anti-human serum albumin single-domain antibody library

[0064] Human serum albumin was mixed with Freund's adjuvant and used to immunize two llamas four times, every two weeks. After immunization, peripheral blood cells were extracted from the camels to separate lymphocytes for the extraction and isolation of single-domain antibody fragments. The steps for single-domain antibody fragment extraction and isolation were as follows: total RNA was extracted, cDNA was synthesized by reverse transcription, single-domain antibody fragments were amplified by nested PCR, digested with restriction endonucleases, ligated into phage display vectors, and electroporated into competent cells. Two independent phage display libraries of anti-human serum albumin single-domain antibodies were successfully constructed. The library size was determined by counting the number of single colonies by serially diluting the libraries on plates; both libraries were >10⁸. Thirty single colonies were randomly selected from each library for colony PCR detection; the empty vector rate of both libraries was 0%.

[0065] Example 2. Screening process for single-domain antibodies against human serum albumin

[0066] Human serum albumin was conjugated onto an ELISA plate and coated overnight. After blocking with blocking buffer, a phage library was added. After multiple washes with PBST, the phages specifically binding to human serum albumin were dissociated using TEA elution buffer and used to infect logarithmically growing *E. coli* cells. The phages were then expanded for the next round of screening. Each library underwent three rounds of bio-panning to enrich specific VHH antibodies. The first and third rounds were screened using human serum albumin-conjugated plates, while the second round was screened using mouse serum albumin-conjugated plates. This achieved the goal of using phage display technology to screen for specific antibodies in the antibody library that bind to human serum albumin and have good species cross-species characteristics.

[0067] Example 3. Screening for specific single positive clones using phage enzyme-linked immunosorbent assay (ELISA).

[0068] Eighty single colonies were randomly selected from two libraries and inoculated. After reaching the logarithmic growth phase, expression was induced by IPTG. After centrifugation and collection of bacterial cells, crude antibody was extracted from the periplasm using the osmotic shock method. The antibody was added to an ELISA plate coated with human HSA protein and incubated. After washing with PBST, mouse anti-HA tag antibody was used as the primary antibody and goat anti-mouse alkaline phosphatase conjugate antibody was used as the secondary antibody. Alkaline phosphatase was added for color development, and absorbance values ​​were read. Sample wells with OD values ​​more than three times that of control wells were identified as positive control wells. All positive clones were cultured, plasmids were extracted, and sequenced to obtain multiple single-domain antibody sequences. One of them was named RYPE182, and its amino acid sequence is shown in SEQ ID NO:7. The RYPE182 single-domain antibody was humanized using the single-domain antibody humanization universal framework transplantation method to obtain multiple humanized variants of the RYPE182 single-domain antibody. One of them was named RYPE182-13, and its amino acid sequence is shown in SEQ ID NO:8. Another variant, named RYPE216, has an amino acid sequence as shown in SEQ ID NO:9. Using the universal framework transplantation method for humanization of single-domain antibodies, the RYPE216 single-domain antibody was humanized to obtain multiple humanized variants of the RYPE216 single-domain antibody. One of these variants was named RYPE216-24, and its amino acid sequence is shown in SEQ ID NO:10.

[0069] The single-domain antibodies prepared by this invention are shown in Table 1 below.

[0070] Table 1. Single-domain antibodies

[0071]

[0072] Example 4. Expression and purification of single-domain antibodies

[0073] (1) Expression: An 8X histidine tag was added to the C-terminus of different single-domain antibody sequences, which were then cloned into the pET28b expression vector and transformed into BL21(DE3)plysS strain. The transformed samples were plated with AMP and incubated overnight at 37°C. Single clones were picked from the AMP plates and incubated at 37°C until the OD600 was approximately 0.4. Then, expression was induced overnight at 28°C with 0.1 mM IPTG at 200 rpm.

[0074] (2) Purification: Take 8000g of bacterial culture, centrifuge for 10min to collect the expression bacteria, and weigh the wet bacterial weight. Perform periplasmic protein extraction at a weight-to-volume ratio of 1:30, and finely purify the extracted protein supernatant by Ni-FF affinity capture, anion and cation purification.

[0075] Example 5. Affinity determination using surface plasmon resonance

[0076] Surface plasmon resonance (SPR) on a Biacore 8K instrument was used to perform kinetic analysis of anti-HSA single-domain antibody fusion constructs with serum albumin from humans, cynomolgus monkeys, rats, and mice. The assay was performed at 25°C using HBS-EP+ as the experimental buffer. Ligands were immobilized onto the surface of a CM5 chip. After the chip surface stabilized, serially diluted analytes were flowed through the immobilized chip surface at a controlled flow rate. Ebolings' HAS nanobody (synthesized and expressed according to its patented sequence, labeled RYPE238) served as a positive control. The experimental buffer served as a blank control, and a single repetition concentration was set. The affinity assay results are shown in Table 2 below.

[0077] Table 2. Affinity Test Results

[0078]

[0079]

[0080] HSA is human serum albumin, RSA is rat serum albumin, MSA is mouse serum albumin, and CynoSA is cynomolgus monkey serum albumin.

[0081] As shown in the table above, the antibody described in this invention is superior to positive antibodies in terms of affinity and species cross-linking, and the affinity of the antibody before and after humanization is comparable.

[0082] Furthermore, under PBST pH 5.5 conditions, the single-domain antibody molecule described in this invention can still bind normally to hFcRn after binding to HSA, indicating that the antibody described in this invention does not interfere with the binding of HSA and FcRn.

[0083] Example 6. Construction, expression, and purification of fusion proteins

[0084] (1) Expression: The Extend-4 sequence and linker (GGGGSGGGGSGGGGS) were fused to the N-terminus of the RYPE182-13 single-domain antibody sequence, and an 8X histidine tag was added to the C-terminus to construct a fusion protein, labeled RYPE351. The amino acid sequence of this fusion protein is shown in SEQ ID NO:11, and the encoding nucleotide sequence is shown in SEQ ID NO:16. The fusion protein was cloned into the pET28b expression vector and transformed into BL21(DE3)plysS strain. The protein was plated with AMP and incubated overnight at 37°C. Single clones were picked from the AMP plates and incubated at 37°C until the OD600 reached approximately 0.4. Then, expression was induced overnight at 28°C with 0.1 mM IPTG at 200 rpm.

[0085] (2) Purification: Take 8000g of bacterial culture, centrifuge for 10min to collect the expression bacteria, and weigh the wet bacterial weight. Perform periplasmic protein extraction at a weight-to-volume ratio of 1:30, and finely purify the extracted protein supernatant by Ni-FF affinity capture, anion and cation purification.

[0086] (3) Analysis: Protein purity was analyzed by SDS-PAGE 4-12% gradient gel and HPLC SEC.

[0087] For details of the SDS-PAGE gel results of the single-domain antibody and fusion protein described in this invention, please refer to [link to relevant documentation]. Figure 1 For detailed HPLC SEC results, please refer to [link / reference]. Figure 2-4 (in, Figure 2 The results are from HPLC SEC analysis of RYPE182. Figure 3 The results are from HPLC SEC analysis of RYPE182-13. Figure 4 (Results of HPLC SEC analysis of RYPE351). Figure 1-4 It is evident that the single-domain antibody described in this invention can achieve high expression and has high purity.

[0088] Example 7. Stability Testing

[0089] Detection method: The target protein is concentrated to a certain concentration by centrifugation. The sample is then loaded onto a UNCLE instrument for TM, Tonset, Tagging266, and particle size distribution analysis.

[0090] The test results are shown in Table 3 below.

[0091] Table 3. Stability test results

[0092]

[0093] As shown in Table 3 above, the single-domain antibody described in this application has good thermal stability.

[0094] Example 8. In vivo PK study in rats

[0095] RYPE182 and RYPE238 were used as subjects in this study. Wistar rats or SD rats aged 4 to 6 weeks and weighing approximately 200g were selected and divided into 4 groups, with 6 rats in each group (half male and half female). Group 1 received a single tail vein administration of 2 mg / kg RYPE238; Group 2 received a single tail vein administration of 10 mg / kg RYPE238; Group 3 received a single tail vein administration of 2 mg / kg RYPE182; and Group 4 received a single tail vein administration of 10 mg / kg RYPE182.

[0096] Blood sample collection times: before medication, 5±1 min and 30±2 min after medication, 2, 6, 10, 24, 48, and 72 h, and 5, 7, 10, 14, 17, 21, 28, and 35 days.

[0097] Blood sample collection method: Collect approximately 0.3 mL of whole blood per animal per time point from the orbital or jugular vein.

[0098] Blood sample processing: Before blood collection, place an EP tube containing 10 μL of heparin sodium working solution (1000 U / mL) in a refrigerator or ice box at 2-8℃; add the collected blood to a centrifuge tube, manually invert at least 5 times until well mixed; centrifuge at 1500g for 10 min at 2-8℃, completing the centrifugation within 2 hours after blood collection. Transfer the centrifuged plasma to newly labeled tubes, aliquot into 3 tubes, and store at -80℃.

[0099] Sample detection: (1) Rabbit-derived anti-camelid VHH antibody was coated onto microplates. The treated serum samples were added to the microplates, and the amount of RYPE182 in the serum samples was detected by HRP-conjugated anti-His-tag antibody.

[0100] (2) HAS protein was coated onto microplates. The treated serum samples were added to the microplates, and the amount of RYPE0003-182 in the serum samples was detected by HRP-conjugated anti-His-tag antibody.

[0101] The test results are shown in Table 4 below.

[0102] Table 4. Results of in vivo PK study in rats

[0103]

[0104]

[0105] As shown in Table 4 above, the half-life of the single-domain antibody RYPE182 described in this application in rats is significantly better than that of the positive control antibody RYPE238.

[0106] Example 9. PK Study in Crab-Eating Monkeys

[0107] Using RYPE351 as the research subject, four cynomolgus macaques aged 3-7 years and weighing approximately 3-6.5 kg were selected and divided into two groups of two, half male and half female. Group 1 received a single intravenous dose of 0.3 mg / kg RYPE351; Group 2 received a single intravenous dose of 1 mg / kg RYPE351.

[0108] Blood sample collection times: 0h (before drug administration), 0.25h (15min), 0.5h (30min, needle removal point), 2h, 8h, 24h (D2), 72h (D4), 168h (D8), 336h (D15), 504h (D22), 672h (D29), 1008h (D43), 1344h (D57) after drug administration.

[0109] Blood sample collection method: Collect approximately 1 mL of whole blood from a vein in the fore / hind limb (excluding sites near the administration site).

[0110] Blood sample processing: Before blood collection, place EP tubes containing 10 μL of heparin sodium working solution (1000 U / mL) and 20 μL of protease inhibitor working solution (50 IU / mL) in a refrigerator at 2-8℃ or an ice bath; add the collected blood to the EP tubes and manually invert at least 5 times until well mixed; centrifuge at 1500g for 10 min at 2-8℃, completing the centrifugation within 2 hours after blood collection. Aliquot the centrifuged plasma into two equal portions, transfer them to newly labeled tubes, and store below -80℃.

[0111] Sample detection: Anti-Exendin 4 antibody was coated onto microplates. Processed plasma samples were added to the microplates and analyzed using MonoRab... TM Rabbit Anti-Camelid VHH Cocktail [HRP] was used to detect the level of RYPE351 in plasma.

[0112] The test results are shown in Table 5 below.

[0113] Table 5. Results of PK study in cynomolgus monkeys

[0114]

[0115]

[0116] As shown in Table 5 above, after the single-domain antibody described in this application forms a fusion protein with Exten4, the half-life of the fusion protein RYPE351 in cynomolgus monkeys is about 6-7 days, which is significantly better than that of antibody NB22 (antibody NB22 is detailed in patent CN201811314177.3, and its half-life in cynomolgus monkeys is about 1.2 days). This indicates that the single-domain antibody described in this application significantly improves the half-life of the fusion protein.

[0117] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. SEQUENCE LISTING <110> Ruiyang (Suzhou) Biotechnology Co., Ltd. <120> Single-domain antibodies targeting human serum albumin and their applications <130> 20220523 <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 11 <212> PRT <213> Artificial sequence <400> 1 Ser Gly Arg Thr Tyr Val Ser Tyr Val Leu Gly 1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial sequence <400> 2 Gly Arg Thr Phe Asp Pro Tyr Ser Met Gly 1 5 10 <210> 3 <211> 9 <212> PRT <213> Artificial sequence <400> 3 Ile Thr Pro Ser Gly Arg Ser Ile Leu 1 5 <210> 4 <211> 8 <212> PRT <213> Artificial sequence (artificial sequence) <400> 4 Ile Thr Trp Ser Gly Arg Asn Thr 1 5 <210> 5 <211> 15 <212> PRT <213> Artificial sequence (artificial sequence) <400> 5 Ala Gly Gly Val Gly Leu Tyr Arg Glu Ala Thr Ala Tyr Asp Tyr 1 5 10 15 <210> 6 <211> 15 <212> PRT <213> Artificial sequence (artificial sequence) <400> 6 Ala Tyr Gly Leu Gly Ser Tyr Val His Ser Tyr Gln Tyr Asp Phe 1 5 10 15 <210> 7 <211> 124 <212> PRT <213> Artificial sequence (artificial sequence) <400> 7 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Tyr Val Ser Tyr 20 25 30 Val Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Thr Ile Thr Pro Ser Gly Arg Ser Ile Leu Tyr Ala Asp Ile Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Ala Ala Gly Gly Val Gly Leu Tyr Arg Glu Ala Thr Ala Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Ser Gln Val Thr Val Ser Ser 115 120 <210> 8 <211> 124 <212> PRT <213> artificial sequence <400> 8 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Tyr Val Ser Tyr 20 25 30 Val Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ala Thr Ile Thr Pro Ser Gly Arg Ser Ile Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Gly Gly Val Gly Leu Tyr Arg Glu Ala Thr Ala Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Ser Gln Val Thr Val Ser Ser 115 120 <210> 9 <211> 124 <212> PRT <213> artificial sequence <400> 9 Glu Val Asp Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Asp Pro Tyr 20 25 30 Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Thr Ile Thr Trp Ser Gly Arg Asn Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Tyr Gly Leu Gly Ser Tyr Val His Ser Tyr Gln Tyr Asp 100 105 110 Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 10 <211> 124 <212> PRT <213> artificial sequence <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Asp Pro Tyr 20 25 30 Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45 Ser Leu Ile Thr Trp Ser Gly Arg Asn Thr Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Tyr Gly Leu Gly Ser Tyr Val His Ser Tyr Gln Tyr Asp 100 105 110 Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 11 <211> 181 <212> PRT <213> Artificial sequence <400> 11 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 35 40 45 Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 50 55 60 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Tyr Val Ser 65 70 75 80 Tyr Val Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe 85 90 95 Val Ala Thr Ile Thr Pro Ser Gly Arg Ser Ile Leu Tyr Ala Asp Ser 100 105 110 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu 115 120 125 Tyr Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 130 135 140 Cys Ala Ala Ala Gly Gly Val Gly Leu Tyr Arg Glu Ala Thr Ala Tyr 145 150 155 160 Asp Tyr Trp Gly Gln Gly Ser Gln Val Thr Val Ser Ser His His His 165 170 175 His His His His His 180 <210> 12 <211> 372 <212> DNA <213> Artificial sequence <400> 12 caacttcaat tggttgaaag cggcggaggg ctggtacaag ctggtgggag ccttcgcctc 60 agttgcgccg ccagtggccg cacctacgtt agctacgttc tgggctggtt ccgccaggcc 120 ccgggcaaag aacgcgaatt cgttgccacc atcaccccga gtggccgcag catcctgtac 180 gccgatatcg ttaaaggccg cttcaccatc agccgcgata ataccaaaaa taccgtttac 240 ctgcagatga ccagcctgaa accggaagat accgccgttt acttctgcgc cgccgccggc 300 ggcgttggcc tgtaccgcga agccaccgcc tacgattact ggggccaggg cagccaggtt 360 accgttagca gc 372 <210> 13 <211> 372 <212> DNA <213> Artificial sequence <400> 13 caagtgcagc tggtggagag tggtggtgga ctggttcagc ctggtggcag tttaagactg 60 agctgtgcag caagtggcag aacctatgtg agctatgtgc tgggctggtt tagacaagcc 120 cctggcaaag gcctggaatt tgtggccacc attaccccaa gtggcagaag cattctgtat 180 gcagatagtg tgaaaggcag atttaccatt agcagagata acaccaaaaa caccctgtat 240 ctgcagatga ctagcctgag agcagaagat actgcagtgt attattgtgc agcagctggt 300 ggtgtgggcc tgtatagaga agccactgcc tatgattatt ggggccaagg cagccaagtg 360 actgtgagca gc 372 <210> 14 <211> 372 <212> DNA <213> Artificial sequence <400> 14 gaagtggatt tagtggagag tggtggtggc ctggtgcaag ctggtggcag cctgagactg 60 agctgtgccg caagtggcag aacctttgat ccatatagca tgggctggtt tagacaagcc 120 cctggcaaag aaagagaatt tgtggccacc attacctgga gtggcagaaa caccaactat 180 gcagatagtg tgaaaggcag atttaccatt agcaaagata atgccaaaaa caccatgtat 240 ctgcagatga acagcctgaa acctgatgat actgcagtgt attattgcgc agcagcctat 300 ggcctgggca gctatgtgca tagctatcag tatgattttt ggggccaagg cacccaagtg 360 actgtgagca gc 372 <210> 15 <211> 372 <212> DNA <213> artificial sequence <400> 15 gaggtgcaac ttgtggaaag tggtggtggc ttagtgcagc ctggtggcag cctgagactg 60 agctgtgcag caagtggcag aacctttgat ccatatagca tgggctggtt tagacaagcc 120 cctggcaaag gcctggaatt tgtgagcctg attacctgga gtggcagaaa cactggctat 180 gcagatagtg tgaaaggcag atttaccatt agcagagata acaccaaaaa caccctgtat 240 ctgcagatga acagcctgaa acctgatgat actgcagtgt attattgtgc agcagcctat 300 ggcctgggca gctatgtgca tagctatcag tatgattttt ggggccaagg cacccaagtg 360 actgtgagca gc 372 <210> 16 <211> 542 <212> DNA <213> artificial sequence <400> 16 catggcgaag gcacctttac gagcgatctg agcaaacaga tggaagagga agcggtgcgc 60 ctgtttattg aatggctgaa aaacggcggc ccgagcagcg gcgcgccgcc accgagcggc 120 ggtggcggca gcggcggtgg cggcagccaa gtgcagctgg tggaaagcgg cggtggcctg 180 gttcagccgg gcggtagctt acgcctgagc tgcgcggcga gtggccgcac ctatgtgagc 240 tatgtgctgg gctggttcg ccaagcgccg ggcaaaggcc tggaatttgt ggcgaccatt 300 accccgagcg gccgcagcat tctgtatgcg gatagcgtga aaggccgctt taccattagc 360 cgcgataaca ccaaaaacac cctgtatctg cagatgacga gcctgcgc ggaatacc 420 gcggtgtatt attgcgcggc cgcgggcggc gtgggcctgt atcgcgaagc gaccgcgtat 480 gattattggg gccaaggcag ccaagtgacc gtgagcagcc atcatcacca ccatcaccat 540 ca 542

Claims

1. A single-domain antibody targeting human serum albumin, characterized in that: The single-domain antibody comprises the following complementarity-determining regions: (1) Contains complementarity determination region 1 CDR1 of the amino acid sequence shown in SEQ ID NO:1, complementarity determination region 2 CDR2 of the amino acid sequence shown in SEQ ID NO:3, and complementarity determination region 3 CDR3 of the amino acid sequence shown in SEQ ID NO:5; or (2) Contains complementarity determination region 1 CDR1 of the amino acid sequence shown in SEQ ID NO:2, complementarity determination region 2 CDR2 of the amino acid sequence shown in SEQ ID NO:4 and complementarity determination region 3 CDR3 of the amino acid sequence shown in SEQ ID NO:

6.

2. The single-domain antibody according to claim 1, characterized in that: The single-domain antibody comprises: (1) CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:3, and CDR3 as shown in SEQ ID NO:5; or (2) CDR1 as shown in SEQ ID NO:2, CDR2 as shown in SEQ ID NO:4, and CDR3 as shown in SEQ ID NO:

6.

3. The single-domain antibody according to claim 2, characterized in that: The single-domain antibody comprises: (1) The amino acid sequence as shown in SEQ ID NO:7; or (2) The amino acid sequence as shown in SEQ ID NO:8; or (3) The amino acid sequence as shown in SEQ ID NO:9; or (4) The amino acid sequence shown in SEQ ID NO:

10.

4. A fusion protein, characterized in that: The fusion protein is prepared by fusing the single-domain antibody according to any one of claims 1-3 with one or more polypeptide molecules having therapeutic functions.

5. The fusion protein according to claim 4, characterized in that: The fusion protein is prepared by directly fusing the single-domain antibody with the therapeutic polypeptide molecule; or by linking the single-domain antibody to a fusion protein containing one or more of the therapeutic polypeptide molecules via a linker peptide.

6. A multispecific molecule, characterized in that: The multispecific molecule comprises the single-domain antibody according to any one of claims 1-3.

7. A coupling agent, characterized in that: The conjugate comprises the single-domain antibody portion and the conjugate portion as described in any one of claims 1-3.

8. The coupling according to claim 7, characterized in that: The single-domain antibody portion and the conjugation portion are conjugated via a linker fragment.

9. A nucleic acid molecule encoding a single-domain antibody according to any one of claims 1-3.

10. A carrier, characterized in that: The carrier comprises the nucleic acid molecule of claim 9.

11. A protein or polypeptide, characterized in that: The protein or polypeptide is obtained by expression using the vector described in claim 10 through an expression system.

12. A host cell comprising the nucleic acid molecule of claim 9 or the vector of claim 10.

13. The use of the single-domain antibody of any one of claims 1-3, the fusion protein of any one of claims 4-5, the multispecific molecule of claim 6, the conjugate of any one of claims 7-8, the nucleic acid molecule of claim 9, the vector of claim 10, the protein or polypeptide of claim 11, and / or the host cell of claim 12 in the preparation of a pharmaceutical composition.

14. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the single-domain antibody of any one of claims 1-3, the fusion protein of any one of claims 4-5, the multispecific molecule of claim 6, the conjugate of any one of claims 7-8, the nucleic acid molecule of claim 9, the vector of claim 10, the protein or polypeptide of claim 11, and / or the host cell of claim 12.

15. The pharmaceutical composition according to claim 14, characterized in that: The pharmaceutical composition may also include, optionally, a pharmaceutically acceptable carrier.

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