Albumin-bound nano-antibody, preparation method thereof, albumin-bound nano-antibody derivative and application of albumin-bound nano-antibody derivative

Abstract

The invention discloses an albumin-binding nano antibody and a preparation method thereof, an albumin-binding nano antibody derivative and applications of the albumin-binding nano antibody derivative and the albumin-binding nano antibody derivative. The method comprises the following steps: constructing a phage surface display VHH antibody library by using human HSA immune alpaca, and obtaining a positive phage clone strain through at least one round of panning process; and when the elutriation round is more than one round, the pH value of the incubation system in the first round of elutriation process is not equal to that of the incubation system in the second round of elutriation process. Two nano antibodies P1D12 and P1B6 which can be simultaneously combined with mouse albumin, monkey albumin and human albumin are obtained through panning, verification is carried out through a fusion protein mode, and the result shows that the two nano antibodies obtained by the invention can remarkably prolong the pharmacokinetic half-life period of recombinant protein, so that the pharmacokinetic half-life period of Slit2D2 is increased to 2-3 times of the original pharmacokinetic half-life period.

Description

Technical Field

[0001] The present invention relates to the field of antibody engineering, and particularly to an albumin-binding nanobody, a preparation method thereof, an albumin-binding nanobody derivative, and their applications. Background Art

[0002] For protein-based biologics and drugs (such as antibodies, antibody fragments, growth factors, enzymes, and peptides), during application, in order to maximize their efficacy and reduce dosage requirements, it is usually necessary to extend the in vivo serum half-life.

[0003] When the molecular weight of a protein is less than 40 - 50 kDa, it will rapidly pass through renal filtration and be cleared from the blood circulation, thus unable to exert the expected efficacy.

[0004] Therefore, currently, scientists have developed various methods for improving the pharmacokinetics of small biologics with short half-lives. For example, through glycosylation modification, polyethylene glycol (PEG)ylation, albumin fusion, transferrin fusion, Fc fusion, etc. Among them, PEGylation extends the half-life by increasing the hydrodynamic radius; albumin fusion, transferrin fusion, and Fc fusion extend the half-life through the FcRn-mediated recycling pathway and increasing the molecular size. Other methods also include inert protein fusions such as XTEN fusion, HAP fusion, ELP fusion, etc., and negatively charged protein fusions such as CTP fusion, etc.

[0005] When using Fc fusion, while extending the serum half-life, effector functions such as ADCC can also be generated. The resulting protein is IgG-like in size and geometry. However, in some cases, such as when it is necessary to maintain a small size (for example, to increase tumor penetration), effector functions are not required (for example, to reduce cytokine release), and non-natural molecular geometry (for example, to improve bispecificity and bivalent engagement), in such cases, using a serum albumin-binding protein to extend the half-life has significant advantages.

[0006] Currently, it has been reported that small serum albumin-binding proteins (mainly antibody fragments) have been successfully used to extend the serum half-life of biologics, including streptococcal albumin-binding domain (ABD), DARPins, immunoglobulin variable heavy chain (VH) and variable light chain (VL) domains, camel / alpaca heavy chain variable region (VHH) domains, shark new antigen receptor variable (VNAR) domains, and antigen-binding fragments (Fab). This is mainly achieved by utilizing the long half-life characteristics of serum albumin.

[0007] However, to achieve the half-life of a drug using this method, there are relatively high performance requirements for the binding protein used to extend the drug's half-life, and multiple requirements need to be met simultaneously: ① having medium to high affinity with albumin of multiple species to better meet preclinical animal studies and human clinical studies; ② binding to albumin without disrupting the interaction between albumin and FcRn; ③ still retaining the binding ability to albumin after fusion with different biological agents; ④ being able to maintain high binding stability to albumin under different environmental conditions (such as different pH values).

[0008] Therefore, it is of great significance to prepare an albumin-binding protein that can efficiently bind to serum albumin and achieve long-term metabolism of drugs. Summary of the Invention

[0009] To achieve the above objectives, the technical solution adopted in the present invention is as follows:

[0010] In the first aspect, the present invention provides an albumin-binding nanobody, which includes at least one albumin-binding domain, and its albumin-binding domain includes at least one group of the following complementarity-determining regions:

[0011] ① VHCDR11, VHCDR12, and VHCDR13,

[0012] The VHCDR11 contains the sequence GGTLSNYA (SEQ ID NO.11);

[0013] The VHCDR12 contains the sequence ITWSVST (SEQ ID NO.12);

[0014] The VHCDR13 contains the sequence AARSRSHYSGTYSGVSGYDN (SEQ ID NO.13);

[0015] ② VHCDR21, VHCDR22, and VHCDR23

[0016] The VHCDR21 contains the sequence GFDFNNFG (SEQ ID NO.14);

[0017] The VHCDR22 contains the sequence INSRGDTT (SEQ ID NO.15);

[0018] The VHCDR23 contains the sequence VIGRGTP (SEQ ID NO.16).

[0019] The heavy chain variable region VHCDR11 comprises the sequence shown by GGTLSNYA (SEQ ID NO.11) or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;

[0020] The heavy chain variable region VHCDR12 comprises the sequence shown by ITWSVST (SEQ ID NO.12) or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;

[0021] The heavy chain variable region VHCDR13 comprises the sequence shown by AARSRSHYSGTYSGVSGYDN (SEQ ID NO.13) or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;

[0022] The heavy chain variable region VHCDR21 comprises the sequence shown by GFDFNNFG (SEQ ID NO.14) or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;

[0023] The heavy chain variable region VHCDR22 comprises the sequence shown by INSRGDTT (SEQ ID NO.15) or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto;

[0024] The heavy chain variable region VHCDR23 comprises the sequence shown by VIGRGTP (SEQ ID NO.16) or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0025] Furthermore, it has an amino acid sequence shown by any one of SEQ ID NO.1 and SEQ ID NO.3. The albumin-binding nanobody of the present invention can bind to the albumin of mice, monkeys and humans simultaneously, thus providing a better basis for clinical application; and after the albumin-binding nanobody of the present invention is fused with Slit2D2, it can effectively extend the pharmacokinetic half-life of Slit2D2, and even reaches an in vivo half-life of 13 - 16 hours.

[0026] In a second aspect, the present invention provides a nucleic acid molecule encoding the aforementioned albumin-binding nanobody.

[0027] In a third aspect, the present invention provides an albumin-binding nanobody having a nucleotide sequence as shown in any one of SEQ ID NO.2 and SEQ ID NO.4.

[0028] In a fourth aspect, the present invention provides a vector containing the aforementioned nucleic acid molecule or albumin-binding nanobody.

[0029] In a fifth aspect, the present invention provides a fusion or conjugate containing the aforementioned isolated antibody.

[0030] In a sixth aspect, the present invention provides an expression cassette or recombinant bacterium containing the aforementioned nucleic acid molecule or nucleotide sequence.

[0031] In a seventh aspect, the present invention provides a composition comprising at least one of the isolated antibody in the first aspect, the nucleic acid molecule in the second aspect, the vector in the fourth aspect, or the fusion or conjugate in the fifth aspect, and at least one of its corresponding pharmaceutically acceptable carriers.

[0032] In an eighth aspect, the present invention provides a method for detecting albumin in a biological sample, comprising contacting the biological sample with the isolated antibody in the first aspect or the fusion or conjugate in the fifth aspect.

[0033] In a ninth aspect, the present invention provides a fusion that simultaneously fuses the albumin-binding nanobody described in any one of the first, third, and fourth aspects with Slit2D2. By fusing the albumin-binding nanobody and Slit2D2, the fusion of the present invention utilizes the high binding affinity and binding stability between the nanobody and albumin, and by taking advantage of the long half-life of albumin itself, the half-life of the entire fusion is significantly increased.

[0034] In a tenth aspect, the present invention provides the use of the antibody or antigen-binding fragment in the first aspect, the fusion or conjugate in the fifth aspect, the composition in the seventh aspect, or the fusion in the ninth aspect in the preparation of a drug and / or the extension of the drug half-life.

[0035] In an eleventh aspect, the present invention provides a method for preparing the albumin-binding nanobody described in any one of the first, third, and fourth aspects, which comprises the following steps:

[0036] S1. Screening of nanobodies that recognize albumin: After immunizing alpacas with human serum albumin, isolating alpaca PBMCs, and constructing a phage library of albumin-binding nanobodies;

[0037] S2. Selection of nanobodies: Through at least one round of selection process, positive phage clones are obtained; when the number of selection rounds is greater than 1, the pH of the incubation system in the first round of selection process is not equal to the pH of the incubation system in the second round.

[0038] S3. Expression and identification of albumin-binding nanobodies.

[0039] By using the technology of immunizing alpacas with albumin and screening phage libraries, the present invention unexpectedly screened two nanobodies. The nanobodies obtained in the present invention all have high affinity with albumin; and they can bind albumin both in acidic environment and neutral environment, meeting the requirement that when albumin enters the acidic environment of endosomes, the nanobodies can still bind to albumin, preventing shedding in the acidic environment and thus not being degraded by lysosomes; through experimental verification, the two nanobodies obtained in the present invention can both bind to the albumin of mice, monkeys and humans, which can meet the verification of the drug activity and safety of the fusion protein drug in mice and monkeys in preclinical studies; through the verification by the fusion protein method, the two nanobodies obtained in the present invention can significantly increase the half-life of Slit2D2 in vivo, achieving the effect of making Slit2D2 long-acting in vivo. Description of the Drawings

[0040] Figure 1 It is the SDS-PAGE reducing electrophoresis diagram of VHH-HIS sample;

[0041] Figure 2 It is the SDS-PAGE reducing electrophoresis diagrams of three recombinant proteins in Example 4.1. In the figure, band 1 is Slit2D2HIS, band 2 is Sli2D2-P1D12-HIS, and band 3 is Slit2D2-P1B6-HIS;

[0042] Figure 3(a) is the drug metabolism curve diagram of Sli2D2-P1D12-HIS and Slit2D2-P1B6-HIS in Example 5.2 of the present invention;

[0043] Figure 3(b) is the drug metabolism curve diagram of Slit2D2HIS in Example 5.2 of the present invention;

[0044] Figure 4(a) is the ELISA curve diagram for detecting the binding of Slit2D2-P1B6 HIS protein to albumin of different species in Example 4 of the present invention;

[0045] Figure 4(b) is the ELISA curve diagram for detecting the binding of Slit2D2-P1D12 HIS protein to albumin of different species in Example 4 of the present invention;

[0046] Figure 5This is the electrophoresis result diagram after the first round of PCR reaction in Example 1.2.2 of the present invention. Detailed implementation manners

[0047] The following elaborates on the preferred embodiments of the present invention in conjunction with the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making the protection scope of the present invention more clearly defined.

[0048] Example 1 Screening of nanobodies that recognize albumin

[0049] In the screening process of nanobodies, the immunized animals selected are camelids, such as alpacas or camels can be selected for immunization. Here, the immunization of alpacas is taken as an example for illustration. However, in practice, it is not limited to only immunizing alpacas.

[0050] 1.1 Animal immunization

[0051] The screening of nanobodies that recognize albumin is mainly completed through the processes of alpaca immunization, alpaca peripheral blood (PBMC) separation, phage library construction, and phage screening.

[0052] In some embodiments, a healthy alpaca is selected for antigen immunization. Before immunization, human serum albumin (HSA) and adjuvant are mixed. The volumes of both the antigen and the adjuvant are 1 ml, and the emulsification of the antigen and the adjuvant is completed by ultrasonic emulsification. A total of four immunizations are carried out. The adjuvant used in the first immunization is Freund's complete adjuvant, and the adjuvants used in the subsequent three immunizations are Freund's incomplete adjuvants. All the adjuvants used are purchased from Sigma Company. The dosage of the antigen (human serum albumin) for each immunization is 1 mg. The interval period between each immunization is 2 weeks, and the immunization site is the subcutaneous area behind the neck.

[0053] 1.2 Nanobody library construction

[0054] 1.2.1 RNA extraction

[0055] After the third and fourth immunizations of the alpaca during the animal immunization process in step 1.1, 50 ml of peripheral blood is collected respectively. The alpaca PBMC is separated using a PBMC separation kit and stored in a -80 °C refrigerator.

[0056] Use Trizol reagent (ThermoFisher, catalog number: 15596026) to extract RNA from PBMC. The experimental steps for RNA extraction are as follows:

[0057] ① After counting the separated PBMC, add 0.75 ml of TRIzol to every 0.25 ml of PBMC (cell amount: 5 - 10×10 6 ) TMReagents are placed at room temperature for 5 min to allow sufficient lysis. Do not directly freeze the samples before sufficient lysis (the criterion for sufficient lysis is: observe the samples under a microscope, all cells are broken, and there are no intact cells). After sufficient lysis, the samples are stored at -80 °C for long term.

[0058] ② Centrifuge at 12,000 rpm for 5 min and discard the pellet.

[0059] ③ Add chloroform at 200 μl chloroform / ml Trizol, mix well by shaking and then place at room temperature for 15 min. Note: Do not use a vortex oscillator to avoid breaking genomic DNA.

[0060] ④ Centrifuge at 12,000 g at 4 °C for 15 min. Pipette the upper aqueous phase into another centrifuge tube.

[0061] ⑤ Add isopropanol at 0.5 ml isopropanol / ml Trizol, mix well and place at room temperature for 5 - 10 min.

[0062] ⑥ Centrifuge at 12,000 g at 4 °C for 10 min, discard the supernatant, and the RNA precipitates at the bottom of the tube.

[0063] ⑦ Add 75% ethanol at 1 ml 75% ethanol / ml Trizol, gently shake the centrifuge tube to suspend the pellet. Centrifuge at 8,000 g at 4 °C for 5 min and discard the supernatant as much as possible.

[0064] ⑧ Air dry at room temperature or dry under vacuum for 5 - 10 min. Note: Do not over - dry the RNA samples, otherwise they will be difficult to dissolve.

[0065] ⑨ The RNA samples can be dissolved in 50 μl H 2 O, TE buffer or 0.5% SDS. After treatment at 55 - 60 °C for 5 - 10 min, perform subsequent quantitative calculation of RNA. (Note: H 2 O, TE or 0.5% SDS must be treated with DEPC and autoclaved.)

[0066] ⑩ Measure the OD value to quantify the RNA concentration.

[0067] 1.2.2 Construction of the phage library of albumin - binding nanobodies

[0068] Use a reverse transcription kit (ThermoFisher, catalog number: K1622) to perform RNA reverse transcription on the RNA samples in step 1.2.1 to obtain cDNA. Design two sets of primers and amplify the nanobody fragment VHH by nested PCR. Using AlpVh - LD / CH2 - R as primers, perform the first - round PCR amplification with cDNA as the template. The amplification system and conditions are as follows:

[0069] 2×bufferMix 125 AlpVh-LD 10 CH2-R 10 cDNA <= 20 μl dNTP mixture (each 10 mM) 5 High-fidelity polymerase 5 <![CDATA[H 2 O]]> 75 μl Total 250 μl

[0070] The reaction conditions for the first round of PCR were as follows: 95°C for 4 min; 95°C for 30 s, 52°C for 30 s, 72°C for 30 s, for 5 cycles; 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, for 20 cycles; 72°C for 10 min; store at 10°C. Electrophoresis on a 1.2% gel (long gel) yielded two bands (approximately 700 bp and 950 bp respectively), and the 700-bp band was recovered as the VHH fragment. The electrophoresis pattern was as shown in Figure 5 shown.

[0071] Using the product of the first round of PCR as a template, the second round of PCR amplification was performed with Camel-Phage-F / Camel-Phage-R. The amplification system was as follows:

[0072] 2×bufferMix 200 μl AlpVh-F1 16 μl AlpVHH-R1 16 μl AlpVHH-R2 16 μl PCR product recovered from the previous round 8 μl (>200 ng) dNTP mixture 4 μl High-fidelity polymerase 4 μl <![CDATA[H 2 O]]> ~136 μl, Up to 200 μl Total 400 μl

[0073] The reaction conditions for the second round of PCR were as follows: 95°C for 4 min; 95°C for 30 s, 56°C for 30 s, 72°C for 30 s, for 10 cycles; 95°C for 20 s, 60°C for 25 s, 72°C for 30 s, for 20 cycles; 72°C for 10 min; store at 10°C.

[0074] The products were detected by 1.2% gel electrophoresis, and the target fragments were recovered; the above-recovered fragments and the vector pComb3XSS were digested with SfiI respectively, and digested overnight at 50°C (about 20 h).

[0075] The digestion system for the above-recovered fragments was as follows:

[0076] VHH target fragment 4 μg SfiI enzyme 8 μl 10*Buffer 8 μl <![CDATA[ddH 2 O]]> Up to 80 μl

[0077] The digestion system for the vector pComb3XSS was as follows:

[0078]

[0079] The digested and purified VHH fragments and the phage vector were ligated using the following ligation system (VHH fragment:vector = 4:1). First, the VHH fragment and the vector were mixed and incubated at 50°C for 3 minutes to dissolve the ends (55°C, 5 min), and then immediately placed on ice for annealing. Then, SolutionI (brand: TAKARA) was added, incubated at room temperature for half an hour, then ligated overnight at 16°C, inactivated the ligase at 70°C for 15 min, and then purified the ligation product through a gel column.

[0080] VHH digested product 1.2 μg Vector digested product 3 μg SolutionI 100 μl <![CDATA[H 2 O]]> Up to 200 μl

[0081] The ligation product of the VHH fragment and the vector was used to prepare a phage library by electrotransforming TG1 (brand: lucigen, catalog number: 60502-2) competent cells. The electrotransformation parameters and experimental procedures were referred to the product manual of TG1 competent cells.

[0082] The primer sequences used in the above experimental process are as follows:

[0083]

[0084] Example 2 Nanobody Panning

[0085] ① First, the HSA protein was biotinylated. The method of protein biotinylation was referred to the biotin reagent instruction manual to obtain the HSA(biotin) protein. Take 100 μl of SA magnetic beads, wash them three times with 0.05% tween20-PBS, and set aside.

[0086] ② Take 100 μl of the phage prepared in Example 1 (1*10E11 CFU HSA-immunized alpaca phage display library), add the HSA(biotin) protein, incubate at room temperature for 2 h. Take 20 μl of SA magnetic beads and add them to the above phage library, invert and mix well on a rotary instrument. Adjust the pH value of the incubation system to 5.5 with acid, and incubate at room temperature for 2 h.

[0087] ③ Place the magnetic bead-EP tube on a magnetic rack to remove the supernatant; wash it 20 times with 0.05% PBS+Tween-20 (pH 7.4) to wash away non-specific phages.

[0088] ④ Add 100 μl of 0.25% trypsin, incubate at 37 °C for 20 min to dissociate the phages specifically binding to HSA, and infect the Escherichia coli TG1 cells in the logarithmic growth phase. Culture at 37 °C for 1 h to produce and purify phages for the next round of screening.

[0089] ⑤ Repeat the same screening process (steps ①-④) for 3 rounds to obtain enriched phage clone strains. The pH value of the incubation system in the first round of screening is 5.5, and the pH values in the subsequent two rounds are 7.4. Therefore, nanobodies that can bind to albumin under acidic conditions can be screened through the incubation system in the first round of screening.

[0090] ⑥ Randomly pick 96 single colonies from the plate for measuring the titer of the eluate in the final round of panning with a sterilized toothpick and inoculate them into a 96-well deep-well plate containing 0.3 mL of 2×YT-A. Incubate at 37 °C and 220 r / min with shaking until the logarithmic growth phase. Transfer 0.1 mL of the bacterial solution to a new 96-well plate, seal the new 96-well plate and place it in a 4 °C refrigerator. Add M13K07 phage to the original 96-well plate at a ratio of cell:phage = 1:10, incubate at 37 °C for 15 min, shake at 220 r / min for 30 min; add an equal volume of medium, incubate at 30 °C with vigorous shaking overnight; place the above culture at 4 °C

[0091] Centrifuge at 5000 rpm for 10 min, collect the supernatant for ELISA identification.

[0092] ⑦ Use ELISA to select positive clones expressing antigen-specific binders from the corresponding third-round panned VHH library. Specifically, coat 96-well plates with human albumin (HSA), mouse albumin (MSA), and rhesus albumin (RSA) respectively, and block with milk powder. Wells with absorbance values at 405 nm twice higher than those of the negative control wells are positive wells, and the corresponding colonies are positive colonies.

[0093] The detection results of ELISA identification are shown in Table 1:

[0094] Table 1 ELISA test results

[0095]

[0096] From the ELISA results: Two nanobodies (samples corresponding to clone numbers 5 and 9) were successfully screened in the present invention. Sequencing was performed on their corresponding clones, and the corresponding clones were respectively named: P1D12 and P1B6.

[0097] Example 3 Expression and identification of albumin nanobodies

[0098] 3.1 Expression of protein nanobodies

[0099] Through the phage screening in the above examples, two nanobodies that can bind three species of albumin were obtained. Sequencing was performed on them, and the amino acid sequences and nucleotide sequences obtained are as follows:

[0100] The amino acid sequence of the albumin nanobody P1D12 obtained in the present invention is shown in SEQ ID NO.1. The variable domain sequence of the albumin nanobody P1D12 of the present invention is shown in Table 2, and the complementarity-determining regions (CDRs) based on Kabat numbering are underlined.

[0101] Table 2 Amino acid sequence of the variable domain of the albumin nanobody P1D12 of the present invention

[0102]

[0103] The sequences of the heavy chain complementarity-determining regions VHCDR11, VHCDR12, and VHCDR13 of the heavy chain variable domain of the albumin nanobody P1D12 of the present invention are respectively:

[0104] VHCDR11 contains the sequence GGTLSNYA (SEQ ID NO.11);

[0105] VHCDR12 contains the sequence ITWSVST (SEQ ID NO.12);

[0106] VHCDR13 contains the sequence AARSRSHYSGTYSGVSGYDN (SEQ ID NO.13).

[0107] The nucleotide sequence of P1D12 obtained in the present invention is as shown in SEQ ID NO.2.

[0108] The amino acid sequence of the albumin nanobody P1B6 obtained in the present invention is as shown in SEQ ID NO.3. For the variable domain sequence of the albumin nanobody P1B6 of the present invention, see Table 3. The complementarity-determining regions (CDRs) based on Kabat numbering are underlined:

[0109] Table 3 Amino acid sequence of the variable domain of the albumin nanobody P1B6 of the present invention

[0110]

[0111] The sequences of the heavy-chain complementarity-determining regions VHCDR21, VHCDR22, and VHCDR23 of the heavy-chain variable domain of the albumin nanobody P1B6 of the present invention are respectively:

[0112] VHCDR21 contains the sequence GFDFNNFG (SEQ ID NO.14);

[0113] VHCDR22 contains the sequence INSRGDTT (SEQ ID NO.15);

[0114] VHCDR23 contains the sequence VIGRGTP (SEQ ID NO.16);

[0115] The nucleotide sequence of P1B6 obtained in the present invention is as shown in SEQ ID NO.4.

[0116] In order to prepare albumin nanobodies, the sequence genes of the above-obtained 2 nanobodies were fused with HIS tags to obtain VHH-HIS genes. The two VHH-HIS genes were respectively cloned into the pcDNA3.4 vector for subsequent antibody expression.

[0117] pcDNA3.4-VHH-his was transiently transfected into HEK293 cells respectively for protein production. The recombinant expression plasmid was diluted with CD05 medium, and PEI (polyethyleneimine) solution was added for transformation; the plasmid / PEI mixtures of each group were respectively added to the cell suspension and incubated at 37 °C, 7% CO 2 2, 120 rpm. After 6 days, the culture supernatant was collected, and the VHH-HIS protein sample was purified by a nickel column for nanobody activity identification. The obtained protein sample was detected by SDS-PAGE, and the results were asFigure 1 As shown in Figure 1 , the target band can be clearly seen, indicating that the two strains of VHH-HIS prepared by the present invention are successfully expressed.

[0118] 3.2 Identification of albumin nanobody

[0119] The antibody affinity was measured using OctetRED384. The his sensor was selected and the Running buffer with pH 7.4 was used.

[0120] The antigen - human HSA sample was diluted to 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM respectively. The Load time was set to 180 s, the Baseline time was 180 s, the binding time was 180 s, the dissociation time was 600 s, and the regeneration buffer was 50 mM pH 1.7 Gly - HCl (glycine - HCl buffer).

[0121] The experimental results are as follows:

[0122] Table 4 Affinity determination results of the two strains of albumin nanobodies obtained in the present invention

[0123]

[0124]

[0125] The long serum half - life of albumin is mainly driven by two characteristics: (i) the large size (65 kDa) of albumin limits its glomerular filtration, and (ii) albumin has a good binding affinity to FcRn at low pH values, and can prevent the degradation of albumin in lysosomes after passive endocytosis by endothelial and epithelial cells by recycling from lysosomes to the extracellular environment through FcRn molecules. In order for albumin - binding nanobodies to confer a long serum half - life through albumin binding and subsequent recycling, they need to maintain binding to albumin in the pH range of 5.0 - 7.4. Therefore, ensuring that albumin nanobodies always maintain a stable binding affinity to albumin in the pH range of 5.0 - 7.4 is the most critical technical requirement for extending the serum half - life of albumin.

[0126] In response to this, the present invention measures the binding effect of the prepared albumin nanobody with albumin in an acidic environment. The present invention uses an Octet instrument to measure the binding and dissociation rates of serum albumin nanobody with HSA at pH 5.2 and pH 7.4 respectively. The results show that there is no significant difference in the binding of the two nanobodies obtained in the present invention with HSA at pH 5.2 and pH 7.4, and the binding force shows an increasing trend under acidic conditions. This is related to the panning carried out under pH conditions during nanobody screening, ensuring that the albumin nanobody of the present invention can still bind tightly to HSA at low pH.

[0127] Example 4 Detection of the binding ability of recombinant protein with albumin of different species

[0128] Human serum albumin, monkey serum and mouse serum were respectively coated on enzyme-linked immunosorbent assay (ELISA) plates to detect whether Slit2D2-P1B6HIS and Slit2D2-P1D12HIS can bind to the corresponding species albumin.

[0129] The corresponding albumin was captured by coating with carbonate / bicarbonate coating buffer (pH 9.8) at a concentration of 1 μg / mL and incubated overnight at 4°C.

[0130] Discard the coating solution and wash the ELISA plate once. Add 250 μL of washing buffer to each well each time. Gently shake the ELISA plate above the sink to remove the solution or washing liquid. Tap the ELISA plate on a paper towel to remove the remaining droplets.

[0131] Before use, mix all reagents well. Do not generate a large amount of foam in the liquid to avoid adding a large number of air bubbles during sample addition, resulting in errors in sample addition.

[0132] Add 250 μL of blocking buffer to each well to block the remaining protein binding sites in the coated wells. The ELISA plate was incubated at 37°C for at least 1 - 2 hours.

[0133] Wash the ELISA plate twice with 250 μL of washing buffer.

[0134] Add 100 μL of samples of Slit2D2-P1B6HIS and Slit2D2-P1D12HIS at different concentrations to each well and incubate at 37°C for 1 h.

[0135] Discard the samples and wash the ELISA plate twice with 250 μL of washing buffer.

[0136] Add 100 μL of Goat anti HIS-HRP to each well. Dilute it 1:10000 with blocking buffer before use, gently shake and mix well, and incubate at 37°C for 30 minutes.

[0137] Wash the ELISA plate three times with washing buffer.

[0138] Add 50 μl of each of chromogenic substrate A and B to each well, gently shake and mix well, and incubate at 37 °C for 5 - 10 minutes. Avoid light.

[0139] Take out the ELISA plate, quickly add 50 μl of stop solution, and measure the results immediately after adding the stop solution. Measure the OD value of each well at a wavelength of 450 nm. The experimental results of the binding effects of Slit2D2-P1B6HIS and Slit2D2-P1D12HIS with albumin of different species are as Figure 4(a) and 4(b) shown.

[0140] The experimental results show that after the two nanobodies P1B6 and P1D12 are respectively fused with the Slit2D2 protein, they can still bind to albumin of three species, namely human, mouse and monkey, and can be used for the subsequent study of the half-life in animals. These two nanobodies (P1D12 and P1B6) can simultaneously bind to albumin of three species, monkey, human and mouse, and both have a high specific binding effect.

[0141] Example 5 Verification of the Effect of Nanobody on Prolonging the Half-Life of Fusion Protein

[0142] 5.1 Preparation of Recombinant Albumin

[0143] To verify whether the nanobodies screened in the present invention can prolong the half-life of the target protein or drug, the second domain (Domain2) of the Slit2 protein was selected for fusion expression with the VHH molecule, and then the half-life of the target molecule was measured in mice.

[0144] The inventors found in the research that the Slit protein plays an important role in angiogenesis, tumor cell migration, leukocyte chemotaxis, etc. The patent (CN108929383A) proves that the second binding domain D2 of the Slit2 protein is the main active part. By fusing the Slit2D2 molecule with the HSA protein, the Slit2D2-HSA fusion protein was prepared. This protein showed excellent anti-fibrotic drug efficacy and the effect of prolonging the drug half-life in animal models. However, due to the large molecular weight of HSA and the presence of a large number of disulfide bonds between its molecules, disulfide bond mismatching is likely to occur during subsequent production, which is not conducive to subsequent process development and quality control.

[0145] In the present invention, the Slit2D2-P1B6HIS and Slit2D2-P1D12HIS fusion proteins were respectively constructed to verify whether the constructed molecules still possess the binding ability to albumin and whether the screened nanobodies possess the ability to prolong the half-life of albumin in vivo.

[0146] The amino acid sequence of the fusion protein Slit2D2-P1B6HIS is designed as shown in SEQ ID NO.5. The fusion protein Slit2D2-P1B6HIS refers to a fusion protein that simultaneously fuses Slit2D2, P1B6, and albumin.

[0147] The nucleotide sequence of the fusion protein Slit2D2-P1B6HIS is designed as shown in SEQ ID NO.6.

[0148] The amino acid sequence of the fusion protein Slit2D2-P1D12HIS is designed as shown in SEQ ID NO.7.

[0149] The nucleotide sequence of the fusion protein Slit2D2-P1D12HIS is designed as shown in SEQ ID NO.8.

[0150] The fusion protein Slit2D2-P1D12HIS refers to a fusion protein that simultaneously fuses Slit2D2, P1D12, and albumin.

[0151] The amino acid sequence of the fusion protein Slit2D2HIS is as shown in SEQ ID NO.9.

[0152] The nucleotide sequence of the fusion protein Slit2D2HIS is as shown in SEQ ID NO.10.

[0153] The genes of the above three fusion proteins (Slit2D2, Slit2D2-P1B6HIS, and Slit2D2-P1D12HIS) were respectively cloned into the pcDNA3.4 vector. The above three plasmids were respectively extracted and transiently transfected into HEK293 cells for protein production. The recombinant expression plasmid was diluted with CD05 medium, and a PEI (polyethyleneimine) solution was added for transformation; the plasmid / PEI mixtures of each group were respectively added to the cell suspension and incubated at 37 °C, 7% CO 2 , 120 rpm. After 6 days, the culture supernatant was collected, and the protein sample was purified by a nickel column. The obtained protein sample was detected by SDS-PAGE. The results are as Figure 2 shown, and the target band can be clearly seen, indicating that the VHH-HIS of the three recombinant proteins were successfully expressed.

[0154] 5.2 Study on the half-life of recombinant proteins in mice

[0155] Eight-week-old female Balb / C mice were selected and divided into three groups: the Slit2D2HIS administration group, the Sli2D2-P1D12-HIS administration group, and the Slit2D2-P1B6-HIS administration group. Each group of animals was intraperitoneally injected with 50 μg of the corresponding recombinant protein, and the volume of the recombinant protein was 200 μl. After injection, mouse serum was collected at different time points, and the concentration of the recombinant protein in the mice was measured by ELISA. The ELISA method was a sandwich method, that is, the coating antibody was an antibody against Slit2 (Domain2), and the detection antibody was AntiHIS-HRP. The specific experimental steps were as follows:

[0156] ①Dilute the antibody against Slit2 (Domain2) with PBS to 5 μg / mL, add 100 μL / well to the enzyme-labeled wells, and coat at 4°C for 12 h;

[0157] ②Discard the coating solution, wash 3 times with PBST, add 300 μL of 3% skim milk to each well, and block at 37°C for 2 h;

[0158] ③Wash 3 times with PBST, add 50 μL of mouse serum diluent (mouse serum diluted 100 times with PBS) or different concentrations of recombinant protein control products, and then add 50 μL of PBS respectively, incubate at 37°C for 1 h;

[0159] ④Wash 5 times with PBST, add horseradish peroxidase-labeled AntiHIS-HRP antibody (diluted 1:5000 with 3% skim milk), 100 μL / well, and react at 37°C for 1 h;

[0160] ⑤Wash the plate 6 times with PBST, add the reaction substrate TMB chromogenic solution for color development, 100 μL / well, at 37°C for 20 min, add the termination solution to terminate the reaction, 50 μL / well, and measure the optical density OD value at 450 nm;

[0161] ⑥According to the concentration and OD value of the control product protein at different concentrations, establish a standard curve, and then calculate the concentration of the corresponding recombinant protein contained in the serum collected at different time points.

[0162] A curve relationship diagram of the concentration values of different time points and different recombinant protein molecules in mouse serum was established (as shown in Figure 3), with the abscissa representing the serum collection time and the ordinate representing the drug concentration in the serum. The results showed that the drug content could still be detected in the mouse serum of the Sli2D2-P1D12-HIS administration group and the Slit2D2-P1B6-HIS administration group at 72 h, while the content in the mouse serum of the Slit2D2HIS administration group was lower than the lowest detection concentration of the experimental detection after 20 h, and the drug could not be detected.

[0163] By calculation, the pharmacokinetic half-life of Sli2D2-P1D12-HIS is: 16 h; the pharmacokinetic half-life of Slit2D2-P1B6-HIS is: 13 h; the pharmacokinetic half-life of Slit2D2HIS is: 4.5 h. The experimental results show that both of the two nanobodies, P1B6 and P1D12, can effectively prolong the half-life of albumin in animals by binding to albumin. The above results also show that the albumin-binding nanobody prepared by the present invention has a significant effect of prolonging the duration of action of recombinant proteins.

[0164] It can be thus shown that the two albumin-binding nanobodies screened by the present invention can effectively prolong the in vivo pharmacokinetic half-life of recombinant proteins, and can increase the in vivo half-life time of Sli2D2 protein by 2-3 times.

[0165] The above embodiments are only for illustrating the technical concept and features of the present invention, and the purpose is to enable those skilled in the art to understand the content of the present invention and implement it. However, the protection scope of the present invention cannot be limited thereby. Any equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. An albumin-binding nanobody, characterized in that: it comprises at least one albumin-binding domain, and the albumin-binding domain comprises at least one group of the following complementarity-determining regions: ① VHCDR11, VHCDR12 and VHCDR13, wherein the VHCDR11 comprises the sequence GGTLSNYA (SEQ ID NO.11); the VHCDR12 comprises the sequence ITWSVST (SEQ ID NO.12); the VHCDR13 comprises the sequence AARSRSHYSGTYSGVSGYDN (SEQ ID NO.13); ② VHCDR21, VHCDR22 and VHCDR23, wherein the VHCDR21 comprises the sequence GFDFNNFG (SEQ ID NO.14); the VHCDR22 comprises the sequence INSRGDTT (SEQ ID NO.15); the VHCDR23 comprises the sequence VIGRGTP (SEQ ID NO.16).

2. The albumin-binding nanobody according to claim 1, characterized in that: it has an amino acid sequence shown in any one of SEQ ID NO.1 and SEQ ID NO.

3.

3. A nucleic acid molecule encoding the albumin-binding nanobody according to claim 1 or 2.

4. An albumin-binding nanobody, characterized in that: it has a nucleotide sequence shown in any one of SEQ ID NO.2 and SEQ ID NO.

4.

5. A vector of an albumin-binding nanobody containing the nucleic acid molecule according to claim 3.

6. A fusion or conjugate comprising the isolated antibody according to claim 1 or 2.

7. An expression cassette or recombinant bacterium containing the nucleic acid molecule according to claim 3.

8. A composition comprising at least one of the isolated antibody of claim 1 or 2, the nucleic acid molecule of claim 3, the vector of claim 5, or the fusion or conjugate of claim 6, and at least one of its corresponding pharmaceutically acceptable carriers.

9. A method for detecting albumin in a biological sample, comprising contacting the biological sample with the isolated antibody according to claim 1 or 2 or the fusion or conjugate according to claim 6.

10. A fusion, characterized in that, it simultaneously fuses the albumin-binding nanobody according to any one of claims 1, 2, 4, and 5 and Slit2D2.

11. Use of the antibody or antigen-binding fragment according to claim 1 or 2, the fusion or conjugate according to claim 6, the composition according to claim 8, or the fusion according to claim 10 in the preparation of a drug and / or the prolongation of the drug half-life.

12. A method for preparing the albumin-binding nanobody according to any one of claims 1, 2, 4, and 5, characterized in that: it comprises the following steps: S1. Screening of nanobodies recognizing albumin: After immunizing alpacas with human serum albumin, isolating alpaca PBMCs, and constructing a phage library of albumin nanobodies; S2. Panning of nanobodies: Through at least one round of panning process, positive phage clones are obtained; when the number of panning rounds is greater than 1, the pH of the incubation system in the first round of panning process is not equal to that in the second round; S3. Expression and identification of albumin-binding nanobodies.

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