An anti-CD16 nanobody and a multispecific antibody containing the nanobody

Abstract

This invention discloses an anti-CD16 nanobody and a multispecific antibody containing the nanobody, belonging to the field of biotechnology. The anti-CD16 nanobody contains a CDR region as shown in SEQ ID NO. 1-3 or as shown in SEQ ID NO. 8-10, preferably A1 as shown in SEQ ID NO. 15 or A2 as shown in SEQ ID NO. 16. This nanobody exhibits high affinity and excellent thermal stability. Based on this nanobody, this invention constructed a trispecific antibody GN-C6-A2 with an asymmetric structure. This antibody expressed at 95.30 mg / L in the HEK293 system, with 100% purity by SEC-HPLC, and its affinity for all three targets reached therapeutic levels. ELISA showed that the EC50 values ​​of GN-C6-A2 binding to the three targets were 0.01678, 0.008873, and 0.006819 μg / mL, respectively. The nanobody and multispecific antibody of this invention can be used for tumor treatment, possessing advantages such as strong penetration, high specificity, and good stability.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an anti-CD16 nanobody and a multispecific antibody containing the nanobody. Background Technology

[0002] CD16 is a surface marker of natural killer (NK) cells and is an IgG Fc receptor III (FcγRIII). Anti-CD16 antibodies bind to CD16 on the surface of NK cells, mediating cytotoxic effects (ADCC) to activate NK cells and enhance their cytotoxic function. Therefore, the CD16 molecule is one of the targets of antibody drugs (including bispecific antibodies and multispecific antibodies).

[0003] While patents and articles have reported on anti-CD16 monoclonal antibodies, traditional monoclonal antibodies (150kD) have excessively large molecular weights, making it difficult to penetrate tissues and resulting in low effective concentrations in tumor areas and insufficient therapeutic effects. Traditional antibodies also exhibit high immunogenicity, while modified antibodies struggle to achieve the same affinity. Furthermore, fully humanized traditional antibodies suffer from long development cycles, high production costs, insufficient stability, and are difficult to genetically engineer, all of which limit their clinical application and widespread use. Moreover, most existing monoclonal antibodies are murine-derived, which can generate human anti-mouse antibodies upon interaction with humans, reducing their therapeutic efficacy. Therefore, there is a need in this field for an anti-CD16 antibody drug with low immunogenicity and good biocompatibility in humans, capable of activating NK cells for tumor treatment.

[0004] In recent years, nanobodies, as a novel form of antibody molecule, have shown great potential in overcoming the limitations of traditional antibody applications. These antibodies are derived from the variable region fragments of naturally occurring heavy-chain antibodies that lack light chains, found in camels. With a molecular weight of only about 15 kDa, roughly one-tenth the molecular weight of traditional antibodies, they possess unique structural and functional characteristics distinct from conventional antibodies. Specifically, their smaller molecular size gives them superior tissue penetration, allowing them to penetrate more uniformly into deeper areas of solid tumors, thereby more effectively blocking tumor-related immune pathways. Simultaneously, although the antigen-binding region of nanobodies has a relatively simple structure, it still possesses high antigen-binding affinity and specificity. This characteristic helps reduce off-target effects and further improves the safety of tumor treatment. In addition, nanobodies offer significant advantages such as good stability, high solubility, ease of modification through genetic engineering, and large-scale production. Not only are their production costs relatively low, but they can also be developed into various dosage forms, including subcutaneous injection and intratumoral administration, which is of great significance for improving patient convenience and treatment adherence.

[0005] Currently, although there are reports on research related to nanobodies targeting CD16, the existing technology system still has many shortcomings that need to be addressed: First, single-target nanobodies are unable to cope with the complexity of tumor immune escape mechanisms and cannot achieve ideal therapeutic effects; second, the construction process of multispecific nanobodies is relatively complicated, and the efficiency of heterodimerization is low, resulting in antibody expression levels that are difficult to meet the needs of clinical applications; most importantly, how to design multispecific nanobodies with stable structures and synergistic effects of various functional domains remains a core technical challenge in this research field. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0008] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide an anti-CD16 nanobody.

[0009] To address the aforementioned technical problems, the present invention provides the following technical solution: the nanobody specifically binds to CD16 and includes a complementarity-determining region (CDR) selected from the following group: (a) CDR1 as shown in SEQ ID NO.1, CDR2 as shown in SEQ ID NO.2, and CDR3 as shown in SEQ ID NO.3; or (b) CDR1 as shown in SEQ ID NO.8, CDR2 as shown in SEQ ID NO.9, and CDR3 as shown in SEQ ID NO.10; Or a variant thereof, wherein the variant has at least 90% sequence identity with the above-described complementarity-determining region sequence and retains binding activity with CD16.

[0010] In a preferred embodiment of the anti-CD16 nanobody of the present invention, the backbone region (FR) sequence of the nanobody is selected from: (a) FR1, FR2, FR3, FR4 as shown in SEQ ID NO. 4, 5, 6, 7; or (b) FR1, FR2, FR3, and FR4 as shown in SEQ ID NO. 11, 12, 13, and 14; Or a variant thereof, wherein the variant has at least 90% sequence identity with the above-described FR sequence.

[0011] As a preferred embodiment of the anti-CD16 nanobody of the present invention, the nanobody comprises, (a) an amino acid sequence as shown in SEQ ID NO.15, A1; or (b) The amino acid sequence is A2 as shown in SEQ ID NO.16; Or its functionally active fragments or derivatives.

[0012] Beneficial effects of this invention: The nanobodies obtained by screening in this invention have high affinity (A2's KD reaches 1.50 × 10⁻⁶). -8 It exhibits excellent thermal stability, maintaining its binding activity to CD16 even after treatment at 80°C for 2 hours, significantly superior to traditional monoclonal antibodies. This high thermal stability is beneficial for antibody production, storage, and transportation, reducing cold chain costs.

[0013] Another object of the present invention is to provide a nucleic acid molecule that encodes the nanobody of claim 3, comprising a nucleotide sequence as shown in SEQ ID NO. 17 or SEQ ID NO. 18.

[0014] Another object of the present invention is to provide an expression vector comprising the aforementioned nucleic acid molecule or a functional variant thereof, wherein the functional variant comprises a nucleotide sequence having at least 95% sequence identity with SEQ ID NO.17 or SEQ ID NO.18.

[0015] Another object of the present invention is to provide a host cell comprising the expression vector described above.

[0016] Another object of the present invention is to provide a method for preparing anti-CD16 nanobodies, wherein the anti-CD16 nanobodies are obtained by culturing the host cells and separating and purifying the culture.

[0017] Another object of the present invention is to provide a multispecific antibody.

[0018] To solve the above-mentioned technical problems, the present invention provides the following technical solution: the multispecific antibody comprises at least the nanobody described above, and further comprises one or more antigen-binding domains; The antigen-binding domain includes a dimerizing domain that promotes the formation of dimers between polypeptide chains.

[0019] As a preferred embodiment of the multispecific antibody of the present invention, the multispecific antibody is a trispecific antibody, comprising, (a) A first polypeptide chain comprising a first antigen-binding domain specifically binding to a first target, a first dimerizing domain and a third antigen-binding domain specifically binding to a third target; (b) A second polypeptide chain comprising a second antigen-binding domain that specifically binds to a second target, a second dimerizing domain, and a third antigen-binding domain that specifically binds to a third target; Wherein, at least one of the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain is a nanobody according to any one of claims 1-3, and the first dimerizing domain and the second dimerizing domain combine with each other to form a dimer.

[0020] As a preferred embodiment of the multispecific antibody of the present invention, wherein: the first antigen-binding domain is a nanobody that specifically binds to GPC3; The second antigen-binding domain is a nanobody that specifically binds to LAG3; The third antigen-binding domain is a nanobody that specifically binds to CD16.

[0021] It should be noted that the trispecific antibody constructed in this application has a "2+1" asymmetric structure, where "2" represents two different targets (GPC3 and LAG3) at the N-terminus of the two polypeptide chains, and "1" represents the same target (CD16) carried by both polypeptide chains at the C-terminus. This target exists in a bivalent form, specifically: As a preferred embodiment of the multispecific antibody of the present invention, wherein: the first antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO.19, and the second antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO.20.

[0022] As a preferred embodiment of the multispecific antibody of the present invention, wherein the first dimerization domain and the second dimerization domain are immunoglobulin Fc regions or their functionally active fragments.

[0023] As a preferred embodiment of the multispecific antibody of the present invention, wherein the immunoglobulin Fc region contains a modification that promotes the formation of heterodimers.

[0024] As a preferred embodiment of the multispecific antibody of the present invention, the modification is selected from spatial complementarity modification, charge complementarity modification, chain exchange modification or a combination thereof.

[0025] As a preferred embodiment of the multispecific antibody of the present invention, the first polypeptide chain further comprises a first linker peptide located between a first antigen-binding domain and a first dimerization domain, and a second linker peptide located between a first dimerization domain and a third antigen-binding domain. The second polypeptide chain further includes a third linker peptide located between the second antigen-binding domain and the second dimerization domain, and a fourth linker peptide located between the second dimerization domain and the third antigen-binding domain.

[0026] In a preferred embodiment of the multispecific antibody described in this invention, the first linker peptide, the second linker peptide, the third linker peptide, and the fourth linker peptide are independently selected from (G4S). n , where n is an integer from 1 to 5.

[0027] Beneficial effects of this invention: (1) Unique “2+1” asymmetric trispecific antibody structure design: This invention successfully constructed an IgG-like trispecific antibody GN-C6-A2 with a “2+1” asymmetric structure, where “2” represents two different targets (GPC3 and LAG3) at the N-terminus of the two polypeptide chains, and “1” represents the same target (CD16) carried by both polypeptide chains at the C-terminus, which exists in a bivalent form. This design places the GPC3 nanobody at the N-terminus of the first polypeptide chain to achieve tumor-targeted enrichment; places the LAG3 nanobody at the N-terminus of the second polypeptide chain to block the immunosuppressive pathway; and places the CD16 nanobody A2 at the C-terminus of both polypeptide chains to achieve bivalent binding of CD16 and efficiently activate NK cells.

[0028] (2) Excellent purity and uniformity: The GN-C6-A2 constructed in this application expressed 95.30 mg / L in the HEK293 system and had a purity of 100% by SEC-HPLC. All binding domains maintained their original affinity, proving that the asymmetric structural design of “N-terminal dual target + C-terminal common target” and the mutations introduced in each Fc region of this invention do not affect the functional activity of each antigen-binding domain.

[0029] (3) Excellent target binding properties: SPR assay showed that GN-C6-A2 had therapeutic affinity for all three targets (GPC3: 8.45 nM, LAG3: 4.72 pM, CD16: 1.25 pM). ELISA showed that the EC50 values ​​for binding to the three targets were 0.01678 μg / mL (GPC3), 0.008873 μg / mL (LAG3), and 0.006819 μg / mL (CD16), respectively. In particular, the bivalent binding to CD16 resulted in a dissociation half-life of up to 1.8 hours, demonstrating a sustained effector cell recruitment ability and efficient activation of NK cells.

[0030] Another object of the present invention is to provide the use of the nanobody or multispecific antibody in the preparation of a medicament for treating tumors.

[0031] Another object of the present invention is to provide a pharmaceutical composition comprising the nanobody or multispecific antibody.

[0032] Another object of the present invention is to provide a fusion protein comprising the rice antibody or the multispecific antibody and a heterologous protein domain, wherein the heterologous protein domain is selected from Fc domains, toxic proteins, cytokines or fluorescent proteins.

[0033] Beneficial effects of this invention: (1) This invention successfully verified the feasibility of constructing multispecific antibodies with CD16 nanobody as the core. This technology platform can be rapidly expanded to other target combinations, providing a technical basis for the development of next-generation multispecific tumor immunotherapy drugs.

[0034] (2) The nanobodies and multispecific antibodies of the present invention can not only be used for systemic drug delivery to treat a variety of solid tumors, but can also be developed into various drug delivery forms such as intratumoral injection formulations and local sustained-release formulations. At the same time, they can be used for in vitro diagnosis and in vivo molecular imaging of CD16 expression, realizing integrated diagnosis and treatment. Attached Figure Description

[0035] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 This is a graph showing the titer of alpaca (Vicugna pacos) immune serum in this invention. The horizontal axis represents the serum dilution factor, and the vertical axis represents the corresponding absorbance OD450 value.

[0036] Figure 2 The results of agarose gel electrophoresis of total RNA from alpaca PBMCs in this invention are shown.

[0037] Figure 3 The agarose gel electrophoresis results of the amplified VHH gene in this invention are shown.

[0038] Figure 4 The results of colony PCR agarose gel electrophoresis in this invention are shown.

[0039] Figure 5 The results of identifying positive clones using the sandwich phage ELISA method in this invention.

[0040] Figure 6This is an SDS-PAGE protein electrophoresis image of CD16 nanobodies A1 and A2 in this invention. In the image, lane M: molecular weight marker; lane 1: nanobodies A1; lane 2: nanobodies A2.

[0041] Figure 7 These are the binding and dissociation kinetic curves of nanobodies A1 and A2 with CD16 protein in this invention. A. Binding and dissociation kinetic curve of nanobodies A1 with CD16 protein. B. Binding and dissociation kinetic curve of nanobodies A2 with CD16 protein. Figure 8 The figure shows the experimental results of the thermal stability of CD16 nanoantibodies A1 and A2 in this invention.

[0042] Figure 9 This is an SDS-PAGE protein electrophoresis image of the asymmetric trispecific nanobody GN2-C6-A2 in this invention. Lane M: molecular weight marker; Lane R: molecular weight of the fusion protein in the reduced state (80 kDa); Lane NR: molecular weight of the fusion protein in the non-reduced state (160 kDa). The structural characteristics of this antibody are: the first polypeptide chain (Knob chain) has a GPC3 nanobody GN at its N-terminus and a CD16 nanobody A2 at its C-terminus; the second polypeptide chain (Hole chain) has a LAG3 nanobody C6 at its N-terminus and a CD16 nanobody A2 at its C-terminus. Both chains have A2 fused to their C-termini, achieving bivalent binding to CD16.

[0043] Figure 10 This is the SEC-HPLC chromatogram of the trispecific nanobody GN2-C6-A2 in this invention, showing the protein peaks at 214 nm and 280 nm, along with their numerical values. The main peak accounts for 100%, indicating high purity of the fusion protein, which mainly exists in the correctly folded antibody form.

[0044] Figure 11 The image shows the ELISA detection results of the trispecific nanobody GN2-C6-A2 in this invention. GN2-C6-A2 can specifically bind to GPC3, CD16 and LAG3 antigens, with EC50 values ​​of 0.01678, 0.008873 and 0.006819 ug / mL, respectively. Detailed Implementation

[0045] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0046] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0047] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0048] Unless otherwise specified, all raw materials used in this invention are commercially available. The phage vector pComb3X was purchased from Beijing Baokewei Food Safety Biotechnology Co., Ltd.; the pPICZαA vector was purchased from Invitrogen.

[0049] The antibody products or primer sequences used in specific embodiments of the present invention are shown in Table 1.

[0050] Table 1

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057] Example 1: Construction of an immune alpaca and nanobody gene library (1) Immunizing alpacas: 1 mg of CD16 protein expressed in HEK293 eukaryotic cells was emulsified with Freund's complete adjuvant, totaling 2 mL, and administered as the first immunization to a healthy adult alpaca via multiple subcutaneous injections. Two weeks later, 0.5 mg of CD16 protein was emulsified with Freund's complete adjuvant, totaling 2 mL, and administered as the second immunization via multiple subcutaneous injections. Subsequently, every two weeks, 0.5 mg of CD16 protein was emulsified with Freund's incomplete adjuvant, totaling 2 mL, for each subsequent immunization. A total of 6 immunizations were administered. Blood was collected on the 7th day after each immunization to determine the titer. After determining the serum titer, 100 mL of peripheral blood was collected. like Figure 1As shown, a positive serum-to-negative serum ratio (positive / negative) ≥ 2.1 is considered positive, indicating that the serum titer of immunized alpacas can reach 1:1024000. This demonstrates a good immunization effect, stimulating alpacas to produce high-titer antibodies and ensuring the diversity of the gene library.

[0058] (2) RNA extraction from peripheral blood lymphocytes: Lymphocytes were isolated using the lymphocyte separation medium following the manufacturer's instructions; RNA was extracted using Trizol, and approximately 10 white blood cells were collected from one tube. 7 Add 2 mL of Trizol and pipette repeatedly until cells are completely lysed. Let stand for 5 min. Add 1 / 5 volume of chloroform to the lysis buffer and shake vigorously for 15 s to emulsify thoroughly. Let stand at room temperature for 5 min. Centrifuge at 12000 g at 4℃ for 15 min. At this point, the homogenate will separate into three layers. Transfer the supernatant to another fresh centrifuge tube. Add an equal volume of isopropanol to the supernatant, invert the centrifuge tube to mix thoroughly, and let stand at room temperature for 10 min. Centrifuge at 12000 g at 4℃ for 10 min, carefully discard the supernatant, and slowly add 75% ethanol (prepared with DEPC-treated water) along the wall of the centrifuge tube. Gently invert the centrifuge tube to wash the wall. Centrifuge at 12000 g at 4℃ for 5 min, and carefully discard the ethanol. Dry the precipitate at room temperature for 2-5 min, add an appropriate amount of RNase-free water to dissolve the precipitate, and perform electrophoresis after the RNA precipitate is completely dissolved. Store the remaining sample at -80℃. Electrophoresis results are shown below. Figure 2 As shown in Table 2, the total RNA volume and detection results are presented.

[0059] Table 2 Total RNA volume and detection results

[0060] Figure 2 Two clear major ribosomal RNA bands, 28S rRNA and 18S rRNA, were observed in the lane. The 28S band was approximately twice as bright as the 18S band (28S:18S ≈ 2:1), indicating that the total RNA was of good integrity and high purity, meeting the requirements for subsequent experiments.

[0061] (3) cDNA synthesis: 190 μg of RNA was reverse transcribed to synthesize cDNA according to the instructions of the SuperScript® III First-Strand Synthesis System for RT-PCR.

[0062] (4) Amplification of the alpaca VHH gene: Using the cDNA strand from step (3) as a template, four PCR reactions were performed using primers HS and Hanti1 (sequences shown in Table 1) to amplify the VHH gene of alpaca heavy chain antibody IgG3. Six PCR reactions were performed using primers HS and Hanti2 (sequences shown in Table 1) to amplify the VHH gene of alpaca heavy chain antibody IgG2. Figure 3 The inventors of this application discovered that the VHH gene originates from heavy chain antibodies IgG2 and IgG3. Therefore, the VHH gene amplified using these two primer pairs includes both the VHH gene of IgG2 and the VHH gene of IgG3, resulting in a more diverse gene library compared to other research institutions that use primers to amplify only the VHH gene of IgG2.

[0063] The reaction system was as follows: 2 μL cDNA; 1.3 μL HS primers; 1.3 μL Hanti1 or Hanti2 primers; 44.4 μL TaqEnzyme Mix; reaction program: 94℃, 3 min; 94℃, 30 s, 55℃, 30 s, 72℃, 1 min, 32 cycles; 72℃, 5 min. PCR products were analyzed by electrophoresis, and the PCR products (approximately 500 bp single band) were recovered by gel excision. The amount of recovered DNA is shown in Table 3 below.

[0064] Table 3 Recovery of PCR Products

[0065] (5) Recombination and ligation of the vector with the VHH gene fragment: The recovered VHH gene was digested with SfiⅠ restriction endonuclease; the phage vector pComb3X was also digested with SfiⅠ restriction endonuclease.

[0066] The inventors discovered that the SfiⅠ enzyme is sensitive to methylated DNA. Therefore, *E. coli* C2925 was chosen as the host bacterium for amplification of this vector. *E. coli* C2925 lacks nonspecific endonuclease I (endA1) activity and methyltransferase activity, so its RNA sequence is not methylated. This allows for the amplification of high-quality pComb3X plasmids without affecting the SfiⅠ digestion efficiency. *E. coli* C2925 competent cells were purchased and transformed into *E. coli* C2925 competent cells using the pComb3X phage vector. 1 μL of the pComb3X phage vector was added to thawed *E. coli* C2925 competent cells, incubated in ice water for 10-20 min, heat-shocked at 42℃ for 60-90 s, and then incubated on ice for 2 min. SOC medium at 25-37℃ was added, and the cells were incubated at 37℃ and 250 rpm for 1 h. 200 μL of the bacterial culture was plated on an ampicillin-resistant plate and incubated overnight at 37℃. The following day, single clones were selected and cultured overnight at 37°C and 250 rpm in ampicillin-resistant LB medium. Plasmids were extracted using a plasmid extraction kit. Enzyme digestion was performed in a 0.2 μL PCR tube: 1 μg of plasmid pComb3X, 4 μL of SfiⅠ enzyme, 4 μL of CutSmart Buffer, and ultrapure water / deionized water were added to a final volume of 50 μL. The tube was placed in a PCR instrument at 50°C for 12 h. The following day, the digestion products were electrophoresed, and the large fragments were recovered by gel extraction.

[0067] Based on the principle of homologous recombination, the VHH gene was ligated into the pComb3X vector: Homologous recombination was performed using the ClonExpress UltraOne Step Cloning Kit according to the following system: 200 ng of the pComb3X vector fragment (digested with SfiI and then gel-recovered), 60 ng of the VHH gene, 5 μL of 2×ClonExpress Mix, and ultrapure water / deionized water to a final volume of 10 μL. The mixture was incubated at 50°C for 5 minutes, and then immediately cooled on ice. The ligation product was purified and recovered using a PCR product purification kit. The recovered product analysis results are shown in Table 4.

[0068] Table 4. Results of enzyme digestion and ligation detection

[0069] The use of homologous recombinase increased the ligation efficiency between the vector and the VHH gene, and the absence of DNA ligase in the entire system reduced the efficiency of vector self-ligation, thus increasing the library capacity. The bacterial library detection results are shown in Table 5 below.

[0070] Table 5. Detection Indicators for Bacterial Library

[0071] (6) Electroporation for gene library construction and library rescue: Add 3 μl of the ligation product from step 5) to 50 μl of *E. coli* ER2738 competent cells, mix thoroughly, transfer to an electroporation cuvette (1 mm cuvette, 1.8 kV), gently shake the cuvette with your wrist to allow the cells to sink to the bottom, immediately place it in an electroporator, electroporation conditions: 1400-1600 V, 200-400 Ω, 10 μF, 3.5-4.5 ms, immediately add 975 μl of preheated SOC medium, mix the cells by blowing up and down three times, transfer to a bacterial culture tube, incubate at 37°C and 250 rpm for 1 h; take 1 μl of bacterial culture and dilute 10... -1 10 -3 1 0-5 10 -7 The library capacity was determined by plating after fold expansion (LB for ampicillin resistance). Forty-eight clones were randomly selected from the plate for colony PCR to verify ligation efficiency. Figure 4 All 48 selected single clones were positive, representing a 100% positivity rate. The primers for colony PCR were primers HS and Hback (Table 1). After sequencing, the 48 randomly selected single clones were transcribed and translated into protein sequences using GENtle software. Sequence diversity alignment showed that all 48 sequences were independent, with 100% diversity, meeting the bacterial library diversity requirements.

[0072] All bacterial cultures were transferred to 200 ml of SB medium (50 μg / ml ampicillin and 20 μg / ml tetracycline) and incubated at 37°C with shaking at 250 rpm for 2 h; 1 ml of helper phage (1 x 10⁻⁶) was added. 13 After incubating at 37°C for half an hour with a concentration of pfu / ml, the culture was incubated at 37°C with shaking at 250 rpm for 2 hours. Cannabis chloride (final concentration 70 μg / ml) was added, and the culture was incubated overnight at 37°C with shaking at 250 rpm. The next day, the overnight culture was centrifuged at 4°C and 10,000 rpm for 15 minutes. The supernatant was transferred to another clean centrifuge bottle, 25 ml of 5× PEG / NaCl was added, and the mixture was incubated on ice for 2 hours. Then, the culture was centrifuged at 4°C and 12,000 rpm for 15 minutes. The precipitate was resuspended in 1 ml of PBS, filtered through a 0.22 μm filter, and 10 μl was used to determine the titer. The remaining nanobody phage library was resuspended in a protective buffer (PBS solution containing 1× protease inhibitor, 0.02% sodium azide, and 0.5% BSA) to obtain 4 mL of nanobody phage library with a titer of 1.0 × 10⁻⁶. 14 pfu / ml.

[0073] Example 2: Panning and Identification of CD16 Nanobodies (1) Selection of CD16 nanobodies: Affinity screening was performed using magnetic beads coupled with streptavidin, specifically: Take 200 μl of magnetic beads, wash twice with 1 mL TBST, add 1 mL blocking buffer (3% BSA), and block at 4℃ for 1 h; simultaneously, add 1% BSA to the phage library and remove impurities at 4℃ for 1 h, then place the magnetic beads on a magnetic rack for 30 s, discard the supernatant, and wash three times with 1 mL TBST; add 200 μL of binding buffer and 30 μL of biotinylated CD16 protein to the washed magnetic beads, and bind at 4℃ for 0.5 h, then add the removed phage library and bind at 4℃ for 1 h; place the reaction solution on a magnetic rack for 30 s, discard the supernatant, add 200 μL of glycine-hydrochloric acid (pH 2.2) to the magnetic beads, shake at 200 rpm for 15 min, place on a magnetic rack for 30 s, discard the supernatant, and immediately add 1.6 μL of Tris base (pH 9.1) to obtain the first output; take 10 μL... The output titers were measured, and the remaining outputs were amplified and then subjected to second and third rounds of screening. The biotinylated CD16 protein was reduced to 6 μL and 1.5 μL respectively. The results of the three rounds of screening are shown in Table 6 below. The enrichment degree is calculated by dividing the screening titer by the input titer. The larger the value, the higher the enrichment degree of the antibody in the input library. The fold difference is calculated by dividing the screening titer by the control titer. The higher the value, the higher the content of the positive antibody in the screening library.

[0074] Table 6 Results of the Three Rounds of Screening

[0075] (2) Screening and identification of positive clones: After the third round of screening, 96 clones were picked from the plates and incubated overnight at 37°C. Positive clones were identified by phage ELISA. The absorbance values ​​of the phage ELISA were obtained. Of the 56 selected clones, 48 ​​were positive, with a positive rate of 85.7%. Figure 5 ).

[0076] For positive clones, plasmids are extracted and sequenced. Based on the sequencing results, the nucleotide sequence of the CD16 nanobody of the present invention (SEQ ID NO.17, SEQ ID NO.18) can be obtained, and the amino acid sequence of the nanobody (SEQ ID NO.15, SEQ ID NO.16) can be obtained according to the codon table.

[0077] Example 3: Expression, purification, and SPR affinity detection of CD16 nanobody For positive strains, plasmids were extracted using a Qiagen plasmid extraction kit, digested with EcoRI and NotI, and ligated into the similarly digested pPICZαA vector. The vector was then electroporated into Pichia pastoris X33, and expression was induced with methanol. Positive clones were selected and added to 30 ml of BMGY medium. The mixture was incubated at 30°C and 250 rpm until an OD600 value of approximately 2–6 (approximately 36 h). After centrifugation at 3000 rpm for 5 min, the supernatant was discarded. The precipitate was resuspended in 5 ml of autoclaved water and centrifuged at 3000 rpm for 5 min, discarding the supernatant. The precipitate was resuspended in 3 mL of BMMY medium, transferred to BMMY medium, and inducing expression at 30°C and 250 rpm to a final concentration of 1% methanol. The supernatant was collected and added to a nickel column. Incubation was performed at 4°C for 3–6 h. Four column volumes were washed with 20 mmol / M imidazole, and 5 mL of 50 mmol / L and 100 mmol / L imidazole wash buffer were collected to obtain the nanobody. The PAGE electrophoresis results of the nanobody are shown below. Figure 6 As shown in the figure, the molecular weights of the nanobodies A1 and A2 prepared in this invention are approximately 15 kDa.

[0078] The affinity of nanobodies A1 and A2 for target proteins was determined using a SPR (Biacore T200) instrument. Biotin-labeled antigen CD16 was loaded onto the streptavidin sensor, blocking unreacted groups. The control channel was directly blocked after activation, without protein coupling. The test antibodies were prepared into a series of solutions at concentrations of 125, 250, 500, and 1000 nM using running buffer. The "binding-dissociation-regeneration-equilibrium" process was repeated sequentially from low to high concentration. Affinity was fitted after double subtraction correction of the data using BIAEvaluation 4.1.

[0079] Figure 7 The affinity of nanobodies A1 and A2 for antigen CD16 was determined using SPR assay. The SPR assay results are shown in Table 7.

[0080] Table 7. Affinity details of CD16 nanobodies

[0081] The results showed that the KD values ​​for A1 and A2 were 4.20E-8 and 1.50E-8, respectively. The koff, kon, and KD values ​​were consistent across all concentrations, indicating good experimental stability. 2 The values ​​are generally high (>0.95), indicating that the fitting curves agree well with the experimental data. Relatively speaking, nanobody A2 has a higher affinity than nanobody A1.

[0082] Example 4: Thermal stability experiment of CD16 nanobody CD16 protein was coated onto ELISA plates at a concentration of 1 μg / mL (100 μL per well), and the plates were incubated overnight at 4°C. After washing three times with PBST, 300 μL of 5% skim milk was added to each well, and the plates were blocked at 37°C for 1 hour. The nanobody of this invention and a commercially available CD16 monoclonal antibody (CD16-mAb, Cell Signaling) were added to each well after being treated at different temperatures (4°C, 37°C, 60°C, 70°C, 80°C, 90°C) for 2 hours. 100 μL of each nanobody was added to each well, and the plates were incubated at room temperature for 1 hour, followed by washing three times with PBST.

[0083] For nanobody groups A1 and A2, because the nanobodies have His tags, HRP-labeled His-mAb (Cell Signaling) was added to each well, incubated at room temperature for 40 minutes, washed 3 times with PBST, and then TMB was added for color development for 10 minutes. After terminating the reaction with 2M sulfuric acid, the UV absorbance (OD450 value) at 450 nm was measured using a microplate reader.

[0084] The CD16mAb group was incubated with HRP-labeled anti-rabbit IgG secondary antibody (Cell Signaling) for 30 minutes at room temperature. After washing the plate three times with PBST, TMB was added for color development for 10 minutes. The reaction was terminated with 2M sulfuric acid, and the UV absorbance (OD450 value) at 450 nm was measured using an ELISA reader.

[0085] The results are as follows Figure 8 As shown, the results indicate that the nanobody structure of this application is stable, and nanobodies A1 and A2 still exhibit CD16 antigen binding activity after treatment at 80°C for 2 hours. This demonstrates that compared to CD16-mAb, the nanobody structure of this invention is more stable and has better heat resistance.

[0086] Example 5: Construction of IgG-like asymmetric trispecific nanobody GN-C6-A2 containing A2 This embodiment constructs a trispecific antibody with a "2+1" asymmetric structure, where "2" represents two different target sites (GPC3 and LAG3) at the N-terminus of the two polypeptide chains, and "1" represents the same target site (CD16) carried by both polypeptide chains at the C-terminus. This target site exists in a bivalent form, specifically: A trispecific nanobody was constructed using nanobodies GN (GPC3), C6 (LAG3), and A2 (CD16). The nanobodies targeting two sites at the N-terminus—GN (GPC3) and C6 (LAG3)—are located on two different polypeptide chains. The nanobodies targeting one site at the C-terminus—A2 (CD16)—are fused to the C-terminus of both Fc regions, achieving bivalent binding of CD16. This asymmetric distribution of "dual N-terminal targets + common C-terminal target" allows a single molecule to simultaneously recognize three different targets, and the bivalent CD16 target enhances NK cell recruitment and efficiently activates NK cells.

[0087] (1) Peptide chain design First polypeptide chain: GN (SEQ ID NO.19) - (G4S)3 linker peptide (SEQ ID NO.24) - First Fc region (containing a mutation, SEQ ID NO.25) - (G4S)3 linker peptide (SEQ ID NO.24) - A2 (SEQ ID NO.16) Second polypeptide chain: C6 (SEQ ID NO.20) - (G4S)3 linker peptide (SEQ ID NO.24) - second Fc region (containing cavity mutation and C220A mutation, SEQ ID NO.27) - (G4S)3 linker peptide (SEQ ID NO.24) - A2 (SEQ ID NO.16) (2) Construction of expression carrier Synthesizing step 1 (1) The coding gene sequences of the two polypeptide chains were obtained, and NotI and XbaI restriction sites were designed at both ends respectively. The synthesized genes were cloned into the pcDNA3.4 eukaryotic expression vector and transformed into Escherichia coli DH5α competent cells. Positive clones were selected, amplified, and then the plasmids were extracted for DNA sequencing identification. The recombinant plasmids were named pcDNA3.4-GN-Fc-A2 and pcDNA3.4-C6-Fc-A2 respectively.

[0088] (3) Cell culture and transfection HEK293T cells were revived and passaged three times under the following conditions: 120 rpm, 8% CO2, 37°C, with a cell density maintained at 0.3 × 10⁻⁶ cells / year. 6 Plasmid transfection was performed when the cell density reached 70% (cells / ml). Add 150 μL of Lipofectamine™ 3000 to 2.5 ml of Opti-MEM medium and name it Solution 1; add 100 μg of plasmid pcDNA3.4-GN-Fc-A2 and 100 μg of plasmid pcDNA3.4-C6-Fc-A2, and 200 μL of P3000 to 2.5 ml of Opti-MEM medium and name it Solution 2; Add solution 2 to solution 1, mix well, incubate at 37°C for 15 minutes, then add the mixed transfection solution dropwise to the cell culture medium while shaking. Place on a shaker at 37°C, 120 rpm, and 8% O2 for 5-7 days. Collect the cell culture medium supernatant and centrifuge (15,000 g, 20-30 minutes) to remove cell debris and particulate matter.

[0089] (4) Protein purification Equilibrate the Protein A column with 1×PBS phosphate buffer (pH 7.2–7.4). Wash the column with at least 20 column volumes of 1×PBS buffer at a flow rate of 1 mL / min until the UV baseline is completely stable. Replace the PBS with the sample to be purified and load the sample at a flow rate of 1 mL / min for 4 min. Then elute contaminating proteins with 1×PBS at a flow rate of 1 mL / min for at least 10 column volumes. Elute the column with sodium acetate buffer (pH 3.4) at a flow rate of 1 mL / min for 5 min. Immediately after elution, collect small aliquots of the collected solution using centrifuge tubes. Measure the absorbance of each aliquot at 280 nm using NanoDrop. Combine the high-concentration protein solutions and place them into pretreated dialysis bags, then seal. Immerse the dialysis bags in a large volume of dialysis buffer (120 mM NaAc-HAc + 70 mM Arginine, pH 5.5). Dialyze at 4°C, changing the buffer solution every 3-4 hours for a total of 3-4 times. After dialysis, remove the protein solution, aliquot it, and store it at -80°C.

[0090] (5) Protein detection The protein concentration obtained by step (4) using NanoDrop assay reached 3.90 mg / mL, and the protein expression level reached 95.30 mg / L. SDS-PAGE was used to detect the expression level. Figure 9 It can be seen that the molecular weight of the protein in the reduced state is 81 / 80 kDa, and the molecular weight of the protein in the non-reduced state is 161 kDa, which is consistent with the expected structure.

[0091] The purity of the fusion protein was determined by SEC-HPLC using a high-performance liquid chromatography (HPLC) system and a gel chromatography column. Experimental conditions: TSKgel G3000SWxl column, 100 mM PB (pH 7.0) mobile phase, flow rate 1 ml / min, injection volume 20 μl (concentration ≤ 4 mg / ml) or 50 μg (concentration > 4 mg / ml), column temperature 35℃, detection wavelengths 214 nm and 280 nm, and acquisition time 15 min. Detection results (…) Figure 10 The study found that the main peak accounted for 100%, the trispecific nanobody had high purity, and it mainly existed in the form of correctly folded antibodies.

[0092] The binding performance of the trispecific nanobody GN-C6-A2 to the target antigen was detected using SPR, and the results are shown in Table 8. The binding performance of the trispecific nanobody to the target antigen was detected using ELISA, and the results are shown in Table 8. Figure 11 show.

[0093] Table 8. Binding of the trispecific nanobody GN-C6-A2 to various targets.

[0094] The results show that GN-C6-A2 specifically binds to GPC3, CD16, and LAG3 antigens, with EC50 values ​​of 0.01678, 0.008873, and 0.006819 ug / mL, respectively. This trispecific antibody achieves therapeutic binding to all three targets, and all binding domains maintain their original affinity. This demonstrates that the "2+1" asymmetric structural design and the introduction of mutations in each Fc region do not affect the functional activity of each antigen-binding domain, while simultaneously improving the uniformity of the fusion protein. This invention successfully constructs an asymmetric trispecific antibody with the CD16 nanobody A2 as its core. This antibody not only maintains the affinity of each binding domain but also exhibits high expression levels and high purity, providing theoretical and technical references for the large-scale production, in vivo diagnostics, and targeted therapy applications of nanobody fusion proteins.

Claims

1. An anti-CD16 nanobody, characterized in that: The nanobody specifically binds to CD16 and contains a complementarity-determining region selected from the group consisting of: (a) CDR1 as shown in SEQ ID NO.1, CDR2 as shown in SEQ ID NO.2, and CDR3 as shown in SEQ ID NO.3; or (b) CDR1 as shown in SEQ ID NO.8, CDR2 as shown in SEQ ID NO.9, and CDR3 as shown in SEQ ID NO.10; Or a variant thereof, wherein the variant has at least 90% sequence identity with the above-described complementarity-determining region sequence and retains binding activity with CD16.

2. The anti-CD16 nanobody as described in claim 1, characterized in that: The backbone region sequence of the nanobody is selected from: (a) FR1, FR2, FR3, FR4 as shown in SEQ ID NO. 4, 5, 6, 7; or (b) FR1, FR2, FR3, and FR4 as shown in SEQ ID NO. 11, 12, 13, and 14; Or a variant thereof, wherein the variant has at least 90% sequence identity with the above-described FR sequence.

3. The anti-CD16 nanobody as described in claim 2, characterized in that: The nanobody comprises, (a) an amino acid sequence as shown in SEQ ID NO.15, A1; or (b) The amino acid sequence is A2 as shown in SEQ ID NO.16; Or its functionally active fragments or derivatives.

4. A nucleic acid molecule, characterized in that: The nanobody encoding claim 3 comprises a nucleotide sequence as shown in SEQ ID NO. 17 or SEQ ID NO.

18.

5. A method for preparing an anti-CD16 nanobody, characterized in that: Culture host cells, isolate and purify the culture to obtain anti-CD16 nanoantibody; The host cell contains an expression vector of the nucleic acid molecule of claim 4 or a functional variant thereof, wherein the functional variant contains a nucleotide sequence having at least 95% sequence identity with SEQ ID NO. 17 or SEQ ID NO.

18.

6. A multispecific antibody, characterized in that: The multispecific antibody is a trispecific antibody, comprising, (a) A first polypeptide chain comprising a first antigen-binding domain specifically binding to a first target, a first dimerizing domain and a third antigen-binding domain specifically binding to a third target; (b) A second polypeptide chain comprising a second antigen-binding domain that specifically binds to a second target, a second dimerizing domain, and a third antigen-binding domain that specifically binds to a third target; Wherein, at least one of the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain is a nanobody according to any one of claims 1-3, and the first dimerizing domain and the second dimerizing domain combine with each other to form a dimer.

7. The multispecific antibody as described in claim 6, characterized in that: The first antigen-binding domain is a nanobody that specifically binds to GPC3 and contains the amino acid sequence shown in SEQ ID NO.19; The second antigen-binding domain is a nanobody that specifically binds to LAG3 and contains the amino acid sequence shown in SEQ ID NO.20; The third antigen-binding domain is a CD16-specific nanobody selected from the nanobody described in claim 3.

8. The multispecific antibody as described in claim 7, characterized in that: The first polypeptide chain further includes a first linker peptide located between a first antigen-binding domain and a first dimerizing domain, and a second linker peptide located between a first dimerizing domain and a third antigen-binding domain; The second polypeptide chain further includes a third linker peptide located between the second antigen-binding domain and the second dimerization domain, and a fourth linker peptide located between the second dimerization domain and the third antigen-binding domain. The first dimerization domain and the second dimerization domain are immunoglobulin Fc regions or their functionally active fragments; the immunoglobulin Fc region contains modifications that promote heterodimer formation; the modifications are selected from spatial complementarity modifications, charge complementarity modifications, chain exchange modifications, or combinations thereof. The first, second, third, and fourth linker peptides are independently selected from (G4S). n , where n is an integer independently selected from 1 to 5.

9. The use of the nanobody according to any one of claims 1-3 or the multispecific antibody according to any one of claims 6-8 in the preparation of a medicament for treating tumors.

10. A fusion protein, characterized in that: The nanobody comprises the nanobody according to any one of claims 1-3 or the multispecific antibody according to any one of claims 6-8 and the heterologous protein domain, wherein the heterologous protein domain is selected from Fc domain, toxic protein, cytokine or fluorescent protein.

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