An antibody targeting cd33 and nk cell engagers in bispecific and trispecific antibody formats

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to an antibody targeting CD33 and an NK cell linker in the form of a bispecific and trispecific antibody of NKG2A. In one aspect, a CD33 monoclonal antibody with stronger binding capacity is designed through sequence optimization. In another aspect, a multifunctional antibody is designed by using the sequence of the CD33 monoclonal antibody. For example, a trispecific antibody that can simultaneously target the NK cell surface activating receptor CD16a and NKG2A and tumor cell CD33. The multifunctional antibody can be used as an NK cell linker, has a wider target coverage and stronger mediation of NK killing of tumor cells, and has a wide application prospect in the preparation of drugs for preventing and / or treating tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antibody targeting CD33 and NK cell connectors in the form of bispecific and trispecific antibodies against NKG2A. Background Technology

[0002] Malignant tumors are a major public health problem worldwide. According to data from the Global Burden of Disease (GBD) study, malignant tumors not only cause extremely high morbidity and mortality rates, but also place a heavy burden on patients' families and social healthcare resources due to their high recurrence rates, easy metastasis, and exorbitant treatment costs. With the increasing aging of the global population, changing lifestyles, and increased environmental exposure, the global cancer burden continues to rise, driving a rigid increase in demand for anti-tumor drugs and making malignant tumors one of the core research directions in the current life sciences and pharmaceutical R&D fields.

[0003] Taking acute myeloid leukemia (AML) as an example, AML is characterized by the malignant clonal expansion of myeloid progenitor cells in the bone marrow and peripheral blood. Approximately 50% of cases relapse after chemotherapy, resulting in a poor prognosis and a clear unmet clinical need. The impaired self-renewal and differentiation of leukemia stem cells (LSCs) is a core mechanism in the occurrence and maintenance of AML. AML immunotherapy targets mainly include CD33, CD123 (IL-3 receptor α chain), CLEC12A (CLL-1), and IL1RAP. Among these, CD33 (Siglec-3) is highly expressed on the surface of AML progenitor cells, but lowly expressed or not expressed on the surface of normal hematopoietic stem cells. This differential expression characteristic makes it one of the most valuable targets for AML immunotherapy. Developing new antibodies with better binding affinity to CD33 is an important research topic in this field.

[0004] CD16a (FcγRIIIa) is a low-affinity IgG Fc receptor expressed on the surface of NK cells and is a core effector molecule mediating NK cell antibody-dependent cytotoxicity (ADCC). However, approximately one-third of patients' blast cells express a high-affinity IgG Fc receptor, CD64 (FcγRI), which can cis-capture antibody Fc fragments and competitively block the trans-binding of CD16a on the NK cell surface, leading to ADCC resistance and significantly limiting the efficacy of traditional antibodies and T-cell adaptors.

[0005] NK cell connectors (NKCEs), as multispecific antibody constructs, mediate immune synapse formation and trigger targeted NK cell killing by simultaneously targeting tumor-associated antigens and NK cell activation receptors (such as CD16a and NKp46). NKG2A (KLRC1) is an important immune checkpoint molecule expressed on the surface of NK cells and effector cell subsets such as activated CD8⁺ T cells. Its ligand is the non-classical MHC-I molecule HLA-E. Blocking antibodies targeting NKG2A can remove the inhibitory regulation of this immune checkpoint on effector cells. Whether used alone or in combination with other immune checkpoint inhibitors, they can significantly enhance the tumor-killing function of CD8⁺ T cells and NK cells. Trifunctional NKCEs (such as SAR443579 targeting CD123×NKp46×CD16a) bypass CD64-mediated ADCC inhibition through NKp46 co-activation mechanism, conditionally inducing NK cell activation and cytokine secretion only upon contact with target cells. They exhibit superior antitumor activity, prolonged pharmacodynamic response, and significantly reduced risk of cytokine release syndrome compared to traditional ADCC-enhancing antibodies, providing a safe and effective new immunotherapy strategy for relapsed / refractory AML.

[0006] It is evident that developing new specific antibodies and constructing more multispecific NKCEs with different forms and design ideas by combining multiple targets mentioned above is an important research topic for breaking through the technical bottlenecks of existing AML immunotherapy and improving the clinical prognosis of patients. It has broad clinical application prospects and significant social value. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides an antibody targeting CD33 and NK cell connectors in the form of bispecific and trispecific antibodies against NKG2A.

[0008] An antibody targeting CD33, the amino acid sequence of which is shown in SEQ ID NO.5.

[0009] Preferably, the nucleotide sequence of the encoding gene of the antibody targeting CD33 is shown in SEQ ID NO. 12.

[0010] The present invention also provides a multifunctional fusion protein comprising: an antibody or fragment thereof targeting NK cells, and the aforementioned antibody or fragment thereof targeting CD33.

[0011] Preferably, the antibody targeting NK cells is selected from at least one of the following antibodies: Antibodies that specifically bind to the inhibitory receptor NKG2A on the surface of NK cells. An antibody that specifically binds to the activating receptor CD16a on the surface of NK cells.

[0012] Preferably, the multifunctional fusion protein is a dimer composed of polypeptide chain 1 and polypeptide chain 2; the structure of polypeptide chain 1 includes a first antigen-binding region, a linker peptide 1, an IL-15 functional region, a Knob-mutated Fc, a linker peptide 2, and a third antigen-binding region connected in sequence; the structure of polypeptide chain 2 includes a second antigen-binding region, a linker peptide 3, an IL-15 functional region, a Hole-mutated Fc, a linker peptide 4, and a fourth antigen-binding region connected in sequence. The first antigen-binding region and the second antigen-binding region are respectively antibodies or fragments thereof targeting NK cells; the third antigen-binding region and the fourth antigen-binding region are respectively antibodies or fragments thereof targeting CD33.

[0013] Preferably, the amino acid sequences of polypeptide chain 1 and polypeptide chain 2 are as shown in SEQ ID NO. 8 and SEQ ID NO. 9, respectively, or the amino acid sequences of polypeptide chain 1 and polypeptide chain 2 are as shown in SEQ ID NO. 8 and SEQ ID NO. 10, respectively, or the amino acid sequences of polypeptide chain 1 and polypeptide chain 2 are as shown in SEQ ID NO. 9 and SEQ ID NO. 11, respectively.

[0014] The present invention also provides a gene fragment encoding the above-mentioned antibody, or encoding the above-mentioned multifunctional fusion protein.

[0015] Preferably, its nucleotide sequence includes at least one of the sequences shown in SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, and SEQ ID NO. 16.

[0016] The present invention also provides a recombinant vector comprising the above-mentioned gene fragment.

[0017] The present invention also provides a host cell comprising the above-described recombinant vector.

[0018] The present invention also provides the use of the above-mentioned multifunctional fusion protein, gene fragment, recombinant vector, or host cell in the preparation of multispecific antibodies and NK cell connectors.

[0019] Preferably, the multispecific antibody and NK cell connector are used to treat acute myeloid leukemia.

[0020] The present invention also provides an NK cell connector comprising the above-described multifunctional fusion protein.

[0021] The present invention also provides an engineered immune cell comprising the above-mentioned multifunctional fusion protein.

[0022] The technical solution of the present invention achieves the following beneficial technical effects by optimizing the structural features of the fusion protein, such as the structure, functional region sequence, functional region connection order, and connection sequence: (1) The present invention first optimizes the sequence of CD33 antibody. The preferred CD33 antibody has a stronger binding ability to the CD33 target, thus having a better effect on the ability of NK to kill tumor cells.

[0023] (2) This invention constructs a class of multispecific (bispecific, trispecific) NKCEs, which incorporate NKG2A blocking antibodies into NKCEs to design NKCEs targeting NKG2A and tumor cell targets, or further incorporate CD16a targets to design trispecific NKCEs simultaneously targeting CD16a, NKG2A, and tumor cell antigens. The NKCEs provided by this invention can more effectively mediate NK cell killing of tumor cells, and are expected to overcome the technical bottlenecks of existing AML immunotherapy, improving the clinical prognosis of patients. Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0024] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0025] Figure 1 Here are structural diagrams of Y24, Y25, and Y26; Figure 2 SDS-PAGE identification results for CD33 monoclonal antibody, Y24, Y25 and Y26; Figure 3 The results show the ability of the CD33 monoclonal antibody to bind to MV-4-11. Figure 4 Flow cytometry plot of CD33 monoclonal antibody-mediated NK cell killing of tumor cells; Figure 5 Flow cytometry and statistical values ​​of Y24, Y25 and Y26-mediated NK cell killing of tumor cells; Figure 6 Differences in NK cell number and functional indicators after the addition of Y24, Y25 and Y26 cells; Figure 7 Differences in antibody-related targets on the surface of NK cells after the addition of Y24, Y25 and Y26; Figure 8 Differences in NK cell activation markers after the addition of Y24, Y25, and Y26. Detailed Implementation

[0026] Unless otherwise specified, all reagents and materials used in the following examples and experimental cases are commercially available.

[0027] In the following embodiments of the present invention, a natural killer cell binder (NKCE) in the form of a bispecific antibody that specifically binds to the NK cell surface inhibitory receptor NKG2A and tumor cell CD33, or a trispecific antibody in the form of an NKCE that simultaneously targets the NK cell surface activating receptor CD16a and NKG2A and tumor cell CD33, is provided. The NKCE comprises an antibody or fragment thereof that specifically binds to the NK cell surface activating receptor CD16a, an antibody or fragment thereof that specifically binds to the NK cell surface inhibitory receptor NKG2A, and an antibody or fragment thereof that specifically binds to CD33. It also comprises an IL-15 NK activation domain operatively linked to the NK binding domain. Related bispecific and trispecific antibodies, as well as related products and applications, are provided.

[0028] This invention relates to single-domain antibody (i.e., VHH antibody) sequences that can specifically bind to the NK cell surface activating receptor CD16a. In this invention, the CD16a sequence can be selected from CD16a single-domain antibodies or their antigen-binding fragments derived from the prior art. For example, fragments or domains of CD16a antibodies containing WO2023 / 129819. Single-domain antibodies that can specifically bind to the NK cell surface activating receptor CD16a can contain or consist of a full-length anti-CD16a antibody or its antigen-binding fragment, as long as they can specifically bind to CD16a, including but not limited to: full-length antibodies that specifically bind to CD16a, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibodies, VHH, or heavy-chain antibodies, etc.

[0029] In the NKCE of this invention, the NK activation domain may include IL-15 or a functional fragment thereof. The NK activation domain includes the amino acid sequence of SEQ ID NO:2 or a functional variant thereof.

[0030] The VHH antibody or its antigen-binding fragment that specifically binds to the inhibitory receptor NKG2A on the surface of NK cells, as described in this invention, can have its amino acid sequence realized based on existing technology, such as the sequence disclosed in patent CN121574251A.

[0031] The VHH antibody or its antigen-binding fragment for CD33, a therapeutic target related to leukemia tumor cells, involved in this patent, has the amino acid and nucleic acid sequences as described in SEQ ID NO:3.

[0032] The VHH monoclonal antibody of this invention can be engineered, for example, to further improve the properties of engineered antibodies (e.g., affinity). It can also be a humanized antibody obtained after antibody humanization, or a functional variant and affinity variant obtained through other optimization schemes such as random or site-directed mutagenesis, or an antibody whose stability has been engineered (e.g., by mutating asparagine in the antibody to eliminate deamidation).

[0033] In some embodiments, the present invention covers multispecific molecules such as monoclonal antibodies, bispecific antibodies, trispecific antibodies, and multispecific antibodies, including NKG2A VHH antibodies and CD33 VHH antibodies or fragments thereof.

[0034] Therefore, in some embodiments, the present invention relates to a trispecific antibody comprising a first antigen-binding region, a second antigen-binding region, and a third antigen-binding region (fourth antigen-binding region). The first antigen-binding region specifically binds to NKG2A. The second antigen-binding region comprises a CD16a antibody or its antigen-binding fragment or domain. The first antigen-binding region of the bispecific / trispecific antibody suitable for the present invention may comprise, or be composed of, a full-length antibody against CD16a or its antigen-binding fragment, as long as it can specifically bind to CD16a, including but not limited to, for example, a full-length antibody specifically binding to CD16a, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibody, VHH, or heavy-chain antibody. The third antigen-binding region (fourth antigen-binding region) specifically binds to tumor-associated antigens or immune checkpoint molecules, specifically, in an embodiment, the CD33 target associated with AML. Preferably, the structure of the trispecific antibody is as follows: Figure 1 As shown in A (Y24).

[0035] In some embodiments, the present invention relates to a bispecific antibody comprising a first antigen-binding region and a second antigen-binding region. The first antigen-binding region comprises a CD16a antibody or its antigen-binding fragment or domain. The first antigen-binding region of the bispecific / trispecific antibody suitable for the present invention may comprise, or be composed of, a full-length antibody against CD16a or its antigen-binding fragment, as long as it can specifically bind to CD16a, including but not limited to, for example, a full-length antibody specifically binding to CD16a, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibody, VHH, or heavy chain antibody. The second antigen-binding region specifically binds to tumor-associated antigens or immune checkpoint molecules, specifically the CD33 target associated with AML in a specific embodiment. Preferably, the structure of the bispecific antibody is as follows: Figure 1 As shown in B (Y25).

[0036] In some embodiments, the present invention relates to a bispecific antibody comprising a first antigen-binding region and a second antigen-binding region. The first antigen-binding region specifically binds to NKG2A. The second antigen-binding region specifically binds to tumor-associated antigens or immune checkpoint molecules, specifically, in an embodiment, to the CD33 target associated with AML. Preferably, the structure of the bispecific antibody is as follows: Figure 1 As shown in C(Y26).

[0037] In the bispecific and trispecific antibodies according to the present invention, the linker peptide is a peptide of about 3 to about 20 amino acids in length. Common linker peptides in the art include KESGSVSSEQLAQFRSLD (SEQ ID NO. 1), EGKSSGSGSESKST, SGGGSGGGGSGGGGSGGTLQ (SEQ ID NO. 2), (GGS)n, (GGGGS)n, etc. Preferably, the linker comprises (GGGGS). n The amino acid sequence, n = 1, 2, 3, 4, or 5, preferably n = 3.

[0038] In the bispecific antibody of the present invention, the heavy chain constant region C1 and Fc region are derived from the same IgG, such as IgG1, IgG2, IgG3, or IgG4, preferably both derived from IgG1 or IgG4. The Fc region fragment suitable for the antibody molecule of the present invention can be any antibody Fc region. The Fc region can include a native sequence Fc region and a variant Fc region. The native sequence Fc domain encompasses various naturally occurring immunoglobulin Fc sequences, such as various Ig isotypes and their allotypes. For example, the Fc region of the antibody of the present invention can contain two or three constant domains, namely a CH2 domain, a CH3 domain, and optionally a CH4 domain. In some embodiments, the antibody Fc region may also have an IgG hinge region or a partial IgG hinge region at the N-terminus, for example, an IgG1 hinge region or a partial IgG1 hinge region. Mutations may be contained in the hinge region. The Fc region of the present invention can be mutated to obtain the desired properties. Mutations of Fc regions are known in the art. Preferably, based on the knot-in-Hole technique, corresponding knot mutations and hop mutations are introduced into the first monomer Fc region and the second monomer Fc region, respectively.

[0039] In the following embodiments, the amino acid sequences involved include: Table 1 Amino acid sequence In the following embodiments, the corresponding gene coding sequences involved include: Table 2. Encoding gene sequences As will be apparent to those skilled in the art, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences. The nucleic acid sequence encoding the molecule of the present invention can be generated using methods well known in the art, such as de novo solid-phase DNA synthesis or by PCR amplification.

[0040] To facilitate production and purification, antibodies can be fused to the N-terminus with a secretory signal peptide and / or a tagged peptide that facilitates purification, such as a hexahistine tag, biotin label, or hFc label.

[0041] In another aspect, the present invention provides nucleic acids encoding any of the above-mentioned bispecific and trispecific antibodies. The nucleic acids encoding the heavy and light chains of the bispecific antibody can be expressed in the same vector or in different vectors, or in the same or different host cells. Therefore, the method for producing the bispecific and trispecific antibodies of the present invention includes the steps of: culturing host cells containing nucleic acids encoding the heavy and light chains under conditions suitable for expressing the heavy and light chains of the molecule, thereby producing the bispecific and trispecific antibodies of the present invention.

[0042] The present invention also includes at least one selected from the group consisting of pharmaceutically acceptable excipients and adjuvants.

[0043] The delivery carrier includes the NKCE. The delivery carrier is a lipid nanoparticle.

[0044] Example 1: Antibody targeting CD33, fusion protein and its preparation method I. Antibodies targeting CD33 This embodiment provides a novel antibody targeting CD33 (CD33 monoclonal antibody), the amino acid sequence of which is shown in SEQ ID NO.5.

[0045] II. Fusion Protein This embodiment provides fusion proteins Y24, Y25, and Y26, whose structures are as follows: Figure 1 As shown.

[0046] The structures of Y24, Y25, and Y26 are dimers composed of polypeptide chain 1 and polypeptide chain 2. The structure of polypeptide chain 1 includes a first antigen-binding region, linker peptide 1, IL-15 functional region, Knob-mutated Fc, linker peptide 2, and third antigen-binding region connected in sequence. The structure of polypeptide chain 2 includes a second antigen-binding region, linker peptide 3, IL-15 functional region, Hole-mutated Fc, linker peptide 4, and fourth antigen-binding region connected in sequence.

[0047] Y24 is a trifunctional fusion protein that specifically binds to the NK cell surface activating receptor CD16a, the NK cell surface inhibitory receptor NKG2A, and the tumor cell CD33 target. The amino acid sequences of its polypeptide chain 1 and polypeptide chain 2 are shown in SEQ ID NO. 8 and SEQ ID NO. 9, respectively. Specifically, the first antigen-binding region of polypeptide chain 1 is an antibody that specifically binds to the NK cell surface activating receptor CD16a (CD16a VHH), denoted as NK1, and the third antigen-binding region is an antibody targeting CD33 (CD33 VHH), denoted as TAA1. The second antigen-binding region of polypeptide chain 2 is an antibody that specifically binds to the NK cell surface inhibitory receptor NKG2A (NKG2A VHH), denoted as 2A2, and the fourth antigen-binding region is an antibody targeting CD33 (CD33VHH), denoted as TAA1.

[0048] Y25 is a bifunctional fusion protein that specifically binds to the NK cell surface activating receptor CD16a and the tumor cell CD33 target. The amino acid sequences of its polypeptide chain 1 and polypeptide chain 2 are shown in SEQ ID NO. 8 and SEQ ID NO. 10, respectively. Specifically, the first antigen-binding region of polypeptide chain 1 is CD16a VHH (NK1), and the third antigen-binding region is CD33 VHH (TAA1). The second antigen-binding region of polypeptide chain 2 is CD16a VHH (NK1), and the fourth antigen-binding region is CD33 VHH (TAA1).

[0049] Y26 is a bifunctional fusion protein that specifically binds to the NK cell surface activating receptor CD16a and the tumor cell CD33 target. The amino acid sequences of its polypeptide chain 1 and polypeptide chain 2 are shown in SEQ ID NO. 9 and SEQ ID NO. 11, respectively. Specifically, the first antigen-binding region of polypeptide chain 1 is NKG2A VHH (2A2), and the third antigen-binding region is CD33 VHH (TAA1). The second antigen-binding region of polypeptide chain 2 is NKG2A VHH (2A2), and the fourth antigen-binding region is CD33 VHH (TAA1).

[0050] III. Preparation method of CD33 monoclonal antibody 1. Constructing an expression carrier (1) Restriction digestion of the backbone vector: The pcDNA3.1-x-IgG1 tool vector was used in this experiment and double digested with BamHI / EcoRI restriction endonuclease from Yisheng Biotechnology. The digestion system was placed at 37℃ for 5 h. After digestion, the digested product was purified by PCR using the OMEGA Cycle-Pure Kit PCR product purification kit (catalog number D6492-01) to obtain the purified linear backbone vector.

[0051] (2) Homologous recombination: The nucleotide fragments used for recombinant expression were diluted 20-fold with sterile double-distilled water (ddH2O). 1 μL of the diluted nucleotide sample was accurately pipetted and mixed with the linear vector recovered by enzyme digestion. The homologous recombination reaction system was constructed using the nearshore protein NovoRec Plus one-step PCR Cloning Kit recombinase (catalog number NR005-01B). The prepared reaction system was placed in a PCR instrument and incubated at 50℃ for 30 min.

[0052] After the reaction, 100 μL of TOP10 competent cells were added to each tube of homologous recombinant product, gently mixed, and then transformed. The cells were first incubated on ice for 20 min, followed by heat shock at 42°C for 90 s, and immediately transferred to ice for 2 min. Then, 800 μL of LB liquid medium was added, and the cells were cultured at 37°C and 220 rpm for 25-45 min with shaking. After culture, the bacterial culture was spread onto solid LB agar plates containing ampicillin (Amp) resistance and incubated overnight at a constant temperature. The recombinant expression nucleotide fragment sequence used in this experiment corresponds to SEQ ID NO. 12.

[0053] (3) Escherichia coli culture PCR identification: Single Escherichia coli colonies were picked from overnight cultured antibiotic plates and inoculated into 200 μL of LB liquid medium. The culture was then incubated at 37°C and 220 rpm for 3 h with shaking. After the culture was completed, 1 μL of fresh culture was used as a template for PCR amplification and preliminary identification by culture PCR was performed. The PCR products of the positive clones obtained by screening were recovered using the OMEGAGel Extraction Kit (catalog number D2500-01). Homologous recombination was then performed again, and the culture PCR identification process was repeated. Finally, the positive clones obtained by screening were sent to a professional institution for sequencing verification.

[0054] 2. Expression and purification of Hek293F nanobody in cells (1) Antibody expression The positive bacterial strains that were verified by sequencing were inoculated into 20 mL of LB liquid medium containing ampicillin and incubated overnight at 37°C with shaking for activation. The next day, the recombinant expression plasmid was extracted using the Plasmid Miniprep Kit II plasmid extraction kit (catalog number BW-PD1213) from Bevo Medical.

[0055] Hek293F cells with excellent growth and a viability higher than 95% were selected for transfection experiments. The cell density was adjusted to 3 × 10⁶ cells / mL before transfection. Separate transfection systems were prepared: 50 μg of recombinant expression plasmid was added to 1 mL of OPM medium and mixed thoroughly; simultaneously, 150 μg of PEI transfection reagent was added to 1 mL of OPM medium and mixed thoroughly. Both systems were incubated at room temperature for 30 min, then mixed thoroughly and added to 50 mL of Hek293F cell culture medium. The cells were then cultured in a CO₂ incubator using standard methods. The following day, 5% OPM medium was added to the system, and culture continued until day 7. After culture, the cells were centrifuged at 10,000 rpm for 20 min, and the cell culture supernatant was collected for subsequent protein purification experiments.

[0056] (2) Antibody purification This experiment used a TH-protein A affinity chromatography column (5 mL column volume, catalog number Y5001) from Chutian Microsphere Biotechnology (Changsha) Co., Ltd. for the purification of the target antibody. First, column equilibration pretreatment was performed: the column was washed sequentially with 1 column volume of ultrapure water, 1 column volume of 0.1 M NaOH solution, and 3 column volumes of PBS buffer to complete column activation and equilibration.

[0057] All the pretreated cell culture supernatant was loaded into a Protein A chromatography column. After loading, the column was washed with 3 column volumes of PBS buffer to remove extraneous proteins and impurities. The target protein was then eluted with 800 μL of 0.1 M Gly-HCl buffer, and the elution was repeated twice. The elution product was collected, which was the target CD33 nanobody.

[0058] Protein concentration was determined using a micro-spectrophotometer by measuring the absorbance at A280, and the purity of the target protein was ensured to be ≥95% in conjunction with purity testing results. The amino acid sequence of the CD33 target protein prepared in this experiment corresponds to SEQ ID NO. 5.

[0059] 3. Identification of CD33 nanobodies by polyacrylamide gel electrophoresis (SDS-PAGE) (1) In this experiment, the 12% acrylamide rapid gel kit was used to prepare the electrophoresis gel. The specific formula of a single gel is as follows: the separating gel system contains 2.5 mL separating gel A solution, 2.5 mL separating gel B solution, 25 μL 10% ammonium persulfate, and 2.5 μL TEMED; the stacking gel system contains 1 mL stacking gel A solution, 1 mL stacking gel B solution, 10 μL 10% ammonium persulfate, and 2 μL TEMED. The gel was poured according to the standard procedure and allowed to stand and solidify for later use.

[0060] (2) Sample preparation: Accurately pipette 5 μg of the protein solution to be tested into a 1.5 mL sterile centrifuge tube, add 5× Loading buffer (reduced or non-reduced) according to the standard ratio, and shake thoroughly to mix. Place the mixed sample in a 100℃ metal bath for 5 min to denature, and cool before loading.

[0061] (3) Sample loading: Add the processed protein samples sequentially into the gel loading wells, set the constant voltage condition to 120V, and continue electrophoresis for 1 hour. (4) Staining: After electrophoresis, carefully remove the gel and place it in a culture dish containing CBB staining solution. Place it on a shaker and shake at a constant speed for 10 minutes to ensure that the gel is fully stained.

[0062] (5) Decolorization: After staining, discard the staining solution, add sufficient decolorization solution, put the culture dish back on the shaker and continue to shake to decolorize, and repeatedly change the decolorization solution until the gel background is completely transparent and the bands are clearly visible.

[0063] (6) After decolorization, the gel was imaged and collected using a Bio-Rad gel imaging system to observe and analyze the protein electrophoresis bands and complete the identification of antibody purity and integrity.

[0064] IV. Preparation methods of three NKCE (Y24, Y25, Y26) fusion proteins 1. Plasmid preparation This experiment used the pcDNA3.4 eukaryotic high-efficiency expression vector to construct the antibody recombinant plasmid. The specific reproducible construction procedure is as follows: 1) Target gene sequence design and synthesis: Based on the nucleic acid sequence of the target antibody mature protein coding region, redundant signal peptide sequences are removed, mammalian cell preferred codons are optimized, and the complete heavy and light chain functional coding nucleic acid sequences are preserved throughout the process, without base truncation or mutation. The entire gene is synthesized by a professional biotechnology company. 2) Vector linearization and homologous recombination: The pcDNA3.4 vector was linearized by single enzyme digestion with restriction endonucleases, and the linear vector backbone was recovered and purified by agarose gel electrophoresis. The recombination reaction system was prepared according to the instructions of the commercial homologous recombination kit: the molar ratio of linear vector fragment to target gene fragment was 1:3, recombinase and buffer were added, and the reaction was carried out at 37℃ for 30 min to complete the directional seamless cloning of the target gene and vector.

[0065] 3) Transformation of competent cells: Escherichia coli DH5α chemically competent cells were used in this experiment. 10 μL of homologous recombination reaction product was added to 100 μL of DH5α competent cells thawed on ice, gently mixed, and incubated on ice for 30 min. The cells were then subjected to precise heat shock at 42℃ for 90 s and immediately transferred to an ice bath for 2 min to complete the chemical heat shock transformation.

[0066] 4) Screening and identification of positive clones: Add 900 μL of antibiotic-free LB liquid medium to the transformation system and incubate at 37℃ and 200 rpm for 60 min with shaking. Spread an appropriate amount of bacterial solution onto LB solid plates containing ampicillin resistance and incubate at 37℃ inverted for 12-16 h. The next day, pick single colonies with rounded edges and uniform size, inoculate them into resistant LB liquid medium, and amplify them by shaking at 37℃. Preliminary identification of positive clones is performed by colony PCR.

[0067] 5) Sequencing Validation and Plasmid Preparation: The screened positive bacterial cultures were sent to a professional sequencing company for full-length nucleic acid sequencing. The sequencing results were compared with the standard coding sequence of the target antibody. After confirming the absence of abnormalities such as base mutations, deletions, frameshift mutations, and reading frame shifts, clones without mismatches were deemed qualified. The qualified strains were then subjected to large-scale amplification and purified using an endotoxin-free plasmid mass extraction kit to obtain high-purity, low-endotoxin antibody heavy and light chain expression plasmids. The plasmid concentration and purity (OD260 / 280 = 1.8-2.0) were measured and the plasmids were stored at -20°C in the dark for subsequent cell transfection experiments.

[0068] 2. Expression and purification The antibody plasmid, verified by sequencing, was introduced into Chinese hamster ovary (CHO) cells via transient transfection. After transfection, the cells were cultured under suitable conditions (maintained at 37°C, 8% CO2, and 125 rpm in a constant temperature and humidity shaker) for 4-6 days, during which cell viability and expression levels were monitored periodically. After culture, the cell culture supernatant was collected, and cell debris was removed by centrifugation to obtain a crude extract containing the target antibody.

[0069] The crude extract was loaded onto a pre-equilibrated Protein A affinity chromatography column for specific capture using the high affinity of Protein A for the antibody Fc fragment (fractions without Fc were captured using His tags). After loading, the sample was thoroughly washed with equilibration buffer to remove non-specifically bound proteins, followed by elution with an acidic elution buffer (e.g., glycine-hydrochloric acid buffer, pH 3.0-3.5), and the pH was immediately adjusted to neutral with neutralization buffer. The elution peak was collected, concentrated by ultrafiltration, aliquoted, and stored at -80°C for later use.

[0070] 3. Preparation of heterodimers CHO cells were cultured in serum-free suspension medium throughout the entire process, with constant environmental parameters: temperature 37 ℃, CO2 concentration 8%, saturated humidity, and shaking speed of 125 rpm. Cells were passaged every 24 h to ensure that the cells were in the logarithmic growth phase at the time of transfection and that the cell viability was ≥95%.

[0071] Taking Y24 cells as an example, the plasmid expressing NK1 VHH-S1-FC(IgG1-LALA-PG-Knob)-TAA1 and the plasmid expressing 2A22A2 VHH-S1-FC(IgG1-LALA-PG-Hole)-TAA1 were mixed at a molar ratio of 1:1 to maximize heterodimer assembly efficiency and reduce homodimer impurity formation. The cell density was adjusted to 4.0 × 10⁻⁶ cells on the day of transfection. 6 -6.0×10 6 Cells / mL; Following the standard CHO transfection kit system, the mixed plasmid, CHO transfection reagent, and dilution medium were thoroughly mixed and incubated at room temperature for 20 min to form a transfection complex. This complex was then slowly added dropwise to the cell culture medium and gently mixed. 24 h after transfection, the kit-supplied cell enhancer and feed medium were precisely added to maintain cell viability and sustained protein expression. Culture duration gradients of 5 or 7 days and culture volume gradients of 30 mL or 100 mL were set, maintaining a constant temperature and humidity of 37 ℃, 8% CO2, and 125 rpm throughout the culture. After culture, the cell supernatant was collected by centrifugation at 4000 g for 30 min and filtered through a 0.45 μm filter to remove impurities. Dimeric proteins were purified using Protein A affinity chromatography, aseptically aliquoted, and stored at low temperature for later use.

[0072] Y25 was prepared using the same method as Y24, except that the plasmid expressing NK1 VHH-S1-FC(IgG1-LALA-PG-Knob)-TAA1 and the plasmid expressing NK1 VHH-S1-FC(IgG1-LALA-PG)-TAA1 were mixed at a molar ratio of 1:1.

[0073] Y26 was prepared using the same method as Y24, except that the plasmid expressing 2A2 VHH-S1-FC(IgG1-LALA-PG-Hole)-TAA1 and the plasmid expressing 2A2 VHH-S1-FC(IgG1-LALA-PG)-TAA1 were mixed at a molar ratio of 1:1.

[0074] III. Identification and Characterization of Target Antibodies 1. SDS-PAGE identification (1) Experimental methods The absorbance of the test sample at a wavelength of 280 nm was determined by ultraviolet spectrophotometry, and the protein concentration was calculated. Preparation of non-reducing samples: Take 1 μg of the test sample, add 4×LDS loading buffer, add iodoacetamide (final concentration 40 mM), and heat in a dry bath at 75℃ for 10 min. Preparation of reduced samples: Take 1 μg of the test sample, add 4×LDS loading buffer and reducing agent, and heat in a dry bath at 100℃ for 10 min.

[0075] The treated samples were added to the gel wells and electrophoresed at a constant voltage of 140 V for 75 min. After electrophoresis, the gels were removed, stained with Coomassie Brilliant Blue R-250, and destained with destaining solution until the background was clear. The electrophoretic patterns were then scanned and recorded using a color scanner. ImageJ software was used to calculate the purity of each band using the peak area normalization method: under non-reducing conditions, the non-reducing purity was calculated using the area of ​​the main band at approximately 150 kDa; under reducing conditions, the reducing purity was calculated using the sum of the areas of the heavy chain band at approximately 55 kDa and the light chain band at approximately 25 kDa.

[0076] System suitability requirements: The antibody expression reference (IPI) has a molecular weight of approximately 150 kDa and a purity greater than 90% under non-reducing conditions; under reducing conditions, the heavy chain molecular weight is approximately 55 kDa, the light chain molecular weight is approximately 25 kDa, and the purity of the sum of the heavy and light chains is greater than 90%.

[0077] (2) Experimental results The SDS-PAGE identification results of CD33 monoclonal antibody, Y24, Y25, and Y26 are as follows: Figure 2 As shown in the figure. The results indicate that the present invention successfully prepared CD33 monoclonal antibodies, Y24, Y25, and Y26.

[0078] 2. Endotoxin testing (1) Experimental methods Preparation of endotoxin standard solution: Dissolve the endotoxin standard in water used for the Limulus Amebocyte Lysate (LAL) test to prepare a standard solution of the required concentration. Sample solution: Dilute the sample to be tested to the appropriate concentration using water used for the LAL test.

[0079] Take four calibrated Limulus ampoule reagents, wipe the neck of each ampoule with a 75% ethanol cotton ball, and then open it. Add 0.1 mL of LAL test water to each ampoule to reconstitute the reagent, and gently mix. Add samples according to the following groups: Negative control tube: Add 100 μL of water for LAL testing; Positive control tube: Add 100 μL of positive control solution (containing known amounts of endotoxin standard); Positive control tube for sample: Add 100 μL of positive solution for test sample (the sample to be tested contains a known amount of endotoxin standard); Test sample tube: Add 100 μL of test sample solution.

[0080] After adding the sample, gently mix each tube and incubate in a constant temperature incubator at (37±1)℃ for 60 ± 2 min. After incubation, slowly invert each tube 180° and observe. Tubes that form a firm gel and do not flow when inverted are considered positive (+), while tubes that do not form a gel or have a loose gel that flows when inverted are considered negative (-).

[0081] Result determination: If the negative control tube is negative, the positive control tube is positive, the sample positive control tube is positive, and the test sample tube is negative, it is considered to meet the requirements.

[0082] (2) Experimental results The results are shown in Table 3. In this invention, the endotoxin test results for Y24, Y25, and Y26 were all <1 EU / mg, which meets the endotoxin qualification standard.

[0083] Table 3. Endotoxicity content of Y24, Y25 and Y26 The fusion proteins Y24, Y25, and Y26 synthesized in this embodiment are multispecific antibodies that can physically link NK cells to tumor cells, thereby activating the killing effect of NK cells on tumor cells and serving as NK cell connectors (NKCell Engager, NKCE).

[0084] The technical solution of the present invention will be further illustrated by the following experiments. The samples CD33 monoclonal antibody, Y24, Y25, and Y26 used in the following experimental examples were all prepared using the method of Example 1.

[0085] Experiment Example 1: Detection of CD33 monoclonal antibody binding and killing ability I. Experimental Methods 1. Nanobody binding ability assay: 10 nM CD33 antibody (CD33 monoclonal antibody H33-13 prepared in Example 1 or control antibody) was mixed with 1×10 5MV-4-11 target cells were incubated at 4°C for 2 hours in IMDM medium with 10% fetal bovine serum. After incubation, cell samples were collected in 1.5 mL EP tubes, washed twice with PBS, the supernatant was discarded, and the cells were resuspended in 100 μL of PBS. Then, 5 μL of PE-labeled anti-human IgG Fc antibody (Biolegend, 410708) was added to each tube. After incubation at 4°C for 40 min, the cells were washed twice, and then resuspended in 100 μL of PBS before flow cytometry was used to detect PE signal.

[0086] 2. CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher, C34557) was used to proliferate human CD33-positive tumor cells (3×10⁻⁶). 5 3 x 10^9 MOLM-13 leukemia cells were stained with RPMI 1640 + 10% fetal bovine serum. Then, 1 nM of CD33 antibody (CD33 monoclonal antibody H33-13 prepared in Example 1 or control antibody) was added. After incubation in an incubator for 30 min, 3 x 10^9 NK cells derived from PBMC isolation culture were added. 5 After co-incubation for 24 hours, cells were collected and apoptosis staining was performed using the APC Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend, 640930). Early apoptotic cells (7-AAD-Annexin V+) and late apoptotic cells (7-AAD+Annexin V+) were counted.

[0087] II. Experimental Results 1. Flow cytometry analysis revealed that H33-13, a negative control (without antibody), and the CD33 control antibody Gemtuzumab (containing only its CD33 sequence) both effectively bound to the CD33 target on the surface of MV-4-11. Figure 3 However, the binding ability of the CD33 monoclonal antibody (H33-13) prepared in Example 1 was better than that of the control antibody.

[0088] 2. Flow cytometry results of CD33 antibody-mediated NK cell killing of tumor cells are shown in [the image]. Figure 4The four figures represent the single tumor cell group, the tumor cell + NK group, the tumor cell + NK + Gemtuzumab group, and the tumor cell + NK + H33-13 group, respectively. The horizontal axis represents the APC-Annexin V area, and the vertical axis represents the 7-AAD area. Q11-4 represents early apoptotic tumor cells, and Q11-2 represents late apoptotic tumor cells. It is evident that in the co-incubation system of NK and MOLM-13, compared to the single tumor cell group and the tumor cell + NK group, the addition of the CD33 monoclonal antibody (H33-13) prepared in Example 1 significantly enhances the ability of NK cells to kill MOLM-13 cells compared to Gemtuzumab. Figure 4 ).

[0089] Therefore, in Example 1 of the present invention, the CD33 sequence H33-13 selected was used to construct the bi / trispecific antibody.

[0090] Experimental Example 2: Y24, Y25, and Y26 antibodies can enhance NK cell killing activity. I. Experimental Methods The CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher, C34557) was used to proliferate human acute promyelocytic leukemia cell lines (3 × 10⁻⁶ cells / year). 5 After staining with HL-60 cells in IMDM + 20% fetal bovine serum, 10 nM NKCE (Y24, Y25, or Y26) was added, and the cells were incubated in an incubator for 30 min. Then, 3 × 10⁶ PBMCs were added to isolate NK cells derived from the culture. 5 After co-incubation for 48 hours, cells were collected and apoptosis staining was performed using the APC Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend, 640930). Early apoptotic cells (7-AAD-Annexin V+) and late apoptotic cells (7-AAD+Annexin V+) were counted.

[0091] II. Experimental Results Figure 5 A shows the flow cytometry results of the HL-60 and NK cell co-incubation system after the addition of PBS, Y24, Y25, and Y26, respectively. The horizontal axis represents the APC-Annexin V flow cytometry antibody staining, and the vertical axis represents the 7-AAD flow cytometry antibody staining. In particular, the Q8-4 region represents tumor cells with early apoptosis, and the Q8-2 region represents tumor cells with late apoptosis. Figure 5 B-5D represent the statistical values ​​for early apoptotic tumor cells, late apoptotic tumor cells, early apoptotic tumor cells, and pre-apoptotic tumor cells, respectively.

[0092] The results showed that, at the same concentration, compared with the HL-60 and NK cell co-incubation system with added PBS, the addition of Y24, Y25, and Y26 all enhanced the killing effect of NK cells on HL-60 cells, specifically in both early-stage and late-stage apoptotic tumor cells. Figure 5 A).

[0093] Experiment Example 3: Differences in NK cell number and functional indicators after the addition of Y24, Y25, and Y26 antibodies. I. Experimental Methods The CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher, C34557) was used to proliferate human acute promyelocytic leukemia cell lines (3 × 10⁻⁶ cells / year). 5 After staining with HL-60 cells in IMDM + 20% fetal bovine serum, 10 nM NKCE (Y24, Y25, or Y26) was added, and the cells were incubated in an incubator for 30 min. Then, 3 × 10⁶ NK cells derived from PBMC isolation culture were added. 5 After co-incubating for 48 h, cells were collected, and 1 μL of GDR-780 cell viability dye (CYTEK, 13-0865-T500) was added to each tube. The cells were incubated at room temperature for 10 min. After washing the cells with PBS, centrifugation was performed, and the supernatant was discarded. Then, 2.5 μL of human Fc receptor blocker (BioLegend, 422302) was added, and the cells were incubated for 15 min. After washing the cells with PBS, centrifugation was performed, and the supernatant was discarded. Then, 5 μL of PERCP-CY5.5-CD56 antibody (BioLegend, 362506) and 5 μL of APC-CD107a (BioLegend, 328606) were added for staining. After staining for 30 min, the cells were washed, and the supernatant was discarded. Cells were fixed and perforated using a fixation and nuclear membrane rupture kit (BioLegend, 424401), and then 5 μL of PE-IFN-γ (BioLegend) was added. After staining (502509) for 45 minutes, the cells were analyzed and the expression of CD56, CD107a and IFN-γ in NK cells was recorded.

[0094] II. Experimental Results Figure 6 AC represent the statistical values ​​of CD56 and CD107a expression on the surface of NK cells and the expression of the cytokine IFN-γ in HL-60 and NK cell co-incubation systems after the addition of PBS, Y24, Y25, and Y26, respectively. Compared with bispecific antibodies containing only CD16a and NKG2A (Y25 and Y26), the addition of a trispecific antibody containing both CD16a and NKG2A (Y24) upregulated the expression of CD56 and CD107a on the surface of NK cells and the expression of the cytokine IFN-γ in NK cells. Figure 6).

[0095] Experiment Example 4: Differential expression of antibody-related target sites on NK cell surface after the addition of Y24, Y25, and Y26 antibodies. I. Experimental Methods The CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher, C34557) was used to proliferate human acute promyelocytic leukemia cell lines (3 × 10⁻⁶ cells / year). 5 After staining with HL-60 cells in IMDM + 20% FBS medium, 10 nM NKCE (Y24, Y25, or Y26) was added, and the cells were incubated in an incubator for 30 min. Then, 3 × 10⁶ PBMC-isolated NK cells were added. 5 After co-incubating for 48 h, cells were collected and incubated for 10 min at room temperature with 1 μL of GDR-780 cell viability dye (CYTEK, 13-0865-T500). Cells were washed with PBS, centrifuged, and the supernatant was discarded. Then, 2.5 μL of human Fc receptor blocker (BioLegend, 422302) was added, and the cells were incubated for 15 min. After washing with PBS, centrifuged, and the supernatant was discarded, 5 μL of BV605-CD16 (BioLegend) was added. Cells were stained with 5 μL of PERCP-CY5.5-CD56 antibody (BioLegend, 362506), 5 μL of BV711-NKG2A antibody (BioLegend, 375156), and 5 μL of APC-hIL15Rα antibody (BioLegend, 330210) for 30 min, washed, resuspended, and analyzed for differences in the expression of NK cell surface antibody-related targets.

[0096] II. Experimental Results Figure 7 AC represents the statistical values ​​of CD16, NKG2A, and IL-15R expression on the surface of NK cells after adding PBS, Y24, Y25, and Y26 to the HL-60 and NK cell co-incubation system, respectively. In Example 1, IL-15 sequences were constructed from Y24, Y25, and Y26. Therefore, the antibody-related NK cell targets are CD16a and NKG2A, and IL-15R. Flow cytometry results showed that the group with only the bispecific antibody containing NKG2A (Y26) had the highest proportion of CD16a-positive NK cells, while the group with only the bispecific antibody containing CD16a (Y25) had the highest proportion of NKG2A-positive NK cells. However, the group with the trispecific antibody containing both CD16a and NKG2A (Y24) showed higher IL-15R expression. The differences in expression are shown in [see attached table]. Figure 7 .

[0097] The results of this experiment show that the trispecific antibody (Y24) containing both CD16a and NKG2A can balance the expression of CD16a and NKG2A on the surface of NK cells in the co-incubation system. At the same time, the Y24 group showed higher expression of IL-15 on the surface of NK cells, which is more conducive to NK cell proliferation and survival.

[0098] Experimental Example 5: Differences in NK cell activation markers after the addition of Y24, Y25, and Y26 antibodies. I. Experimental Methods The CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher, C34557) was used to proliferate human acute promyelocytic leukemia cell lines (3 × 10⁻⁶ cells / year). 5 After staining with HL-60 cells in IMDM + 20% fetal bovine serum, 10 nM NKCE (Y24, Y25, or Y26) was added, and the cells were incubated in an incubator for 30 min. Then, 3 × 10⁶ NK cells derived from PBMC isolation culture were added. 5 After co-incubating with 1 μL of GDR-780 cell viability dye (CYTEK, 13-0865-T500) for 48 h, cells were collected and incubated at room temperature for 10 min. After washing the cells with PBS, centrifuging and discarding the supernatant, 2.5 μL of human Fc receptor blocker (BioLegend, 422302) was added and incubated for 15 min. After washing the cells with PBS, centrifuging and discarding the supernatant, 5 μL of PERCP-CY5.5-CD56 antibody (BioLegend) was added. d, 362506), after staining with 5 μL of BV711 human CD69 antibody (BioLegend, 310944) for 30 min, the cells were washed, fixed and pored using a fixation and nuclear membrane rupture kit (BioLegend, 424401), and then 5 μL of BV650-Ki67 (BioLegend, 350538) was added for staining for 30 min. After washing the cells, the cells were resuspended and analyzed to count the differences in expression of NK cell surface antibody-related targets.

[0099] II. Experimental Results Figure 8 A and B represent the statistical values ​​of CD69 and Ki67-positive NK cell expression in the HL-60 and NK cell co-incubation systems after the addition of PBS, Y24, Y25, and Y26, respectively. In the co-incubation system of this invention, the proportion of CD69-positive NK cells is Y25 > Y24 > Y26, and the proportion of Ki67-positive NK cells is Y24, Y26 > Y25. Figure 8 This indicates that the CD16a-related signaling pathway can increase CD69 expression in NK cells, while the NKG2A signaling pathway has a greater impact on Ki67.

[0100] CD69 is a marker molecule for early NK cell activation, and is almost not expressed by resting NK cells. Its high expression demonstrates that NK cells in the co-incubation system have been effectively activated, emerging from the resting state and possessing the ability to kill cells and initiate immune responses; it is a core marker of NK cell immune effects. Ki67 is a specific marker of cell proliferation activity, expressed only in cells during the division and proliferation phase. Its high expression indicates that activated NK cells enter a rapid proliferation cycle, with a positive increase in cell number and an enhanced immune cell population. These experimental results demonstrate that Y24 can effectively activate the immune function of NK cells, successfully initiating the anti-tumor immune response of NK cells; it can also promote NK cell proliferation and expansion, possessing good immune activation activity and application potential.

[0101] In summary, in the above embodiments, on the one hand, a CD33 monoclonal antibody with stronger binding ability was designed through sequence optimization. On the other hand, multifunctional antibodies Y24, Y25, and Y26 were designed using the sequence of this CD33 monoclonal antibody. In the in vitro cytotoxicity detection system of flow cytometry, a fixed number of NK cells were co-incubated with a quantitative number of tumor target cells. Due to the upper limit of the total number of tumor target cells in the reaction system, under the preset incubation time, the proportions of early and late apoptosis of target cells induced by the two molecular constructs Y24 and Y26 both reached the target cell killing saturation limit corresponding to the detection system. Multiple flow cytometry data of NK cell function confirmed that, compared with the Y25 and Y26 molecular constructs, Y24 can significantly upregulate the expression abundance of NK cell surface functional markers CD56, degranulation marker CD107a, effector cytokine IFN-γ, and IL-15 receptor IL-15R, and strongly activate the cytotoxicity function of NK cells. This suggests that in in vivo and in vitro applications with sustained tumor target cell replenishment, long treatment cycles, or high tumor burden, Y24 can enrich and stably retain a large number of functional NK cell subsets with complete cytotoxic activity, exhibiting superior long-lasting antitumor cytotoxic activity compared to the Y25 and Y26 molecular constructs. These results indicate that the trispecific antibody molecular construct (Y24) simultaneously targeting CD16a, NKG2A, and CD33 possesses superior antitumor activity and shows great promise for application.

Claims

1. An antibody targeting CD33, characterized in that, The amino acid sequence of the antibody targeting CD33 is shown in SEQ ID NO.

5.

2. The antibody according to claim 1, characterized in that: The nucleotide sequence of the encoding gene of the antibody targeting CD33 is shown in SEQ ID NO.

12.

3. A multifunctional fusion protein, characterized in that, It includes: Antibodies or fragments thereof that target NK cells, and antibodies or fragments thereof that target CD33 as described in claim 1 or 2.

4. The multifunctional fusion protein according to claim 3, characterized in that: The multifunctional fusion protein is a dimer composed of polypeptide chain 1 and polypeptide chain 2; the structure of polypeptide chain 1 includes a first antigen-binding region, linker peptide 1, IL-15 functional region, Knob-mutated Fc, linker peptide 2 and third antigen-binding region connected in sequence; the structure of polypeptide chain 2 includes a second antigen-binding region, linker peptide 3, IL-15 functional region, Hole-mutated Fc, linker peptide 4 and fourth antigen-binding region connected in sequence. The first antigen-binding region and the second antigen-binding region are respectively antibodies or fragments thereof targeting NK cells; the third antigen-binding region and the fourth antigen-binding region are respectively antibodies or fragments thereof targeting CD33; The antibody targeting NK cells is selected from at least one of the following antibodies: Antibodies that specifically bind to the inhibitory receptor NKG2A on the surface of NK cells. Antibodies that specifically bind to the activating receptor CD16a on the surface of NK cells; Preferably, the amino acid sequences of polypeptide chain 1 and polypeptide chain 2 are as shown in SEQ ID NO. 8 and SEQ ID NO. 9, respectively, or the amino acid sequences of polypeptide chain 1 and polypeptide chain 2 are as shown in SEQ ID NO. 8 and SEQ ID NO. 10, respectively, or the amino acid sequences of polypeptide chain 1 and polypeptide chain 2 are as shown in SEQ ID NO. 9 and SEQ ID NO. 11, respectively.

5. A gene fragment, characterized in that: It encodes the antibody as described in claim 1 or 2, or the multifunctional fusion protein as described in claim 3 or 4.

6. A recombinant vector, characterized in that: It contains the gene fragment as described in claim 5.

7. A host cell, characterized in that: It comprises the recombinant vector as described in claim 6.

8. Use of the multifunctional fusion protein of claim 3 or 4, the gene fragment of claim 5, the recombinant vector of claim 6, or the host cell of claim 7 in the preparation of multispecific antibodies or NK cell connectors. Preferably, the multispecific antibody and NK cell connector are used to treat acute myeloid leukemia.

9. An NK cell connector, characterized in that: It includes the multifunctional fusion protein as described in claim 3 or 4.

10. An engineered immune cell, characterized in that: The engineered immune cells comprise the multifunctional fusion protein as described in claim 3 or 4.

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