Construction method and application of a novel long-acting multifunctional antibody

Abstract

The application discloses a long-acting multifunctional antibody as well as a construction method and application thereof, and belongs to the technical field of biotechnology. The multifunctional antibody molecule takes a Fab fragment of a monoclonal antibody as a first domain, and connects a second domain, a third domain and a fourth domain through a connecting peptide on the basis of the first domain, wherein the second domain and the third domain are respectively a binding domain targeting a tumor-related antigen or a T cell costimulatory domain, and the fourth domain is an albumin binding domain. The application realizes the prolongation of the half-life of the antibody in the body while maintaining the multi-target function activity through a modular structure design, thereby reducing the administration frequency and improving the treatment effect. The multifunctional antibody can be used for preparing a medicine for treating tumors.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for constructing and applying a novel long-acting multifunctional antibody. Background Technology

[0002] In recent years, immunotherapy, as an emerging anti-cancer method following surgery, radiotherapy, chemotherapy, and targeted therapy, has achieved significant breakthroughs in the field of tumor treatment. Its core is to mobilize or enhance the body's immune system to fight diseases. It has not only been remarkably effective in cancer treatment, but has also provided new directions for the intervention of autoimmune diseases and infectious diseases (Zhang, Y.; Zhang, Z., The history and advances in cancer immunotherapy: understanding the characteristics of tumor-infiltrating immune cells and their therapeutic implications. Cell Mol Immunol 2020, 17 (8), 807-821.). In the field of antibody therapy, bispecific antibodies have stronger therapeutic potential than traditional monoclonal antibodies because they can simultaneously target two different molecules and achieve synergistic anti-disease effects through multiple mechanisms. Moreover, with continuous innovation in target selection and structural design, more than 100 different types of bispecific antibodies have entered the clinical development stage, with very broad application prospects (Brinkmann, U.; Kontermann, RE, Bispecific antibodies. Science 2021, 372 (6545), 916-917.).

[0003] Although bispecific antibodies have demonstrated unique advantages in the treatment of malignant tumors, such as strong targeting synergy and precise anti-tumor effects, their clinical application and efficacy are still limited by the key scientific challenge of tumor immune escape. Tumor cells can evade the surveillance and clearance of the immune system through multiple strategies, ultimately achieving uncontrolled proliferation and metastasis. This has become the core bottleneck restricting the clinical translation of bispecific antibodies and maximizing their therapeutic value (Wu, J.; Fu, J.; Zhang, M.; Liu, D., Blinatumomab: a bispecific T cell engager (BiTE) antibody against CD19 / CD3 for refractory acute lymphoid leukemia. J Hematol Oncol 2015, 8,104.). Based on whether they depend on the expression and recognition process of tumor antigens, tumor immune escape mechanisms can be divided into antigen-dependent and antigen-independent immune escape. In clinical settings, these two types of immune escape mechanisms often exhibit a synergistic effect, which is not only an important cause of resistance to bispecific antibody therapy but also a key pathological basis for cancer recurrence after treatment and remains a critical scientific challenge that urgently needs to be overcome in clinical practice.

[0004] Providing T cells with the co-stimulatory signal (second signal) required for activation can further enhance the effector function and survival of T cells, reduce T cell exhaustion and anergic state, and thus provide an effective strategy for solving the problem of antigen-independent immune escape (Einsele, H.; Borghaei, H.; Orlowski, RZ; Subklewe, M.; Roboz, GJ; Zugmaier, G.; Kufer, P.; Iskander, K.; Kantarjian, HM, TheBiTE (bispecific T‐cell engager) platform: Development and future potential of a targeted immuno‐oncology therapy across tumor types. Cancer 2020, 126(14), 3192-3201.). In the molecular mechanism of T cell activation, the co-stimulatory molecule CD28 plays an indispensable regulatory role as the core mediator of the second signal (Li, Y.-R.; Halladay, T.; Yang, L., Immuneevasion in cell-based immunotherapy: unraveling challenges and novel strategies. Journal of Biomedical Science 2024, 31 (1).).CD28 can mediate the transcription of interleukin-2 (IL-2) by activating NFAT, AP-1, and NF-κB family transcription factors (Warda, W.; DaRocha, MN; Trad, R.; Haderbache, R.; Salma, Y.; Bouquet, L.; Roussel, X.; Nicod, C.; Deschamps, M.; Ferrand, C., Overcoming target epitope masking resistance that can occur on low-antigen-expresser AML blasts after IL-1RAPchimeric antigen receptor T cell therapy using the inducible caspase 9suicide gene safety switch. Cancer Gene Therapy 2021, 28 (12), 1365-1375.). In addition, 4-1BB is an important co-stimulatory molecule in the T cell activation process. With the gradual maturation of the research and development of 4-1BB bispecific antibodies and the continuous expansion of application scenarios, the research and development value and clinical potential of multifunctional antibodies with 4-1BB as a co-stimulatory signal have begun to receive widespread attention (Alegre ML, Frauwirth KA, Thompson CB. T-CELL REGULATIONBY 4-1BB AND CTLA-4[J]. Nature Reviews Immunology, 2001, 1(3):220-228.). 4-1BB signaling can be regulated by the core transcription factor NF-κB, ultimately promoting the production and secretion of cytokines (Bartkowiak, Todd, and Michael A Curran. “4-1BB Agonists: Multi-PotentPotentiators of Tumor Immunity.” Frontiers in oncology vol. 5117. 8 Jun.2015).

[0005] To overcome tumor immune escape and T cell activation limitations, the design and development of trispecific antibodies have gained attention in recent years (Runcie K, et al. Bi-specific and tri-specific antibodies - the next big thing in solid tumor therapeutics. Mol Med. 2018 Sep 24; 24(1): 50.). T cell-conjugating trispecific antibodies represent a highly effective way to redirect activated cytotoxic T cells to tumors (Runcie K, et al. Bi-specific and tri-specific antibodies - the next big thing in solid tumor therapeutics. Mol Med. 2018 Sep 24; 24(1): 50., Mullard A. Trispecific antibodies take to the clinic. Nat Rev Drug Discov. 2020 Oct;19(10): 657-658.). T cell-conjugating trispecific antibodies can simultaneously bind to surface tumor antigens and the CD3ε subunit of T cell receptors, providing a physical link between T cells and tumor cells, thereby effectively activating quiescent T cells to kill tumor cells and achieve the therapeutic effect of tumor treatment. Because of the co-stimulatory requirements of T cell bispecific bypass TCR antigen recognition and T cell activation, they overcome many obstacles faced by T cells in the tumor microenvironment.

[0006] However, these existing technologies have the following problems: the currently reported CD28 (such as SAR443216, CD38 / CD3 / CD28) and 4-1BB (such as DLL3 / CD3 / 4-1BB, WT1 / CD3 / 4-1BB, PSMA / CD3 / 4-1BB) trispecific antibodies still face three major bottlenecks in the clinical translation process: First, the structural design is rigid, and most of them adopt the traditional tandem single-chain variable fragment or IgG-like constant region fusion mode, which makes it difficult to flexibly adjust the valence state and spatial conformation according to the target characteristics; Second, the titer regulation is singular, relying only on a single signal for activation, and cannot achieve customized immune activation for different immunosuppressive states of tumor microenvironments; Third, there is non-specific toxicity mediated by Fc fragments. The intact Fc region is easy to bind through antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) or Fcγ receptor (FcγR), causing non-target damage to normal tissues.

[0007] To overcome the aforementioned limitations, our research team first developed a series of optimized trispecific antibodies through a structural optimization and reconstruction strategy: For solid tumors, we constructed novel Her2 / CD3 / CD28 and Her2 / CD3 / 4-1BB trispecific antibodies, optimizing antigen-binding kinetics and co-stimulatory signal transduction efficiency by adjusting the linkage sequence and hinge region flexibility; For hematologic malignancies, we designed CD19 / CD22 / CD3 trispecific antibodies, employing a dual-target mode to enhance tumor recognition specificity, while simultaneously deleting the constant region to completely eliminate Fc-mediated nonspecific effects, successfully solving the problems of "rigid structure, single potency, and toxicity risk" of traditional trispecific antibodies. However, these optimized trispecific antibodies have a small molecular weight and a rapid metabolic rate in vivo, with a half-life of only 10+ hours, far shorter than traditional IgG antibodies (19-21 days). This rapid clearance rate necessitates frequent dosing (e.g., 1-2 times daily) to maintain effective therapeutic concentrations, affecting patient compliance and potentially impacting the stability of antitumor efficacy and the therapeutic window due to fluctuations in blood drug concentrations.

[0008] Albumin-binding domain conjugates (ABDCon) represent a novel strategy for extending half-life. Their core mechanism involves introducing a high-affinity albumin-binding domain to achieve specific and reversible binding to endogenous serum albumin. This, combined with the long-circulating nature of albumin in vivo, significantly prolongs the in vivo retention time of substances like antibodies (Li S, Shi L, Guo Q, Zhao L, Qi X, Liu Z, Guo Z, Cao YJ. BAFF-based trifunctional T-cell engagers trigger robust tumor immunity against B-cell malignancies. Protein Cell. 2025Jun 27:pwaf054. doi: 10.1093 / procel / pwaf054. Epub ahead of print. PMID:40577379.). From a molecular design perspective, ABDCon is typically composed of a "functionally active unit" coupled to an "albumin-binding domain" via a flexible linker. The albumin-binding domain often originates from mutated, optimized sequences of natural proteins such as Streptococcus protein G. Its dissociation constant (Kd) for serum albumin (HSA) can be as low as nanomolar or even picomolar levels, enabling it to efficiently and competitively bind to specific domains of serum albumin (such as subdomain IIIA) without affecting the normal physiological functions of albumin (such as substance transport and osmotic pressure maintenance). (Wysocki J, Ye M, Hassler L, Gupta AK, Wang Y, Nicoleascu V, Randall G, Wertheim JA, Batlle D. A Novel Soluble ACE2 Variant with ProlongedDuration of Action Neutralizes SARS-CoV-2 Infection in Human Kidney Organoids. J Am Soc Nephrol. 2021 Apr;32(4):795-803. doi: 10.1681 / ASN.2020101537. Epub 2021 Feb 1. PMID: 33526471; PMCID: PMC8017551.).At the metabolic level in vivo, small molecules or short-lived biomolecules are normally easily cleared by renal filtration (e.g., proteins with a molecular weight <60 kDa are easily cleared by glomerular filtration) or enzymatic pathways. However, after ABDCon binds to serum albumin, the molecular weight of the resulting complex is significantly increased (HSA has a molecular weight of approximately 66.5 kDa, and the overall molecular weight after binding usually exceeds 100 kDa), effectively circumventing renal filtration. Simultaneously, HSA has a highly prolonged natural half-life in vivo and can be widely distributed throughout the body via the circulatory system. ABDCon, leveraging this "hitchhiking" effect, can follow serum albumin for a long time, significantly reducing the clearance rate (Zhang Q, Qian M, Wu Y, Wang Y, Shangguan W, Lu J, Zhao W, Feng J. Design and biological evaluation of novel long-acting adalimumab Fabconjugated with the albumin binding domain. Eur J Pharmacol. 2021 Aug 5;904:174152. doi: (10.1016 / j.ejphar.2021.174152. Epub 2021 May 5. PMID: 33964292.). Furthermore, the half-life extension effect of ABDCon is highly controllable. By optimizing the affinity of the albumin-binding domain through targeted mutations (such as adjusting amino acid residues at key binding sites) or altering the length and flexibility of the linker, the binding strength and dissociation kinetics of ABDCon with serum albumin can be precisely controlled. This allows for customized design of the target molecule's in vivo half-life, meeting the long-acting requirements of clinical treatment while avoiding the risk of drug accumulation due to excessively long half-lives. Currently, the ABDCon strategy has been widely applied to optimize the half-life of antibody drug fragments, cytokines, and other biologics, providing crucial technical support for improving drug efficacy and reducing dosing frequency.

[0009] Therefore, our team further optimized the half-life of the multifunctional antibody platform: by using genetic engineering, a flexible linker (containing a (G4S)3 repeat sequence) was directionally coupled to a specific site of the optimized trispecific antibody, and then the albumin-binding domain conjugate was bound to the other end of the linker, ultimately constructing a multifunctional antibody molecule that integrates "targeted binding - immune activation - long-term circulation".

[0010] In summary, we further optimized the antibody design platform. Based on the structurally optimized novel Her2 / CD3 / CD28 trispecific antibody, CD19 / CD22 / CD3 trispecific antibody, and Her2 / CD3 / 4-1BB trispecific antibody, we successfully constructed a multifunctional antibody molecule with targeted binding, immune activation, and long-lasting circulating function by directionally conjugating ABDCon to specific regions of the above-mentioned trispecific antibodies through a linker. This significantly improved the in vivo half-life of the drug, reduced the dosage and frequency of administration, and enhanced the efficacy. It also preserved the function of other structural domains of the antibody to the greatest extent, enabling the expansion of treatment for various tumor types, including solid tumors and hematological malignancies. This greatly enhances the clinical translational potential and is expected to be developed into a universal drug for tumor immunotherapy. Summary of the Invention

[0011] To address the shortcomings of the existing technologies, effectively and conveniently extending the half-life of multi-target drugs to reduce dosage and frequency while ensuring efficacy and safety is one of the challenges in antibody drug development. This invention provides a novel method for constructing and designing multispecific antibodies, which will offer patients more treatment options.

[0012] This invention employs genetic engineering methods to fuse and express different multivalent co-stimulatory domain antibodies and various tumor-associated antigen-targeting antibodies at different positions on the antibody backbone. Furthermore, albumin-binding domains of different titers are introduced at different positions to effectively prolong the half-life. By comparing the relationship between different structural designs and efficacy, and further screening for the optimal and reasonable structure, a multifunctional antibody design and construction strategy with the best efficiency in prolonging the half-life is established.

[0013] In a first aspect, the present invention provides a multifunctional antibody molecule. The multifunctional antibody molecule uses the Fab fragment of a monoclonal antibody as its structural basis as a first domain, and additionally includes a second, third, and fourth domain. The second domain is a T-cell activation co-stimulatory domain, or a first antibody targeting tumor-specific or related antigens. The third domain is a T-cell activation co-stimulatory domain, or a second antibody targeting tumor-specific or related antigens. The fourth domain is an albumin-binding domain. The second, third, and fourth domains are respectively linked to the Fab fragment via linking peptides, or the fourth domain is linked to the second or third domain via linking peptides, thereby forming a multifunctional antibody molecule with multi-target binding capability and enhanced in vivo stability.

[0014] A second aspect of the present invention provides a method for constructing the multifunctional antibody molecule, specifically, the method comprising the following steps: (1) The first domain is CD3 monoclonal antibody Fab; (2) Connecting the second structural domain or the second structural domain / third structural domain to the heavy chain structural domain of the first structural domain Fab; (3) Connecting the second structural domain or the second structural domain / third structural domain to the light chain structural domain of the first structural domain Fab; (4) Connect the fourth structural domain to the heavy chain structural domain or light chain structural domain of the first structural domain Fab; (5) Connect the fourth structural domain to the second or third structural domain; (6) The heavy chain and light chain combine through disulfide bonds in CH1 and CL to form a heterodimer trispecific antibody.

[0015] Furthermore, step (2) can be constructed or connected in the following manner: (a) The second / third domain is linked to the C-terminal 228C region of the SP34 Fab heavy chain constant region (CH1) domain via a linker peptide; or (b) The second / third domain is linked to the C-terminal 228C region of the SP34 Fab heavy chain constant region (CH1) domain via a linker peptide, and the fourth domain is linked to the C-terminus of the second or third domain via a linker peptide.

[0016] Step (3) can be constructed or connected in one of the following ways: (a) The third domain is linked to the C-terminus 217C of the SP34 Fab light chain constant region (CL) domain via a linker peptide; or (b) By linking the third domain to the C-terminus 217C of the SP34 Fab light chain constant region (CL) domain via a linker peptide and linking the fourth domain to the C-terminus of the third domain; or (c) A third domain is linked to the N-terminus of the SP34 Fab light chain variable region (VL) domain via a linker peptide, and then the third domain is sequentially linked to the third domain region at the N-terminus of the SP34 Fab light chain variable region (VL) domain via a linker peptide, and again the third domain is sequentially linked to the third domain region at the N-terminus of the SP34 Fab light chain variable region (VL) domain via a linker peptide, forming a recombinant light chain with the structure of third domain—linker peptide—third domain—linker peptide—third domain—linker peptide—light chain variable region—light chain constant region from the N-terminus to the C-terminus; or (d) The third domain is linked to the N-terminus of the variable region (VL) domain of the SP34 Fab light chain via a linker peptide. Then, the third domain is sequentially linked to the N-terminus of the third domain region of the SP34 Fab light chain variable region (VL) domain via a linker peptide. Again, the third domain is sequentially linked to the N-terminus of the third domain region of the SP34 Fab light chain variable region (VL) domain via a linker peptide. Finally, the fourth domain is linked to the C-terminus of the recombinant light chain. From the N-terminus to the C-terminus, a recombinant light chain is formed with the structure: third domain—linker peptide—third domain—linker peptide—third domain—linker peptide—light chain variable region—light chain constant region—linker peptide—fourth domain; or (e) The third domain is linked to the N-terminus of the variable region (VL) domain of the SP34 Fab light chain via a linker peptide, and then the third domain is sequentially linked to the third domain region at the N-terminus of the SP34 Fab light chain variable region (VL) domain via a linker peptide. Again, the third domain is sequentially linked to the third domain region at the N-terminus of the SP34 Fab light chain variable region (VL) domain via a linker peptide, and a tandemly linked bivalent fourth domain is attached to the C-terminus of the recombinant light chain. From the N-terminus to the C-terminus, a recombinant light chain is formed with the structure: third domain—linker peptide—third domain—linker peptide—third domain—linker peptide—light chain variable region—light chain constant region—linker peptide—fourth domain—linker peptide—fourth domain; or (f) The third domain is linked to the N-terminus of the variable region (VL) domain of the SP34 Fab light chain via a linker peptide. Then, the third domain is sequentially linked to the third domain region at the N-terminus of the SP34 Fab light chain variable region (VL) domain via a linker peptide. Again, the third domain is sequentially linked to the third domain region at the N-terminus of the SP34 Fab light chain variable region (VL) domain via a linker peptide. Finally, a tandem trivalent fourth domain is linked to the C-terminus of the recombinant light chain, forming a recombinant light chain with the following structure from N-terminus to C-terminus: third domain—linker peptide—third domain—linker peptide—third domain—linker peptide—light chain variable region—light chain constant region—linker peptide—fourth domain—linker peptide—fourth domain—linker peptide—fourth domain; or (g) A third domain is linked to the C-terminus of the constant region (CL) domain of the SP34 Fab light chain via a linker peptide; a second third domain is tandemly linked to the C-terminus of the third domain via a linker peptide; and a third third domain is tandemly linked to the C-terminus of the third domain via a linker peptide, forming a recombinant light chain with the structure from N-terminus to C-terminus as follows: light chain variable region—light chain constant region—linker peptide—third domain—linker peptide—third domain—linker peptide—third domain; or (h) The third domain is linked to the C-terminus of the constant region (CL) domain of the SP34 Fab light chain via a linker peptide. The second third domain is linked to the C-terminus of the third domain via a linker peptide. The third third domain is linked to the C-terminus of the third domain via a linker peptide. The fourth domain is linked to the C-terminus of the recombinant light chain. From the N-terminus to the C-terminus, a recombinant light chain is formed with the following structure: light chain variable region—light chain constant region—linker peptide—third domain—linker peptide—third domain—linker peptide—third domain—linker peptide—fourth domain.

[0017] In steps (1), (2), and (3), the first, second, and third domains each independently have the ability to select Her2, VEGFR1 / 2, CD3, CD19, CD22, EGFR, EGFRvIII, HER3, HER4, IGF1R, c-Met, MUC-1, MUC-16, IL13R, Trop-2, GPC2, GPC3, GD2, CEA, PSMA, PSCA, EpCAM, CD79, ROR1, AXL, CD133, CD171, BCMA, CD20, CD123, Claudin 6, Claudin 18.2, CD38, CD30, CD33, CD138, CD56, CS1, CLL1, CD7, CD4, CD8, Lewis The binding specificity of related target antigens such as Y, ALK, KRAS mutants, MYD88 mutants, IDH1 mutants, P53 mutants, NY-ESO-1, NKG2D, CD16, CD56, CD64, PD-1, PD-L1, B7-H3, B7-H4, TGF-beta, CTLA-4, LAG-3, TIM-3, TIGHT, VISTA, ICOS, GITR, CD28, 4-1BB, OX40, CSD27, CD24, CD47, CXCR4, DLL3, Integrin, etc.

[0018] In steps (2) and (3), the second and third domains can be in the form of Adnectin (human fibronectin), affinity protein, anti-carrier protein, bicyclic peptide, DARPin (natural ankyrin repeat sequence), E7 immunoprotein, lymphocyte receptor variable region, single-domain antibody, whole antibody, antibody fragment, single-chain antibody, and nucleic acid aptamer, etc.

[0019] The Fab fragment of the first domain may be derived from, but is not limited to, anti-CD3 monoclonal antibodies.

[0020] Preferably, in step (1), the first domain is an anti-CD3 monoclonal antibody; in steps (2) and (3), the second and third domains and in steps (4) and (5), the fourth domain each independently has binding specificity for Her2, CD19, CD22, CD28, 4-1BB, and HSA / MSA.

[0021] The linker peptide described in this invention is selected from one or more of flexible linker peptides, rigid linker peptides, or helical linker peptides; The flexible linker peptide is selected from one or more of (G4S)3 Linker, 218 Linker, or 212 Linker; The rigid linker peptide is selected from PD Linker; In a preferred embodiment, step (2) may be constructed or connected in one of the following ways: (a) Connecting the second / third domain to the C-terminal 228C region of the SP34 Fab heavy chain constant region (CH1) domain via the (G4S)3 Linker; or (b) The second / third domain is connected to the C-end 228C region of the SP34 Fab heavy chain constant region (CH1) domain via (G4S)3 Linker, and the fourth domain is connected to the C-end of the second domain via 218 Linker.

[0022] Furthermore, step (3) can be constructed or connected in the following manner: (a) The third structural domain is connected to the C-end 217C of the SP34 Fab light chain constant region (CL) structural domain via the (G4S)3 Linker; or (b) The third structural domain is connected to the C-end 217C of the SP34 Fab light chain constant region (CL) structural domain via (G4S)3 Linker, and the fourth structural domain is connected to the C-end of the third structural domain via 218 Linker; or (c) The third structural domain is connected to the N end of the SP34 Fab light chain variable region (VL) structural domain via a PD Linker, and then the third structural domain is sequentially connected to the third structural domain region at the N end of the SP34 Fab light chain variable region (VL) structural domain via a 218 Linker. Again, the third structural domain is sequentially connected to the third structural domain region at the N end of the SP34 Fab light chain variable region (VL) structural domain via a 212 Linker. From the N end to the C end, a recombinant light chain is formed with the structure: third structural domain—212 Linker—third structural domain—218 Linker—third structural domain—PD Linker—light chain variable region—light chain constant region; or (d) The third structural domain is connected to the N end of the variable region (VL) structural domain of the SP34 Fab light chain via a PD Linker, and then the third structural domain is sequentially connected to the third structural domain region at the N end of the SP34 Fab light chain variable region (VL) structural domain via a 218 Linker. The third structural domain is then sequentially connected to the third structural domain region at the N end of the SP34 Fab light chain variable region (VL) structural domain via a 212 Linker. Finally, the fourth structural domain is connected to the C end of the light chain via a (G4S)3 Linker. From the N end to the C end, a recombinant light chain is formed with the structure: third structural domain—212 Linker—third structural domain—218 Linker—third structural domain—PD Linker—light chain variable region—light chain constant region—(G4S)3 Linker—fourth structural domain; or (e) The third structural domain is connected to the N end of the variable region (VL) structural domain of the SP34 Fab light chain via a PD Linker, and then the third structural domain is sequentially connected to the third structural domain region at the N end of the SP34 Fab light chain variable region (VL) structural domain via a 218 Linker. The third structural domain is then sequentially connected to the third structural domain region at the N end of the SP34 Fab light chain variable region (VL) structural domain via a 212 Linker. Finally, the tandemly connected bivalent fourth structural domain is connected to the C end of the light chain via a (G4S)3 Linker. From the N end to the C end, a recombinant light chain is formed with the structure: third structural domain—212 Linker—third structural domain—218 Linker—third structural domain—PD Linker—light chain variable region—light chain constant region—(G4S)3 Linker—fourth structural domain—(G4S)3 Linker—fourth structural domain; or (f) The third structural domain is connected to the N end of the variable region (VL) structural domain of the SP34 Fab light chain via a PD Linker, and then the third structural domain is sequentially connected to the third structural domain region at the N end of the SP34 Fab light chain variable region (VL) structural domain via a 218 Linker. The third structural domain is then sequentially connected to the third structural domain region at the N end of the SP34 Fab light chain variable region (VL) structural domain via a 212 Linker, and then the tandem trivalent fourth structural domain is connected to the C end of the light chain via a (G4S)3 Linker. From the N end to the C end, a recombinant light chain is formed with the structure: third structural domain—212 Linker—third structural domain—218 Linker—third structural domain—PD Linker—light chain variable region—light chain constant region—(G4S)3 Linker—fourth structural domain—(G4S)3 Linker—fourth structural domain—(G4S)3 Linker—fourth structural domain; or (g) The third structural domain is connected to the C-end of the constant region (CL) structural domain of the SP34 Fab light chain via a (G4S)3 Linker. A second third structural domain is connected in series to the C-end of the third structural domain via a 218 Linker. Then, a third third structural domain is connected in series to the C-end of the third structural domain via a 212 Linker. From the N-end to the C-end, a recombinant light chain is formed with the structure: light chain variable region—light chain constant region—(G4S)3 Linker—third structural domain—218 Linker—third structural domain—212 Linker—third structural domain; or (h) The third structural domain is connected to the C end of the SP34 Fab light chain constant region (CL) structural domain via (G4S)3 Linker. The second third structural domain is connected in series to the C end of the third structural domain via 218 Linker. The third third structural domain is connected in series to the C end of the third structural domain via 212 Linker. The fourth structural domain is connected to the C end of the third structural domain via (G4S)3 Linker. From the N end to the C end, a recombinant light chain is formed with the structure of light chain variable region—light chain constant region—(G4S)3 Linker—third structural domain—218 Linker—third structural domain—212 Linker—third structural domain—(G4S)3 Linker—fourth structural domain.

[0023] The present invention further discloses a method for constructing a dimeric multifunctional antibody molecule with an extended half-life that targets the immune effector T cell antigen CD3 and mediates T cell relocalization to tumor cells.

[0024] The method for constructing the multifunctional antibody is based on the first domain—the Fab of the anti-CD3 monoclonal antibody (clone SP34)—as the structural basis. The second domain consists of a nanobody (VHH) targeting Her2 positive targets and an scFv targeting CD19 positive targets. The third domain consists of a nanobody that co-stimulates CD28 molecules on the surface of T cells, a nanobody that co-stimulates 4-1BB molecules on the surface of T cells, and a nanobody targeting CD22 positive targets. The fourth domain is an albumin-binding domain (ABDCon). The three domains are respectively linked to the SP34 Fab fragment or the second and third domains via a linker.

[0025] The construction method, taking the anti-CD28-VHH domain as an example, includes the following steps: (1) The first domain is CD3 monoclonal antibody Fab; (2) The heavy chain structural domain of the Her2-VHH connected to the SP34 Fab structure.

[0026] (3) Anti-CD28-VHH is connected to the N-terminus of the light chain in the SP34 Fab structure in a series of three valences; (4) Single, double, and triple albumin-binding domains are linked to the C-terminus of the light chain of the SP34 Fab structure; (5) The recombinant heavy and light chains obtained in steps (2) and (3) are bound together by disulfide bonds in CH1 and CL of SP34-Fab to form a heterodimer, namely the anti-Her2 / CD3 / CD28-ABDCon multifunctional antibody.

[0027] In a preferred embodiment, step (2) can be constructed or linked in the following manner: anti-Her2-VHH is linked to the C-terminal 228C region of the SP34 Fab heavy chain constant region (CH1) domain via a flexible linker (G4S)3 Linker.

[0028] In a preferred embodiment, step (3) can be constructed or linked as follows: anti-CD28-VHH is linked to the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a rigid linker PD Linker, and anti-CD28-VHH is tandemly linked to the anti-CD28-VHH region at the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a flexible linker 218 Linker, and then anti-CD28-VHH is tandemly linked to the anti-CD28-VHH region at the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a flexible linker 212 Linker. The recombinant light chain forms a recombinant light chain from the N-terminus to the C-terminus consisting of anti-CD28-VHH—212 Linker—anti-CD28-VHH—218 Linker—anti-CD28-VHH—PD Linker—SP34 Fab light chain variable region—SP34 Fab light chain constant region.

[0029] In a preferred embodiment, step (4) may be constructed or connected in one of the following ways: (a) Anti-CD28-VHH is linked to the N-terminus of the variable region (VL) domain of the SP34 Fab light chain via a rigid linker PD Linker, and anti-CD28-VHH is tandemly linked to the anti-CD28-VHH region at the N-terminus of the SP34 Fab light chain variable region (VL) domain via a flexible linker 218 Linker. Then, anti-CD28-VHH is tandemly linked to the anti-CD28-VHH region at the N-terminus of the SP34 Fab light chain variable region (VL) domain via a flexible linker 212 Linker, and ABDCon is linked to the C-terminus of the light chain via a (G4S)3 Linker. The structure formed sequentially from the N-terminus to the C-terminus is: anti-CD28-VHH—212 Linker—anti-CD28-VHH—218 Linker—anti-CD28-VHH—PD Linker—SP34 Fab light chain variable region—SP34 Fab light chain constant region—(G4S)3 Linker—ABDCon; or; (b) Anti-CD28-VHH was linked to the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a rigid linker PD Linker. Anti-CD28-VHH was then tandemly linked to the anti-CD28-VHH region at the N-terminus of the SP34 Fab light chain variable region (VL) domain using a flexible linker 218 Linker. Furthermore, anti-CD28-VHH was tandemly linked to the anti-CD28-VHH region at the N-terminus of the SP34 Fab light chain variable region (VL) domain using a flexible linker 212 Linker. Finally, a tandem bivalent ABDCon was linked to the C-terminus of the light chain using a (G4S)3 Linker. The resulting structure from N-terminus to C-terminus was: anti-CD28-VHH—212 Linker—anti-CD28-VHH—218 Linker—anti-CD28-VHH—PD Linker—SP34 Fab light chain variable region—SP34 Fab The structure of the light chain constant region —(G4S)3Linker—ABDCon—(G4S)3Linker—ABDCon; or; (c) Anti-CD28-VHH is linked to the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a rigid linker PD Linker. Anti-CD28-VHH is then linked in series to the anti-CD28-VHH region at the N-terminus of the SP34 Fab light chain variable region (VL) domain using a flexible linker 218 Linker. Furthermore, anti-CD28-VHH is linked in series to the anti-CD28-VHH region at the N-terminus of the SP34 Fab light chain variable region (VL) domain using a flexible linker 212 Linker. Finally, a trivalent ABDCon is linked to the C-terminus of the light chain using a (G4S)3 Linker. The resulting structure from N-terminus to C-terminus is: anti-CD28-VHH—212 Linker—anti-CD28-VHH—218 Linker—anti-CD28-VHH—PD Linker—SP34 Fab light chain variable region—SP34 Fab The structure of the light chain constant region —(G4S)3Linker—ABDCon—(G4S)3Linker—ABDCon—(G4S)3Linker—ABDCon.

[0030] In a preferred embodiment, the Her2 / CD3 / CD28-ABDCon multifunctional antibody described in step (5) is constructed using an anti-CD3 Fab structure as the backbone, based on the construction method shown in step (3), in combination with the construction method described in step (2), to obtain Her2 / CD3 / CD28-ABDCon multifunctional antibodies with different ABDCon titers, i.e., different half-life extension effects, as shown below: (a) Anti-CD28-VHH was linked to the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a rigid linker PD Linker, and anti-CD28-VHH was linked to the anti-CD28-VHH region at the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a flexible linker 218 Linker. Then, anti-CD28-VHH was linked to the anti-CD28-VHH region at the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a flexible linker 212 Linker, and monovalent ABDCon was linked to the C-terminus of the light chain using a (G4S)3 Linker, labeled Her2 / CD3 / CD28-ABDCon1. (b) Anti-CD28-VHH was linked to the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a rigid linker PD Linker, and anti-CD28-VHH was tandemly linked to the anti-CD28-VHH region at the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a flexible linker 218 Linker. Then, anti-CD28-VHH was tandemly linked to the anti-CD28-VHH region at the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a flexible linker 212 Linker, and tandemly linked to the C-terminus of the light chain using a (G4S)3 Linker, labeled Her2 / CD3 / CD28-ABDCon2. (c) Anti-CD28-VHH was linked to the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a rigid linker PD Linker, and anti-CD28-VHH was tandemly linked to the anti-CD28-VHH region at the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a flexible linker 218 Linker. Then, anti-CD28-VHH was tandemly linked to the anti-CD28-VHH region at the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a flexible linker 212 Linker, and tandemly linked to the C-terminus of the light chain using a (G4S)3 Linker, labeled Her2 / CD3 / CD28-ABDCon3.

[0031] In a preferred embodiment, using the method, a multifunctional antibody was constructed based on structure optimization design. The CD3-HC (SP34) sequence is shown in SEQ ID NO: 1, 2; the CD3-LC (SP34) sequence is shown in SEQ ID NO: 3, 4; the Her2-VHH sequence is shown in SEQ ID NO: 5, 6; the CD28-VHH sequence is shown in SEQ ID NO: 7, 8; and the ABDCon sequence is shown in SEQ ID NO: 9, 10.

[0032] In a preferred embodiment, the amino acid sequence of the linker peptide is shown in Table 1: Table 1

[0033] In a preferred embodiment, the Her2 / CD3 / CD28 trispecific antibody was constructed based on structural optimization design using the method described above. The Her2 / CD3 / CD28 trispecific antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO: 15 and the heavy chain DNA sequence shown in SEQ ID NO: 16, and the light chain amino acid sequences shown in SEQ ID NO: 17, 19, 21 and the light chain DNA sequences shown in SEQ ID NO: 18, 20, 22.

[0034] More preferably, the Her2 / CD3 / CD28-ABDCon multifunctional antibody comprises heavy chain amino acids and DNA sequences as shown in SEQ ID NO: 15, 16, and light chain amino acids and DNA sequences as shown in SEQ ID NO: 17, 18; and / or The Her2 / CD3 / CD28-ABDCon multifunctional antibody comprises heavy chain amino acids and DNA sequences as shown in SEQ ID NO: 15, 16, and light chain amino acids and DNA sequences as shown in SEQ ID NO: 19, 20; and / or The Her2 / CD3 / CD28-ABDCon multifunctional antibody comprises heavy chain amino acids and DNA sequences as shown in SEQ ID NO: 15, 16, and light chain amino acids and DNA sequences as shown in SEQ ID NO: 21, 22.

[0035] This invention further discloses a method for constructing a CD19 / CD22 / CD3-ABDCon multifunctional antibody molecule. The construction method includes the following steps: (1) The anti-CD22-VHH was connected to the C-terminus of the SP34 Fab light chain constant region (CL) domain via (G4S)3 Linker, and ABDCon was connected to the C-terminus of CD22-VHH via 218 Linker. The structure formed from the N-terminus to the C-terminus was SP34 Fab light chain variable region—SP34 Fab light chain constant region—(G4S)3 Linker—anti-CD22-VHH—218 Linker—ABDCon. The heavy chain was expressed as SP34 Fab heavy chain variable region—SP34 Fab heavy chain constant region—(G4S)3 Linker—CD19-scFv, labeled as CD19 / CD22 / CD3-ABDCon LC; (2) The anti-CD19-scFv was linked to the C-terminus of the constant region (CH) domain of the SP34 Fab heavy chain via the (G4S)3 Linker, and then the ABDCon was linked to the C-terminus of the anti-CD19-scFv via the 218 Linker. The structure formed from the N-terminus to the C-terminus was SP34Fab heavy chain variable region—SP34 Fab heavy chain constant region—(G4S)3 Linker—CD19-scFv—218 Linker—ABDCon. The light chain was expressed as SP34 Fab light chain variable region—SP34 Fab light chain constant region—(G4S)3 Linker—anti-CD22-VHH, and labeled as CD19 / CD22 / CD3-ABDCon HC.

[0036] In a preferred embodiment, a multifunctional antibody was constructed using the method based on structure optimization design. The CD3-HC (SP34) sequence is shown in SEQ ID NO: 1, 2; the CD3-LC (SP34) sequence is shown in SEQ ID NO: 3, 4; the CD19-scFv sequence is shown in SEQ ID NO: 23, 24; the CD22-VHH sequence is shown in SEQ ID NO: 25, 26; and the ABDCon sequence is shown in SEQ ID NO: 9, 10.

[0037] In a preferred embodiment, the CD19 / CD22 / CD3-ABDCon multifunctional antibody was constructed based on structural optimization design using the method described above. The CD19 / CD22 / CD3-ABDCon multifunctional antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO: 27, 31 and the heavy chain DNA sequence shown in SEQ ID NO: 28, 32, and the light chain amino acid sequence shown in SEQ ID NO: 29, 33 and the light chain DNA sequence shown in SEQ ID NO: 30, 34.

[0038] More preferably, the CD19 / CD22 / CD3-ABDCon multifunctional antibody comprises heavy chain amino acids and DNA sequences as shown in SEQ ID NO: 27, 28, and light chain amino acids and DNA sequences as shown in SEQ ID NO: 29, 30; and / or The CD19 / CD22 / CD3-ABDCon multifunctional antibody comprises heavy chain amino acids and DNA sequences as shown in SEQ ID NO: 31, 32, and light chain amino acids and DNA sequences as shown in SEQ ID NO: 33, 34.

[0039] This invention further discloses a method for constructing a Her2 / CD3 / 4-1BB-ABDCon multifunctional antibody molecule. The construction method includes the following steps: (1) Anti-4-1BB-VHH is linked to the C-terminus of the constant region (CL) domain of the SP34 Fab light chain via (G4S)3 Linker. The second anti-4-1BB-VHH is tandemly linked to the C-terminus of the first anti-4-1BB-VHH via 218 Linker. The third anti-4-1BB-VHH is then tandemly linked to the C-terminus of the second anti-4-1BB-VHH via 212 Linker. The albumin-binding domain is then linked to the C-terminus of anti-4-1BB-VHH via GS(G4S)3 Linker. The structure formed from the N-terminus to the C-terminus is SP34 Fab light chain variable region—SP34 Fab light chain constant region—(G4S)3 Linker—anti-4-1BB-VHH—218 Linker—anti-4-1BB-VHH—212 Linker—anti-4-1BB-VHH—GS(G4S)3 Linker—ABDCon. The heavy chain is SP34. Fab heavy chain variable region—SP34 Fab heavy chain constant region—(G4S)3 Linker—expressed against Her2-VHH, labeled Her2 / CD3 / 4-1BB-ABDCon LC; (2) ABDCon is connected to the C-terminus of anti-Her2-VHH via GS(G4S)3 Linker, and anti-Her2-VHH is connected to the C-terminus of the SP34 Fab heavy chain constant region (CH) domain via (G4S)3 Linker. The structure formed from the N-terminus to the C-terminus is SP34 Fab heavy chain variable region—SP34 Fab heavy chain constant region—(G4S)3 Linker—anti-Her2-VHH—GS(G4S)3 Linker—ABDCon. The light chain is SP34 Fab light chain variable region—SP34 Fab light chain constant region—(G4S)3 Linker—anti-4-1BB-VHH—218 Linker—anti-4-1BB-VHH—212 Linker—anti-4-1BB-VHH for expression, labeled as Her2 / CD3 / 4-1BB-ABDCon HC.

[0040] In a preferred embodiment, a multifunctional antibody was constructed using the method based on structure optimization design. The CD3-HC (SP34) sequence is shown in SEQ ID NO: 1, 2; the CD3-LC (SP34) sequence is shown in SEQ ID NO: 3, 4; the Her2-VHH sequence is shown in SEQ ID NO: 5, 6; the 4-1BB-VHH sequence is shown in SEQ ID NO: 35, 36; and the ABDCon sequence is shown in SEQ ID NO: 9, 10.

[0041] In a preferred embodiment, the Her2 / CD3 / 4-1BB-ABDCon multifunctional antibody was constructed based on structural optimization design using the method described above. The Her2 / CD3 / 4-1BB-ABDCon multifunctional antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO: 15,39 and the heavy chain DNA sequence shown in SEQ ID NO: 16,40, and the light chain amino acid sequence shown in SEQ ID NO: 37,41 and the light chain DNA sequence shown in SEQ ID NO: 38,42.

[0042] More preferably, the Her2 / CD3 / 4-1BB-ABDCon multifunctional antibody comprises heavy chain amino acids and DNA sequences as shown in SEQ ID NO: 15, 16, and light chain amino acids and DNA sequences as shown in SEQ ID NO: 37, 38; and / or The Her2 / CD3 / 4-1BB-ABDCon multifunctional antibody comprises heavy chain amino acids and DNA sequences as shown in SEQ ID NO: 39, 40, and light chain amino acids and DNA sequences as shown in SEQ ID NO: 41, 42.

[0043] A fifth aspect of the present invention also provides a method for preparing the long-acting multifunctional antibody of the present invention. This method includes: (1) Obtain the fusion gene of the long-acting multifunctional antibody and construct the expression vector of the long-acting multifunctional antibody; (2) The above expression vector was transfected into host cells using genetic engineering methods; (3) The host cells described above are cultured under conditions that allow the production of the long-acting multifunctional antibody; (4) The antibody protein produced is separated and purified.

[0044] The expression vector in step (1) can be a eukaryotic expression vector such as pFuse, pSeqtag, pCMV, pcDNA, pFastBac1, pPIC9K, pcAGGS, etc., or a prokaryotic expression vector such as pET, pGEX, pMAL, pQE, pTrc, pBV, pTXB, with eukaryotic expression vectors being preferred.

[0045] The host cell in step (2) can be Escherichia coli, Bacillus thuringiensis, Pichia pastoris, insect cells, 293 suspension cells, and Chinese insect ovarian cells, with 293 suspension cells being preferred.

[0046] A sixth aspect of the invention provides the use of the antibody in the manufacture of a medicament for treating cancer. The cancers include, but are not limited to, breast cancer, colorectal cancer, anal cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, head and neck cancer, nasopharyngeal cancer, skin cancer, melanoma, ovarian cancer, prostate cancer, urethral cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, brain tumors, gliomas, neuroblastomas, esophageal cancer, gastric cancer, liver cancer, kidney cancer, bladder cancer, cervical cancer, endometrial cancer, thyroid cancer, eye cancer, sarcoma, bone cancer, leukemia, myeloma, or lymphoma.

[0047] Compared with the prior art, the present invention has the following beneficial effects: This invention further optimizes the half-life of the multifunctional antibody platform: by using genetic engineering, a flexible linker (containing a (G4S)3 repeat sequence) is directionally coupled to a specific site of an optimized trispecific antibody, and then an albumin-binding domain conjugate is bound to the other end of the linker, ultimately constructing a multifunctional antibody molecule that integrates "targeted binding - immune activation - long-term circulation".

[0048] The design advantages of this multifunctional antibody are reflected in three aspects: ① Long-lasting effect: ABDCon can specifically bind to endogenous serum albumin (HSA) with a nanomolar dissociation constant (Kd) to form a complex with a large molecular weight, effectively avoiding renal filtration clearance, extending the antibody's in vivo half-life to 100+ hours, and significantly reducing the dosing frequency (it is expected that clinical administration can be adjusted to once every 3 to 7 days). ② Functional integrity: The flexible design of the linker and the selection of directional coupling sites can maximize the preservation of the activity of each functional domain of the antibody—the Her2 / CD19 / CD22 targeting domain maintains high specificity binding to tumor antigens, the CD3 targeting domain ensures T cell recruitment, and the CD28 / 4-1BB co-stimulatory domain transmits activation signals without significant functional interference; ③ Cross-tumor applicability: The platform's differentiated target design for solid tumors (Her2-positive breast cancer, gastric cancer) and hematologic malignancies (CD19 / CD22-positive B-cell lymphoma, leukemia) gives it the potential for cross-tumor treatment.

[0049] This multifunctional antibody platform, through a step-by-step development strategy of "structural optimization - toxicity elimination - half-life extension," not only solves the core defects of traditional trispecific antibodies but also achieves a synergistic improvement in antitumor activity and pharmacokinetic performance. It lays the foundation for building a universal antibody platform for tumor immunotherapy and is expected to provide a new technical solution for the precision treatment of solid tumors and hematological malignancies.

[0050] In summary, this invention further optimizes the antibody platform. By using a linker to directionally conjugate ABDCon to specific regions of the novel Her2 / CD3 / CD28, CD19 / CD22 / CD3, and Her2 / CD3 / 4-1BB trispecific antibodies with optimized structures, a multifunctional antibody molecule with targeted binding, immune activation, and long-lasting circulating functions is successfully constructed. This significantly improves the in vivo half-life of the drug, reduces the dosage and frequency of administration, and enhances efficacy. It also preserves the functions of other antibody domains to the greatest extent, enabling the expansion of treatment for various tumor types, including solid tumors and hematological malignancies. It is expected to be developed into a universal drug for tumor immunotherapy. Attached Figure Description

[0051] Figure 1 Schematic diagram of multifunctional antibody designed based on structure optimization and activity enhancement with extended half-life; Figure 2 Multifunctional antibodies with different structures of Her2 / CD3 / CD28-ABDCon showed their effects on CD8+ in co-incubation of Her2-positive K562-Her2 tumor cells in PBMCs. + T cells and CD4 + Comparison of T cell activation function; Figure 3 Comparison of the tumor cell killing ability of Her2 / CD3 / CD28-ABDCon multifunctional antibodies with different structures; Figure 4 Pharmacokinetic assay of Her2 / CD3 / CD28-ABDCon multifunctional antibodies with different structures; Figure 5 Comparison of the in vivo antitumor activity of Her2 / CD3 / CD28-ABDCon1 multifunctional antibody with extended half-life and optimized structure with Her2 / CD3 / CD28 LTN and SAR442316 in HCC1954 loaded mice; Figure 6 A multifunctional antibody with an extended half-life and optimized structure, Her2 / CD3 / CD28-ABDCon1, and Her2 / CD3 / CD28 LTN, SAR442316, demonstrated specific CD8-specific antitumor therapy in HCC1954-loaded mice. + T cells and CD4 + T cell content detection; Figure 7 Multifunctional antibodies with different structures of CD19 / CD22 / CD3-ABDCon showed effects on CD8+ in co-incubation of CD19 / CD22-positive tumor cells in PBMCs. + T cells and CD4 + Comparison of T cell activation function; Figure 8 Comparison of the tumor cell killing ability of multifunctional antibodies with different structures of CD19 / CD22 / CD3-ABDCon; Figure 9 Pharmacokinetic assay of CD19 / CD22 / CD3-ABDCon multifunctional antibodies with different structures; Figure 10 In vivo imaging of the antitumor effects of CD19 / CD22 / CD3-ABDCon multifunctional antibodies with different structures in Nalm6 loaded mice; Figure 11 Statistical analysis of the average fluorescence intensity of the in vivo antitumor effects of CD19 / CD22 / CD3-ABDCon multifunctional antibodies with different structures in Nalm6 loaded mice; Figure 12 Body weight monitoring in Nalm6 loaded mice using CD19 / CD22 / CD3-ABDCon multifunctional antibodies with different structures; Figure 13 A multifunctional CD19 / CD22 / CD3-ABDCon antibody with an extended half-life and optimized structure demonstrated specific CD8+ antitumor therapy in Nalm6-loaded mice. + T cells and CD4 + T cell content detection; Figure 14 Comparison of the binding ability of Her2 / CD3 / 4-1BB-ABDCon multifunctional antibodies with different structures; Figure 15 Comparison of the tumor cell killing ability of Her2 / CD3 / 4-1BB-ABDCon multifunctional antibodies with different structures; Figure 16 Pharmacokinetic assay of Her2 / CD3 / 4-1BB-ABDCon multifunctional antibodies with different structures; Figure 17 Comparison of the in vivo antitumor activity of Her2 / CD3 / 4-1BB-ABDCon multifunctional antibodies with different structures and Her2 / CD3 / 4-1BB LTC in tumor-bearing mice; Figure 18 Her2 / CD3 / 4-1BB-ABDCon multifunctional antibodies with different structures and Her2 / CD3 / 4-1BB LTC in tumor-bearing mice with specific CD8 + T cells and CD4 + T-cell content detection. Detailed Implementation

[0052] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0053] The present invention will be further explained and described below with reference to the embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention.

[0054] In the following embodiments, all materials used in the experiments can be purchased or prepared with reference to existing publicly available techniques; materials whose source and specifications are not specified are commercially available; and various processes and methods not described in detail are conventional methods known in the art.

[0055] Example 1 Construction of multifunctional antibodies 1.1 Carrier Construction Construction of a single-valent albumin-binding domain Her2 / CD3 / CD28-ABDCon1 expression vector: The heavy chain of the Her2 / CD3 / CD28-ABDCon1 multifunctional antibody is composed of CD3-HC (SP34) and anti-Her2-VHH (2Rs15d) linked in the following manner: anti-Her2-VHH is linked to the C-terminus of the CD3-HC constant region (CH1) domain via a flexible linker (G4S)3 Linker; the light chain is composed of CD3-LC (SP34), anti-CD28-VHH, and the albumin-binding domain linked in the following manner: anti-CD28-VHH is linked to the N-terminus of the SP34 Fab light chain variable region (VL) domain via a rigid linker (PD Linker), and anti-CD28-VHH is tandemly linked to SP34 via a flexible linker (218 Linker). The anti-CD28-VHH region at the N-terminus of the variable region (VL) domain of the Fab light chain is then linked to the anti-CD28-VHH region at the N-terminus of the Fab light chain variable region (VL) domain via a flexible linker 212 linker, and ABDCon is linked to the C-terminus of the light chain via a (G4S)3 linker. The encoding genes for CD3-HC-Her2-VHH and CD28-VHH-CD28-VHH-CD28-VHH-CD3-LC-ABDCon were synthesized using conventional molecular biology methods. The synthesized genes were then inserted into the pCAGGS eukaryotic expression vector with Amp resistance via homologous recombination. Depending on the linking method, the multifunctional antibody expressed later was labeled Her2 / CD3 / CD28-ABDCon1. The anti-Her2-VHH region can be linked to CD3-HC via the (G4S)3 linker selected in this experiment, or alternative linkers known to those skilled in the art can be used. The relevant sequences are shown in Table 2: Table 2

[0056] Construction of the dual-valent albumin-binding domain Her2 / CD3 / CD28-ABDCon2 expression vector: The heavy chain of the Her2 / CD3 / CD28-ABDCon2 multifunctional antibody is composed of CD3-HC (SP34) and anti-Her2-VHH (2Rs15d) linked in the following manner: anti-Her2-VHH is linked to the C-terminus of the CD3-HC constant region (CH1) domain via a flexible linker (G4S)3 Linker; the light chain is composed of CD3-LC (SP34), anti-CD28-VHH, and the albumin-binding domain linked in the following manner: anti-CD28-VHH is linked to the N-terminus of the SP34 Fab light chain variable region (VL) domain via a rigid linker (PD Linker), and anti-CD28-VHH is tandemly linked to SP34 via a flexible linker (218 Linker). The anti-CD28-VHH region at the N-terminus of the variable region (VL) domain of the Fab light chain is then linked to the anti-CD28-VHH region at the N-terminus of the VL domain of the SP34 Fab light chain via a flexible linker 212 linker, and a tandem dual-valent ABDCon is linked to the C-terminus of the light chain via a (G4S)3 linker. The encoding genes for CD3-HC-Her2-VHH and CD28-VHH-CD28-VHH-CD28-VHH-CD3-LC-ABDCon-ABDCon were synthesized using conventional molecular biology methods. These synthesized genes were then inserted into the pCAGGS eukaryotic expression vector with Amp resistance via homologous recombination. Based on the linking method, the multifunctional antibody expressed later was labeled Her2 / CD3 / CD28-ABDCon2. The anti-Her2-VHH linker, in addition to being linked to CD3-HC via the (G4S)3 linker selected in this experiment, can also be replaced with a linker peptide known to those skilled in the art. The relevant sequences are shown in Table 3. Table 3

[0057] Construction of a triple-valent albumin-binding domain Her2 / CD3 / CD28-ABDCon3 expression vector: The heavy chain of the Her2 / CD3 / CD28-ABDCon3 multifunctional antibody is composed of CD3-HC (SP34) and anti-Her2-VHH (2Rs15d) linked in the following manner: anti-Her2-VHH is linked to the C-terminus of the CD3-HC constant region (CH1) domain via a flexible linker (G4S)3 Linker; the light chain is composed of CD3-LC (SP34), anti-CD28-VHH, and the albumin-binding domain linked in the following manner: anti-CD28-VHH is linked to the N-terminus of the SP34 Fab light chain variable region (VL) domain via a rigid linker (PD Linker), and anti-CD28-VHH is tandemly linked to SP34 via a flexible linker (218 Linker). The anti-CD28-VHH region at the N-terminus of the variable region (VL) domain of the Fab light chain is then linked to the anti-CD28-VHH region at the N-terminus of the VL domain of the SP34 Fab light chain via a flexible linker 212 linker. A tandemly linked trivalent ABDCon is then attached to the C-terminus of the light chain via a (G4S)3 linker. The encoding genes for CD3-HC-Her2-VHH and CD28-VHH-CD28-VHH-CD28-VHH-CD3-LC-ABDCon-ABDCon-ABDCon were synthesized using conventional molecular biology methods. These synthesized genes were then inserted into the Amp-resistant pCAGGS eukaryotic expression vector via homologous recombination. Based on the linking method, the multifunctional antibody expressed later was labeled Her2 / CD3 / CD28-ABDCon3. The anti-Her2-VHH linker, in addition to being linked to CD3-HC via the (G4S)3 linker selected in this experiment, can also be replaced with a linker peptide known to those skilled in the art. The relevant sequences are shown in Table 4. Table 4

[0058] Construction of a single-valent albumin-binding domain CD19 / CD22 / CD3-ABDCon expression vector: (1) The heavy chain of the CD19 / CD22 / CD3-ABDCon multifunctional antibody is composed of CD3-HC (SP34) and anti-CD19-scFv or CD3-HC (SP34), anti-CD19-scFv and albumin binding domain are linked in the following ways: anti-CD19-scFv is linked to the C-terminus of the CD3-HC constant region (CH1) domain by a flexible linker (G4S)3 Linker; the light chain is composed of CD3-LC (SP34), anti-CD22-VHH and albumin binding domain are linked in the following ways: anti-CD22-VHH is linked to the C-terminus of the SP34 Fab light chain constant region (CL) domain by (G4S)3 Linker, and the albumin binding domain is linked to the C-terminus of anti-CD22-VHH by 218 Linker. The encoding genes for CD3-HC-CD19-scFv and CD3-LC-CD22-VHH-ABDCon were synthesized using conventional molecular biology methods. These synthesized genes were then inserted into the Amp-resistant pCAGGS eukaryotic expression vector via homologous recombination. Based on the ligation method, the multifunctional antibody expressed later was labeled CD19 / CD22 / CD3-ABDCon LC. The anti-CD19-scFv and anti-CD22-VHH antibodies could be ligated at the C-terminus of CD3 using the (G4S)3 linker selected in this experiment, or alternatively using linker peptides known to those skilled in the art. The relevant sequences are shown in Table 5. Table 5

[0059] (2) The heavy chain of the CD19 / CD22 / CD3-ABDCon multifunctional antibody is composed of CD3-HC (SP34) and anti-CD19-scFv or CD3-HC (SP34), anti-CD19-scFv and albumin binding domain are linked in the following ways: anti-CD19-scFv is linked to the C-terminus of the CD3-HC constant region (CH1) domain by a flexible linker (G4S)3 Linker, and the albumin binding domain is linked to the C-terminus of anti-CD19-scFv by a 218 Linker; the light chain is composed of CD3-LC (SP34) and anti-CD22-VHH are linked in the following ways: anti-CD22-VHH is linked to the C-terminus of the SP34 Fab light chain constant region (CL) domain by a (G4S)3 Linker. The encoding genes for CD3-HC-CD19-scFv-ABDCon and CD3-LC-CD22-VHH were synthesized using conventional molecular biology methods. These synthesized genes were then inserted into the Amp-resistant pCAGGS eukaryotic expression vector via homologous recombination. Based on the ligation method, the multifunctional antibody expressed later was labeled CD19 / CD22 / CD3-ABDCon HC. The anti-CD19-scFv and anti-CD22-VHH antibodies could be ligated at the C-terminus of CD3 using the (G4S)3 linker selected in this experiment, or alternatively using linker peptides known to those skilled in the art. The relevant sequences are shown in Table 6. Table 6

[0060] Construction of the Her2 / CD3 / 4-1BB-ABDCon expression vector with a single albumin binding domain: (1) The heavy chain of the Her2 / CD3 / 4-1BB-ABDCon multifunctional antibody is composed of CD3-HC (SP34) and anti-Her2-VHH (2Rs15d) or CD3-HC (SP34), anti-Her2-VHH (2Rs15d) and albumin-binding domain are linked in the following ways: anti-Her2-VHH is linked to the C-terminus of the CD3-HC constant region (CH1) domain via a flexible linker (G4S)3 Linker; the light chain is composed of CD3-LC (SP34), anti-4-1BB-VHH and albumin-binding domain are linked in the following ways: anti-4-1BB-VHH is linked to the C-terminus of the SP34 Fab light chain constant region (CL) domain via (G4S)3 Linker, and the second anti-4-1BB-VHH is tandemly linked to the C-terminus of the first anti-4-1BB-VHH via a 218 Linker, and then linked to the C-terminus of the first anti-4-1BB-VHH via a 212 Linker. The linker tandemly links the third anti-4-1BB-VHH to the C-terminus of the second anti-4-1BB-VHH, and then uses a (G4S)3 linker to link the albumin-binding domain to the C-terminus of the anti-4-1BB-VHH. The encoding genes for CD3-HC-Her2-VHH and CD3-LC-4-1BB-VHH-4-1BB-VHH-4-1BB-VHH-ABDCon were synthesized using conventional molecular biology methods. The synthesized genes were then inserted into the pCAGGS eukaryotic expression vector with Amp resistance via homologous recombination. Depending on the linking method, the multifunctional antibody expressed later was labeled Her2 / CD3 / 4-1BB-ABDCon LC. The anti-Her2-VHH can be linked to CD3-HC using either the (G4S)3 linker selected in this experiment or a linker known to those skilled in the art. The relevant sequences are shown in Table 7. Table 7

[0061] (2) The heavy chain of the Her2 / CD3 / 4-1BB-ABDCon multifunctional antibody is composed of CD3-HC (SP34) and anti-Her2-VHH (2Rs15d) or CD3-HC (SP34), anti-Her2-VHH (2Rs15d) and albumin-binding domain are linked in the following ways: anti-Her2-VHH is linked to the C-terminus of the CD3-HC constant region (CH1) domain via a flexible linker (G4S)3 Linker, and then the albumin-binding domain is linked to the C-terminus of anti-Her2-VHH via a (G4S)3 Linker; the light chain is composed of CD3-LC (SP34) and 4-1BB-VHH are linked in the following ways: anti-4-1BB-VHH is linked to the C-terminus of the SP34Fab light chain constant region (CL) domain via a (G4S)3 Linker, and then the albumin-binding domain is linked to the C-terminus of anti-Her2-VHH via a 218 The linker tandemly links the second anti-4-1BB-VHH to the C-terminus of the first anti-4-1BB-VHH, and then the 212 linker tandemly links the third anti-4-1BB-VHH to the C-terminus of the second anti-4-1BB-VHH. The encoding genes for CD3-HC-Her2-VHH-ABDCon and CD3-LC-4-1BB-VHH-4-1BB-VHH-4-1BB-VHH were synthesized using conventional molecular biology methods. The synthesized genes were then inserted into the pCAGGS eukaryotic expression vector with Amp resistance via homologous recombination. Based on the linking method, the multifunctional antibody expressed later was labeled Her2 / CD3 / 4-1BB-ABDCon HC. The anti-Her2-VHH can be linked to CD3-HC using the (G4S)3 linker selected in this experiment, or alternatively using a linker peptide known to those skilled in the art. The relevant sequences are shown in Table 8. Table 8

[0062] This invention relates to a trispecific antibody structure based on structure optimization and activity design, such as... Figure 1 As shown.

[0063] Example 2: Biological function evaluation of Her2 / CD3 / CD28-ABDCon multifunctional antibody with extended half-life 2.1 Different structural Her2 / CD3 / CD28-ABDCon multifunctional antibodies against CD8 + T cells and CD4 + Comparison of T cell activation capacity PBMCs were thawed in liquid nitrogen and the cell density was adjusted to 1 million / mL. K562-Her2 tumor cells were centrifuged and the density was adjusted to 0.2 million / mL. After mixing with an equal volume, 1 mL / well was seeded back into a 24-well plate. Her2 / CD3 / CD28-ABDCon multifunctional antibodies with different structures were added at a concentration of 100 pM, and the concentration of human serum albumin (HSA) in the culture system was brought to a physiological concentration of 35 g / L. After 24 hours, the cells were collected by centrifugation. Flow cytometry staining was performed using PerCP cy5.5-anti-human CD4; Pacific blue anti-human CD8; APC anti-human CD69; and FITC anti-human CD25. CD8 staining was analyzed by flow cytometry. + T cells and CD4 + T cell CD69 + CD25 + Percentage, comparing activation effects.

[0064] Activation results as follows Figure 2 As shown in the figure, CD8 is represented by CD8. + T cells and CD4 + T cell activation results, compared to the Her2 / CD3 / CD28 LTN trispecific antibody, showed that in the absence of HSA, Her2 / CD3 / CD28-ABDCon1 was less effective against CD8. + T cells and CD4 + T cells showed comparable activation after co-culturing with target cells, and other multifunctional antibodies with different structures of Her2 / CD3 / CD28-ABDCon all showed activity against CD8. + T cells and CD4 + The activation effect of T cells was weakened after co-culturing with target cells. At physiological HSA concentrations, multifunctional antibodies with different structures of Her2 / CD3 / CD28-ABDCon all showed activity against CD8. + T cells and CD4 + The activation effect of T cells was weakened after co-culturing with target cells. Among them, Her2 / CD3 / CD28-ABDCon1 showed a greater effect on CD8 compared to other Her2 / CD3 / CD28-ABDCon structures. + T cells and CD4 + T cells showed the highest level of activation.

[0065] 2.2 In vitro tumor-killing activity of Her2 / CD3 / CD28-ABDCon multifunctional antibodies with different structures Activated T cells were centrifuged once, counted, and their density adjusted. K562-Her2 tumor cells were used and their density adjusted. K562-Her2 cells were labeled with CFSE, and the T cell to K562-Her2 effector-target ratio was adjusted to 5:1. A positive control group was prepared by mixing K562-Her2 with an equal volume of culture medium. The concentration of human serum albumin in the culture system reached a physiological concentration of 35 g / L. 200 μL of 100 nM antibody was prepared in a U-shaped plate, diluted 10-fold to 6 concentration gradients, and 10 μL / well was transferred to each well of a 96-well plate. 100 μL / well of the density-adjusted T cells and tumor cells were added, and 100 μL / well of K562-Her2 mixed with an equal volume of culture medium was added as a positive control group. After incubation for 24 h, the number of viable cells was detected. The percentage of tumor cell killing was calculated by comparing the number of CFSE-positive cells in 7-AAD negative cells with the negative control group.

[0066] The results are as follows Figure 3 The results show the tumor-killing effects of TCE in both TCE-only and physiological HSA concentration environments. Comparison of the percentage of tumor kill revealed that, compared to the Her2 / CD3 / CD28 LTN trispecific antibody, in the absence of HSA, the multifunctional antibodies of different structures of Her2 / CD3 / CD28-ABDCon exhibited better cytotoxicity against Her2-positive cells K562-Her2, comparable to the Her2 / CD3 / CD28 LTN trispecific antibody. However, when HSA reached physiological concentrations, the cytotoxicity of the multifunctional antibodies of different structures of Her2 / CD3 / CD28-ABDCon against Her2-positive cells K562-Her2 decreased, with the Her2 / CD3 / CD28-ABDCon3 showing the most significant decrease in cytotoxicity.

[0067] 2.3 Pharmacokinetic Detection of Her2 / CD3 / CD28-ABDCon Multifunctional Antibodies with Different Structures Pharmacokinetic studies were conducted using 6-8 week old female BALB / c mice to investigate the effects of different structural Her2 / CD3 / CD28-ABDCon multifunctional antibodies (Her2 / CD3 / CD28-ABDCon1, Her2 / CD3 / CD28-ABDCon2, Her2 / CD3 / CD28-ABDCon3, and Her2 / CD3 / CD28 LTN). Animals were randomly assigned to different treatment groups and intravenously injected with a single dose of 5 mg / kg Her2 / CD3 / CD28-ABDCon1, Her2 / CD3 / CD28-ABDCon2, Her2 / CD3 / CD28-ABDCon3, or 1 mg / kg Her2 / CD3 / CD28 LTN. Serum samples were collected at different time points within 15 days post-injection for bioanalysis. Antibody levels in mouse serum were quantified using a sandwich ELISA method as described above. Antibodies were captured using human Her2-Fc and detected with biotinylated goat anti-human κ polyclonal antibody. The dynamic range of this assay was 0.5 ng / mL to 500 ng / mL. The cyclic half-life was calculated by fitting the data to a single-phase exponential decay equation for the determination of the pharmacokinetics of the multifunctional antibody.

[0068] The results are as follows Figure 4 The pharmacokinetic analysis of four different multifunctional antibody molecules is shown. Comparison of the in vivo metabolic half-life of the antibody drugs revealed that, compared with the Her2 / CD3 / CD28 LTN trispecific antibody, the half-life of the Her2 / CD3 / CD28-ABDCon multifunctional antibodies with different structures was significantly prolonged, increasing from 14.72 h to over 70 h. Among them, Her2 / CD3 / CD28-ABDCon1 showed the most significant increase in half-life, directly increasing to 108.46 h. This demonstrates that Her2 / CD3 / CD28-ABDCon1 is the optimal antibody structure for extending half-life.

[0069] 2.4 Validation of the in vivo tumor therapy efficacy of Her2 / CD3 / CD28-ABDCon1 multifunctional antibody The above experimental results demonstrate that Her2 / CD3 / CD28-ABDCon1 is the optimal antibody structure for extended half-life. Six- to eight-week-old female (NCG) mice were subcutaneously implanted with HCC1954 cells and PBMCs on day 0 of the study. Each mouse was implanted with a mixture of 5 × 10^6 HCC1954 cells and 5 × 10^6 PBMCs, using 50% matrix gel. When the tumor volume reached 80 to 120 mm³, the mice were intravenously injected every five days with 20 nmol / kg of Her2 / CD3 / CD28-ABDCon1, Her2 / CD3 / CD28 LTN, and SAR443216, for a total of four administrations. Saline was used as a control. Starting from day 1 of administration, blood samples were collected every three days to detect CD4 levels in the mice. + With CD8 + T cell count. We set a tumor burden of ≥1500 mm³ as the upper limit surrogate. Tumor burden was systematically monitored using calipers, and tumor volume was calculated using the formula: width × length × height. Peripheral blood samples were analyzed by flow cytometry at specified time points.

[0070] The results are as follows Figure 5 The image shows the therapeutic effects of Her2 / CD3 / CD28-ABDCon1 on tumors. Figure 6 The image shows Her2 / CD3 / CD28-ABDCon1 paired with CD8. + T cells and CD4 + T cell in vivo expansion effect Figure 5 The results showed that, compared with the saline treatment group, the Her2 / CD3 / CD28-ABDCon1, Her2 / CD3 / CD28 LTN, and SAR443216 groups all inhibited tumor growth, with the Her2 / CD3 / CD28-ABDCon1 multifunctional antibody showing a more significant inhibitory effect. Furthermore, with prolonged treatment, the Her2 / CD3 / CD28 LTN and SAR443216 groups showed significant relapse after drug withdrawal, while Her2 / CD3 / CD28-ABDCon1 treatment demonstrated long-term tumor control, indicating that this antibody, compared to other structures, mediates a more significant T-cell killing effect on tumor cells and can achieve optimal long-term therapeutic efficacy. Figure 6 As shown, the results indicate that, while killing tumor cells, the Her2 / CD3 / CD28-ABDCon1, Her2 / CD3 / CD28 LTN, and SAR443216 groups all amplified tumor-specific CD4 during treatment. + With CD8 +T cells, particularly tumor-specific CD4 in the Her2 / CD3 / CD28-ABDCon1 treatment group. + With CD8 + The number of T cells was significantly higher than in other groups. By comparing PBMC activation and tumor cell killing activity, as well as the effect of half-life extension, it can be concluded that the Her2 / CD3 / CD28-ABDCon1 multifunctional antibody with optimized structure design has a significant inhibitory and killing effect on solid tumors.

[0071] Example 3: Biological function evaluation of CD19 / CD22 / CD3-ABDCon multifunctional antibody with extended half-life 3.1 Different structural CD19 / CD22 / CD3-ABDCon multifunctional antibodies against CD8 + T cells and CD4 + Comparison of T cell activation capacity PBMCs were thawed in liquid nitrogen and the cell density was adjusted to 1 million / mL. Nalm6 tumor cells were centrifuged and the density was adjusted to 0.2 million / mL. After mixing with an equal volume, 1 mL / well was seeded back into a 24-well plate. Multifunctional antibodies of different structures (CD19 / CD22 / CD3-ABDCon) were added at a concentration of 100 pM, and the concentration of human serum albumin (HSA) in the culture system was brought to a physiological concentration of 35 g / L. After 24 hours, cells were collected by centrifugation. Flow cytometry staining was performed using PerCP cy5.5-anti-human CD4; Pacific blue anti-human CD8; APC anti-human CD69; and FITC anti-human CD25. CD8 staining was analyzed by flow cytometry. + T cells and CD4 + T cell CD69 + CD25 + Percentage, comparing activation effects.

[0072] Activation results as follows Figure 7 The image shows D8. + T cells and CD4 + T cell activation results showed that, compared to the CD19 / CD22 / CD3 CC trispecific antibody, in the absence of HSA, all CD19 / CD22 / CD3-ABDCon multifunctional antibodies exhibited activity against CD8. + T cells and CD4 + The activation effect of T cells was weakened after co-culturing with target cells, while at physiological HSA concentrations, different structural CD19 / CD22 / CD3-ABDCon multifunctional antibodies showed varying effects on CD8. + T cells and CD4+ The activation effect of T cells was significantly reduced after co-culturing with target cells. Among them, CD19 / CD22 / CD3-ABDCon LC showed a greater effect on CD8 activation compared to other CD19 / CD22 / CD3-ABDCon structures. + T cells and CD4 + T cells showed the highest level of activation.

[0073] 3.2 In vitro tumor-killing activity of multifunctional antibodies with different structures of CD19 / CD22 / CD3-ABDCon Activated T cells were centrifuged once, counted, and their density adjusted. Nalm6 and K562-CD19 tumor cells were used to adjust their density, and CFSE-labeled Nalm6 and K562-CD19 tumor cells were used to adjust the T cell to tumor cell effector-to-target ratio to 5:1. A positive control group was prepared by mixing tumor cells with an equal volume of corresponding culture medium. The concentration of human serum albumin in the culture system reached a physiological concentration of 35 g / L. 200 μL of 100 nM antibody was prepared in a U-shaped plate, diluted 10-fold to 6 concentration gradients, and 10 μL / well was transferred to a 96-well plate. 100 μL / well of the density-adjusted T cells and tumor cells were added, and 100 μL / well of the tumor cells were mixed with an equal volume of culture medium as a positive control group and incubated for 24 h. The number of viable cells was detected. The percentage of tumor cell killing was calculated by comparing the number of CFSE-positive cells in 7-AAD negative cells with the negative control group.

[0074] The results are as follows Figure 8 The results show the killing effects of TCE on different tumors in both TCE and physiological concentration HSA environments. Comparison of the percentage of killing revealed that, compared to CD19 / CD22 / CD3 CC trispecific antibodies, the cytotoxicity of different structural CD19 / CD22 / CD3-ABDCon multifunctional antibodies against CD19 / CD22 double-positive cells or CD19 single-positive tumor cells was reduced. Specifically, when HSA reached physiological concentrations, the decrease in cytotoxicity of different structural CD19 / CD22 / CD3-ABDCon multifunctional antibodies against CD19 / CD22 double-positive cells or CD19 single-positive tumor cells was more significant. At high concentrations, CD19 / CD22 / CD3-ABDCon LC exhibited higher cytotoxicity against tumor cells compared to other CD19 / CD22 / CD3-ABDCon structures.

[0075] 3.3 Pharmacokinetic Detection of Multifunctional Antibodies with Different Structures of CD19 / CD22 / CD3-ABDCon Pharmacokinetic studies of different structural CD19 / CD22 / CD3-ABDCon multifunctional antibodies (CD19 / CD22 / CD3-ABDCon LC, CD19 / CD22 / CD3-ABDCon HC, and CD19 / CD22 / CD3 CC) were conducted using 6-8 week old female BALB / c mice. Animals were randomly assigned to different treatment groups and intravenously injected with a single dose of 5 mg / kg CD19 / CD22 / CD3-ABDCon LC, CD19 / CD22 / CD3-ABDCon HC, or 1 mg / kg CD19 / CD22 / CD3 CC. Serum samples were collected at different time points within 15 days post-injection for bioanalytical assays. Antibody levels in mouse serum were quantified using a sandwich ELISA method as described above. Antibodies were captured using human CD3-Fc and detected using a biotinylated goat anti-human κ polyclonal antibody. The dynamic range of this assay was 0.5 ng / mL to 500 ng / mL. Cyclic half-life is calculated by fitting data to a single-phase exponential decay equation and is used for the determination of pharmacokinetics of multifunctional antibodies.

[0076] The results are as follows Figure 9 The diagram shows the pharmacokinetic analysis of three multifunctional antibody molecules with different structures. Comparison of the in vivo metabolic half-life of the antibody drugs revealed that, compared with the CD19 / CD22 / CD3 CC trispecific antibody, the half-life of the CD19 / CD22 / CD3-ABDCon multifunctional antibodies with different structures was significantly prolonged, increasing from 11.34 h to over 30 h. Among them, the CD19 / CD22 / CD3-ABDCon LC showed the most significant increase in half-life, directly increasing to 93.33 h. This demonstrates that CD19 / CD22 / CD3-ABDCon LC is the optimal antibody structure for extending half-life.

[0077] 3.4 In vivo antitumor activity of multifunctional antibodies with different structures of CD19 / CD22 / CD3-ABDCon in Nalm6 loaded mice Nalm6 cells were removed from liquid nitrogen and rapidly thawed in a 37°C water bath. After culturing to a sufficient number in RPMI 1640 medium, the cell density was adjusted, and 0.5 million tumor cells were injected via tail vein into 6-8 week old NSG mice. Four days later, activated T cells were removed, the cell density was adjusted, and 30 million cells per mouse were injected via tail vein into the mice. Two treatments were administered: 200 μL / mouse of CD19 / CD22 / CD3-ABDCon LC, CD19 / CD22 / CD3-ABDCon HC, and CD19 / CD22 / CD3 CC at a concentration of 18.6 nmol / kg, administered once every five days for a total of two treatments. The fluorescence intensity of Nalm6 tumor cells in mice was counted every four days using a small animal in vivo imaging system. The tumor treatment effect was estimated by calculating the average fluorescence intensity, and CD4+ in peripheral blood samples was analyzed by flow cytometry at specified time points. + With CD8 + T cell content and mouse body weight measurement.

[0078] The results are as follows Figures 10-13 The image shows the in vivo therapeutic effect of CD19 / CD22 / CD3-ABDCon LC. Figure 10-11 The results showed that CD19 / CD22 / CD3-ABDCon LC had a significant advantage in in vivo treatment efficacy compared to CD19 / CD22 / CD3-ABDCon HC and CD19 / CD22 / CD3 CC treatment group without prolonged half-life, had a better ability to inhibit tumor growth, and significantly prolonged the survival of mice. Figure 12 This indicates that extending the drug's half-life did not cause a significant increase in toxicity in mice, and its safety is guaranteed. Figure 13 This indicates that, while killing tumor cells, the CD19 / CD22 / CD3-ABDCon LC, CD19 / CD22 / CD3-ABDCon HC, and CD19 / CD22 / CD3 CC groups all amplified tumor-specific CD4 during treatment. + With CD8 + T cells, particularly tumor-specific CD4 in the CD19 / CD22 / CD3-ABDCon LC treatment group. + With CD8 + The number of T cells was significantly higher than in other groups. By comparing PBMC activation and tumor cell killing activity, as well as the effect of half-life extension, it can be concluded that the CD19 / CD22 / CD3-ABDCon LC multifunctional antibody with optimized structure design has significant inhibitory and killing effects on hematological malignancies.

[0079] Example 4: Biological function evaluation of the Her2 / CD3 / 4-1BB-ABDCon multifunctional antibody with extended half-life 4.1 Comparison of binding affinity of Her2 / CD3 / 4-1BB-ABDCon multifunctional antibodies with different structures Her2 ECD-Fc, CD3 ECD-Fc, and 4-1BB ECD-Fc antigen proteins were diluted to 10 µg / mL with PBS and coated into 96-well ELISA plates at 100 µL / well, incubated overnight at 4°C. The liquid in the plate was discarded, and the plate was washed twice with PBST. Then, 200 µL / well of 5% skim milk PBST solution was added, and the plates were blocked at room temperature for 2 h. The blocking solution was then drained from the wells. Her2 / CD3 / 4-1BB antibodies with different structures were diluted 10-fold in eight gradients with blocking buffer (or human serum HSA), starting at 100 nM. Each concentration gradient was applied in two replicates, with 100 µL / well added to each well, and incubated at room temperature for 2 h. After washing the plate three times with PBST and draining, HRP-labeled anti-kappa chain antibody was diluted 1:5000 with 5% skim milk PBST and added to each well at 100 µL / well, and incubated at room temperature for 1 h. After washing the plate three times with PBST and draining it, add 100µL of TMB chromogenic solution to each well and incubate at room temperature in the dark for 10 min. Then, add 50µL of 2M H2SO4 to each well to stop the chromogenic reaction and measure the absorbance at OD450nm using a microplate reader.

[0080] Combining the results as follows Figure 14 The results show the binding ability of three different multifunctional antibody molecules with antigens. Compared with the Her2 / CD3 / 4-1BB LTC trispecific antibody, Her2 / CD3 / 4-1BB-ABDCon showed comparable binding abilities for CD3, Her2, and 4-1BB antigens in the absence of HSA. At physiological HSA concentrations, the binding ability of the Her2 / CD3 / 4-1BB-ABDCon HC multifunctional antibody was no different from that of Her2 / CD3 / 4-1BB LTC, but the binding peak of Her2 / CD3 / 4-1BB-ABDCon LC was lower than that of the Her2 / CD3 / 4-1BB LTC structure.

[0081] 4.2 In vitro tumor-killing activity of Her2 / CD3 / 4-1BB-ABDCon multifunctional antibodies with different structures Activated T cells were centrifuged once, counted, and their density adjusted. CFSE-labeled HCC1954 or SKOV3 tumor cells were used, and their density was adjusted to 0.2 million / mL. The T cell to tumor cell effector-to-target ratio was adjusted to 5:1, and a positive control group was prepared by mixing tumor cells with an equal volume of culture medium. The concentration of human serum albumin in the culture system reached a physiological concentration of 35 g / L. 200 μL of 100 nM antibody was prepared in a U-shaped plate, diluted 10-fold in six concentration gradients, and 10 μL / well was transferred to each well of a 96-well plate. Then, 100 μL / well of the density-adjusted T cells and tumor cells were added, and the tumor cells were mixed with an equal volume of culture medium as a positive control group (100 μL / well). Incubation was performed for 24 h, and the number of viable cells was measured. The percentage of tumor cell killing was calculated by comparing the number of CFSE-positive cells in the 7-AAD negative cells with the negative control group.

[0082] The results are as follows Figure 15 The results show the killing effects of TCE on different tumors in both TCE-only and HSA-containing environments. Comparison of the percentage of killing revealed that, compared to the Her2 / CD3 / 4-1BB LTC trispecific antibody, the Her2 / CD3 / 4-1BB-ABDCon multifunctional antibodies with different structures exhibited better cytotoxicity against Her2-positive tumor cells in the absence of HSA, comparable to the Her2 / CD3 / 4-1BB LTC trispecific antibody. However, when HSA reached physiological concentrations, the cytotoxicity of the Her2 / CD3 / 4-1BB-ABDCon multifunctional antibodies against Her2-positive tumor cells decreased, with the Her2 / CD3 / 4-1BB-ABDCon HC showing the most significant decrease in cytotoxicity.

[0083] 4.3 Pharmacokinetic Detection of Her2 / CD3 / 4-1BB-ABDCon Multifunctional Antibodies with Different Structures Pharmacokinetic studies of Her2 / CD3 / 4-1BB-ABDCon multifunctional antibodies with different structures (Her2 / CD3 / 4-1BB-ABDCon HC, Her2 / CD3 / 4-1BB-ABDCon LC) and Her2 / CD3 / 4-1BB LTC were conducted using 6-8 week old female BALB / c mice. Animals were randomly assigned to different treatment groups and intravenously injected with a single dose of 5 mg / kg of Her2 / CD3 / 4-1BB-ABDCon HC, Her2 / CD3 / 4-1BB-ABDCon LC, or Her2 / CD3 / 4-1BB LTC. Serum samples were collected at different time points within 15 days post-injection for bioanalysis. Antibody levels in mouse serum were quantified using a sandwich ELISA method as described above. Antibodies were captured using human Her2-Fc and detected using a biotinylated goat anti-human κ polyclonal antibody. The dynamic range of this assay was 0.5 ng / mL to 500 ng / mL. Cyclic half-life is calculated by fitting data to a single-phase exponential decay equation and is used for the determination of pharmacokinetics of multifunctional antibodies.

[0084] The results are as follows Figure 16 The results of pharmacokinetic analysis of three multifunctional antibody molecules with different structures are shown. By comparing the in vivo metabolic half-life of the antibody drugs, it was found that compared with the Her2 / CD3 / 4-1BB LTC trispecific antibody, the half-life of Her2 / CD3 / 4-1BB-ABDCon multifunctional antibodies with different structures was significantly prolonged, from 15h to more than 90h. 4.4 Validation of the in vivo tumor therapy efficacy of Her2 / CD3 / 4-1BB-ABDCon multifunctional antibody Six- to eight-week-old female (NCG) mice were subcutaneously implanted with HCC1954 cells on day 0. Each mouse received 5 × 10^6 HCC1954 cells mixed with 50% matrix gel for tumor inoculation. Tumors were inoculated when the tumor volume reached 80 to 120 mm. 3 Mice were intravenously injected every five days with 5 nmol / kg of Her2 / CD3 / 4-1BB LTC, Her2 / CD3 / 4-1BB-ABDCon HC, and Her2 / CD3 / 4-1BB-ABDCon LC for a total of four administrations, with physiological saline serving as a control. Blood samples were collected every three days starting from the first day of administration to measure CD4 levels in the mice. + With CD8 + T cell count. We will have ≥1500 mm 3Tumor burden was set as an upper limit surrogate indicator. Tumor burden was systematically monitored using calipers, and tumor volume was calculated using the formula: width × length × height. Peripheral blood samples were analyzed by flow cytometry at specified time points.

[0085] The results are as follows Figure 17 and Figure 18 (The in vivo therapeutic effects of Her2 / CD3 / 4-1BB-ABDCon HC are shown in the figure.) Figure 17 The results showed that, compared with the saline treatment group, Her2 / CD3 / 4-1BB LTC, Her2 / CD3 / 4-1BB-ABDCon HC, and Her2 / CD3 / 4-1BB-ABDCon LC groups all inhibited tumor growth. The inhibitory effect of the multifunctional antibody Her2 / CD3 / 4-1BB-ABDCon HC was more significant. Furthermore, with prolonged treatment, both Her2 / CD3 / 4-1BB LTC and Her2 / CD3 / 4-1BB-ABDCon LC groups showed significant relapse after drug withdrawal, while Her2 / CD3 / 4-1BB-ABDCon HC treatment demonstrated long-term tumor control, indicating that this antibody, compared to other structures, maintained a more durable anti-tumor ability of T cells. Figure 18 As shown, the results indicate that, while killing tumor cells, the Her2 / CD3 / 4-1BBLTC, Her2 / CD3 / 4-1BB-ABDCon HC, and Her2 / CD3 / 4-1BB-ABDCon LC groups all amplified tumor-specific CD4 during treatment. + With CD8 + T cells, particularly tumor-specific CD4 in the Her2 / CD3 / 4-1BB-ABDCon HC treatment group. + With CD8 + The number of T cells was significantly higher in the later stages of treatment than in other groups. Therefore, by comparing the ability to prolong half-life and the long-term anti-tumor effect in vivo, it can be concluded that the optimized structural design of the Her2 / CD3 / 4-1BB-ABDCon HC multifunctional antibody has significant advantages in inhibiting solid tumors and long-term efficacy.

Claims

1. A multifunctional antibody molecule, characterized in that, The multifunctional antibody molecule uses the Fab fragment of a monoclonal antibody as its structural basis as a first domain, and adds a second, third, and fourth domain. The second domain is a T-cell activation co-stimulatory domain, or a first antibody targeting tumor-specific or related antigens. The third domain is a T-cell activation co-stimulatory domain, or a second antibody targeting tumor-specific or related antigens. The fourth domain is an albumin-binding domain. The second, third, and fourth domains are respectively linked to the Fab fragment via linker peptides, or the fourth domain is linked to the second or third domain via linker peptides, thereby forming a multifunctional antibody molecule with multi-target binding ability and enhanced in vivo stability.

2. The method for constructing the multifunctional antibody molecule according to claim 1, characterized in that, The construction method specifically includes the following steps: (1) The Fab domain of the CD3 monoclonal antibody is used as the first domain; (2) Connecting the second structural domain or the second structural domain / third structural domain to the heavy chain structural domain of the first structural domain Fab; (3) Connecting the second structural domain or the second structural domain / third structural domain to the light chain structural domain of the first structural domain Fab; (4) Connect the fourth structural domain to the heavy chain structural domain or light chain structural domain of the first structural domain Fab; (5) Connect the fourth structural domain to the second or third structural domain; (6) The heavy chain and light chain combine through disulfide bonds in CH1 and CL to form a heterodimer trispecific antibody.

3. The method for constructing the multifunctional antibody molecule according to claim 2, characterized in that, Step (2) is performed in one of the following ways: (a) The second / third domain is linked to the C-terminal 228C region of the SP34 Fab heavy chain constant region (CH1) domain via a linker peptide; or (b) The second / third domain is linked to the C-terminal 228C region of the SP34 Fab heavy chain constant region (CH1) domain via a linker peptide, and the fourth domain is linked to the C-terminus of the second or third domain via a linker peptide; The construction or connection in step (3) is performed in one of the following ways: (a) The third domain is linked to the C-terminus 217C of the SP34 Fab light chain constant region (CL) domain via a linker peptide; or (b) By linking the third domain to the C-terminus 217C of the SP34 Fab light chain constant region (CL) domain via a linker peptide and linking the fourth domain to the C-terminus of the third domain; or (c) A third domain is linked to the N-terminus of the SP34 Fab light chain variable region (VL) domain via a linker peptide, and then the third domain is sequentially linked to the third domain region at the N-terminus of the SP34 Fab light chain variable region (VL) domain via a linker peptide, and again the third domain is sequentially linked to the third domain region at the N-terminus of the SP34 Fab light chain variable region (VL) domain via a linker peptide, forming a recombinant light chain with the structure of third domain—linker peptide—third domain—linker peptide—third domain—linker peptide—light chain variable region—light chain constant region from the N-terminus to the C-terminus; or (d) The third domain is linked to the N-terminus of the variable region (VL) domain of the SP34 Fab light chain via a linker peptide. Then, the third domain is sequentially linked to the N-terminus of the third domain region of the SP34 Fab light chain variable region (VL) domain via a linker peptide. Again, the third domain is sequentially linked to the N-terminus of the third domain region of the SP34 Fab light chain variable region (VL) domain via a linker peptide. Finally, the fourth domain is linked to the C-terminus of the recombinant light chain. From the N-terminus to the C-terminus, a recombinant light chain is formed with the structure: third domain—linker peptide—third domain—linker peptide—third domain—linker peptide—light chain variable region—light chain constant region—linker peptide—fourth domain; or (e) The third domain is linked to the N-terminus of the variable region (VL) domain of the SP34 Fab light chain via a linker peptide, and then the third domain is sequentially linked to the third domain region at the N-terminus of the SP34 Fab light chain variable region (VL) domain via a linker peptide. Again, the third domain is sequentially linked to the third domain region at the N-terminus of the SP34 Fab light chain variable region (VL) domain via a linker peptide, and a tandemly linked bivalent fourth domain is attached to the C-terminus of the recombinant light chain. From the N-terminus to the C-terminus, a recombinant light chain is formed with the structure: third domain—linker peptide—third domain—linker peptide—third domain—linker peptide—light chain variable region—light chain constant region—linker peptide—fourth domain—linker peptide—fourth domain; or (f) The third domain is linked to the N-terminus of the variable region (VL) domain of the SP34 Fab light chain via a linker peptide. Then, the third domain is sequentially linked to the third domain region at the N-terminus of the SP34 Fab light chain variable region (VL) domain via a linker peptide. Again, the third domain is sequentially linked to the third domain region at the N-terminus of the SP34 Fab light chain variable region (VL) domain via a linker peptide. Finally, a tandem trivalent fourth domain is linked to the C-terminus of the recombinant light chain, forming a recombinant light chain with the following structure from N-terminus to C-terminus: third domain—linker peptide—third domain—linker peptide—third domain—linker peptide—light chain variable region—light chain constant region—linker peptide—fourth domain—linker peptide—fourth domain—linker peptide—fourth domain; or (g) A third domain is linked to the C-terminus of the constant region (CL) domain of the SP34 Fab light chain via a linker peptide; a second third domain is tandemly linked to the C-terminus of the third domain via a linker peptide; and a third third domain is tandemly linked to the C-terminus of the third domain via a linker peptide, forming a recombinant light chain with the structure from N-terminus to C-terminus as follows: light chain variable region—light chain constant region—linker peptide—third domain—linker peptide—third domain—linker peptide—third domain; or (h) The third domain is linked to the C-terminus of the constant region (CL) domain of the SP34 Fab light chain via a linker peptide. The second third domain is linked to the C-terminus of the third domain via a linker peptide. The third third domain is linked to the C-terminus of the third domain via a linker peptide. The fourth domain is linked to the C-terminus of the recombinant light chain. From the N-terminus to the C-terminus, a recombinant light chain is formed with the following structure: light chain variable region—light chain constant region—linker peptide—third domain—linker peptide—third domain—linker peptide—third domain—linker peptide—fourth domain. The first, second, and third domains each independently have the ability to select Her2, VEGFR1 / 2, CD3, CD19, CD22, EGFR, EGFRvIII, HER3, HER4, IGF1R, c-Met, MUC-1, MUC-16, IL13R, Trop-2, GPC2, GPC3, GD2, CEA, PSMA, PSCA, EpCAM, CD79, ROR1, AXL, CD133, CD171, BCMA, CD20, CD123, Claudin 6, and Claudin 18.2 Binding specificity of target antigens of CD38, CD30, CD33, CD138, CD56, CS1, CLL1, CD7, CD4, CD8, LewisY, ALK, KRAS mutant, MYD88 mutant, IDH1 mutant, P53 mutant, NY-ESO-1, NKG2D, CD16, CD56, CD64, PD-1, PD-L1, B7-H3, B7-H4, TGF-beta, CTLA-4, LAG-3, TIM-3, TIGHT, VISTA, ICOS, GITR, CD28, 4-1BB, OX40, CSD27, CD24, CD47, CXCR4, DLL3, and Integrin; The second and third domains are Adnectin (human fibronectin), affinity protein, anticalin (anti-carrier protein), bicyclic peptide, DARPin (natural ankyrin repeat sequence), E7 immunoprotein, lymphocyte receptor variable region, single-domain antibody, whole antibody, antibody fragment, single-chain antibody, and nucleic acid aptamer. The second, third, and fourth domains each independently possess binding specificity for Her2, CD19, CD22, CD28, 4-1BB, and HSA / MSA. The linker peptide is selected from one or more of flexible linker peptides, rigid linker peptides, or helical linker peptides; the flexible linker peptide is selected from one or more of (G4S)3 Linker, 218 Linker, or 212 Linker; the rigid linker peptide is selected from PD Linker.

4. The method for constructing the multifunctional antibody molecule according to claim 3, characterized in that, Step (2) is performed in one of the following ways: (a) Connecting the second / third domain to the C-terminal 228C region of the SP34 Fab heavy chain constant region (CH1) domain via the (G4S)3 Linker; or (b) The second / third domain is connected to the C-end 228C region of the SP34 Fab heavy chain constant region (CH1) domain via (G4S)3 Linker, and the fourth domain is connected to the C-end of the second domain via 218 Linker; The construction or connection in step (3) is performed in one of the following ways: (a) The third structural domain is connected to the C-end 217C of the SP34 Fab light chain constant region (CL) structural domain via the (G4S)3 Linker; or (b) The third structural domain is connected to the C-end 217C of the SP34 Fab light chain constant region (CL) structural domain via (G4S)3 Linker, and the fourth structural domain is connected to the C-end of the third structural domain via 218 Linker; or (c) The third structural domain is connected to the N end of the SP34 Fab light chain variable region (VL) structural domain via a PD Linker, and then the third structural domain is sequentially connected to the third structural domain region at the N end of the SP34 Fab light chain variable region (VL) structural domain via a 218 Linker. Again, the third structural domain is sequentially connected to the third structural domain region at the N end of the SP34 Fab light chain variable region (VL) structural domain via a 212 Linker. From the N end to the C end, a recombinant light chain is formed with the structure: third structural domain—212 Linker—third structural domain—218 Linker—third structural domain—PD Linker—light chain variable region—light chain constant region; or (d) The third structural domain is connected to the N end of the variable region (VL) structural domain of the SP34 Fab light chain via a PD Linker, and then the third structural domain is sequentially connected to the third structural domain region at the N end of the SP34 Fab light chain variable region (VL) structural domain via a 218 Linker. The third structural domain is then sequentially connected to the third structural domain region at the N end of the SP34 Fab light chain variable region (VL) structural domain via a 212 Linker. Finally, the fourth structural domain is connected to the C end of the light chain via a (G4S)3 Linker. From the N end to the C end, a recombinant light chain is formed with the structure: third structural domain—212 Linker—third structural domain—218 Linker—third structural domain—PD Linker—light chain variable region—light chain constant region—(G4S)3 Linker—fourth structural domain; or (e) The third structural domain is connected to the N end of the variable region (VL) structural domain of the SP34 Fab light chain via a PD Linker, and then the third structural domain is sequentially connected to the third structural domain region at the N end of the SP34 Fab light chain variable region (VL) structural domain via a 218 Linker. The third structural domain is then sequentially connected to the third structural domain region at the N end of the SP34 Fab light chain variable region (VL) structural domain via a 212 Linker, and then the tandem bivalent fourth structural domain is connected to the C end of the light chain via a (G4S)3 Linker. From the N end to the C end, a recombinant light chain is formed with the structure: third structural domain—212 Linker—third structural domain—218 Linker—third structural domain—PD Linker—light chain variable region—light chain constant region—(G4S)3 Linker—fourth structural domain—(G4S)3 Linker—fourth structural domain; or (f) The third structural domain is connected to the N end of the variable region (VL) structural domain of the SP34 Fab light chain via a PD Linker, and then the third structural domain is sequentially connected to the third structural domain region at the N end of the SP34 Fab light chain variable region (VL) structural domain via a 218 Linker. The third structural domain is then sequentially connected to the third structural domain region at the N end of the SP34 Fab light chain variable region (VL) structural domain via a 212 Linker, and then the tandem trivalent fourth structural domain is connected to the C end of the light chain via a (G4S)3 Linker. From the N end to the C end, a recombinant light chain is formed with the structure: third structural domain—212 Linker—third structural domain—218 Linker—third structural domain—PD Linker—light chain variable region—light chain constant region—(G4S)3 Linker—fourth structural domain—(G4S)3 Linker—fourth structural domain—(G4S)3 Linker—fourth structural domain; or (g) The third structural domain is connected to the C-end of the constant region (CL) structural domain of the SP34 Fab light chain via a (G4S)3 Linker. A second third structural domain is connected in series to the C-end of the third structural domain via a 218 Linker. Then, a third third structural domain is connected in series to the C-end of the third structural domain via a 212 Linker. From the N-end to the C-end, a recombinant light chain is formed with the structure: light chain variable region—light chain constant region—(G4S)3 Linker—third structural domain—218 Linker—third structural domain—212 Linker—third structural domain; or (h) The third structural domain is connected to the C end of the SP34 Fab light chain constant region (CL) structural domain via (G4S)3 Linker. The second third structural domain is connected in series to the C end of the third structural domain via 218 Linker. The third third structural domain is connected in series to the C end of the third structural domain via 212 Linker. The fourth structural domain is connected to the C end of the third structural domain via (G4S)3 Linker. From the N end to the C end, a recombinant light chain is formed with the structure of light chain variable region—light chain constant region—(G4S)3 Linker—third structural domain—218 Linker—third structural domain—212 Linker—third structural domain—(G4S)3 Linker—fourth structural domain.

5. The method for constructing a multifunctional antibody molecule according to claim 2, characterized in that, The multifunctional antibody molecule is based on the Fab fragment of an anti-CD3 monoclonal antibody (clone SP34) as its first structural domain. The second structural domain comprises a nanobody (VHH) targeting Her2 and an scFv targeting CD19. The third structural domain comprises a nanobody co-stimulating CD28 molecules on the T cell surface, a nanobody co-stimulating 4-1BB molecules on the T cell surface, and a nanobody targeting CD22. The fourth structural domain is an albumin-binding domain (ABDCon). These three domains are linked to the Fab fragment of clone SP34 or the second and third structural domains, respectively, via linker peptides. The method for constructing the anti-CD28-VHH domain includes the following steps: (1) The first domain is CD3 monoclonal antibody Fab; (2) Heavy chain structural domains of the Her2-VHH linked to the SP34 Fab structure; (3) Anti-CD28-VHH is connected to the N-terminus of the light chain in the SP34 Fab structure in a series of three valences; (4) Single, double, and triple albumin-binding domains are linked to the C-terminus of the light chain of the SP34 Fab structure; (5) The recombinant heavy and light chains obtained in steps (2) and (3) are bound together by disulfide bonds in CH1 and CL of SP34-Fab to form a heterodimer, namely Her2 / CD3 / CD28-ABDCon multifunctional antibody.

6. The method for constructing a multifunctional antibody molecule according to claim 5, characterized in that, The construction or linking in step (2) is carried out in the following manner: anti-Her2-VHH is linked to the C-terminal 228C region of the SP34 Fab heavy chain constant region (CH1) domain by a flexible linker peptide (G4S)3 Linker; The construction or linking in step (3) is carried out in the following manner: anti-CD28-VHH is linked to the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a rigid linker PD Linker, and anti-CD28-VHH is linked to the anti-CD28-VHH region at the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a flexible linker 218 Linker, and then anti-CD28-VHH is linked to the anti-CD28-VHH region at the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a flexible linker 212 Linker. The recombinant light chain forms a recombinant light chain from the N-terminus to the C-terminus consisting of anti-CD28-VHH—212 Linker—anti-CD28-VHH—218 Linker—anti-CD28-VHH—PD Linker—SP34 Fab light chain variable region—SP34 Fab light chain constant region. The construction or connection in step (4) is performed in one of the following ways: (a) Anti-CD28-VHH is linked to the N-terminus of the variable region (VL) domain of the SP34 Fab light chain via a rigid linker PD Linker, and anti-CD28-VHH is tandemly linked to the anti-CD28-VHH region at the N-terminus of the SP34 Fab light chain variable region (VL) domain via a flexible linker 218 Linker. Then, anti-CD28-VHH is tandemly linked to the anti-CD28-VHH region at the N-terminus of the SP34 Fab light chain variable region (VL) domain via a flexible linker 212 Linker, and ABDCon is linked to the C-terminus of the light chain via a (G4S)3 Linker. The structure formed sequentially from the N-terminus to the C-terminus is: anti-CD28-VHH—212 Linker—anti-CD28-VHH—218 Linker—anti-CD28-VHH—PD Linker—SP34 Fab light chain variable region—SP34 Fab light chain constant region—(G4S)3 Linker—ABDCon; or; (b) Anti-CD28-VHH was linked to the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a rigid linker PD Linker. Anti-CD28-VHH was then tandemly linked to the anti-CD28-VHH region at the N-terminus of the SP34 Fab light chain variable region (VL) domain using a flexible linker 218 Linker. Furthermore, anti-CD28-VHH was tandemly linked to the anti-CD28-VHH region at the N-terminus of the SP34 Fab light chain variable region (VL) domain using a flexible linker 212 Linker. Finally, a tandem bivalent ABDCon was linked to the C-terminus of the light chain using a (G4S)3 Linker. The resulting structure from N-terminus to C-terminus was: anti-CD28-VHH—212 Linker—anti-CD28-VHH—218 Linker—anti-CD28-VHH—PD Linker—SP34 Fab light chain variable region—SP34 Fab The structure of the light chain constant region —(G4S)3Linker—ABDCon—(G4S)3Linker—ABDCon; or; (c) Anti-CD28-VHH is linked to the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a rigid linker PD Linker. Anti-CD28-VHH is then linked in series to the anti-CD28-VHH region at the N-terminus of the SP34 Fab light chain variable region (VL) domain using a flexible linker 218 Linker. Furthermore, anti-CD28-VHH is linked in series to the anti-CD28-VHH region at the N-terminus of the SP34 Fab light chain variable region (VL) domain using a flexible linker 212 Linker. Finally, a trivalent ABDCon is linked to the C-terminus of the light chain using a (G4S)3 Linker. The resulting structure from N-terminus to C-terminus is: anti-CD28-VHH—212 Linker—anti-CD28-VHH—218 Linker—anti-CD28-VHH—PD Linker—SP34 Fab light chain variable region—SP34 Fab The structure of the light chain constant region —(G4S)3Linker—ABDCon—(G4S)3Linker—ABDCon—(G4S)3Linker—ABDCon; The Her2 / CD3 / CD28-ABDCon multifunctional antibody described in step (5) is constructed using an anti-CD3 Fab structure as its backbone, based on the construction method shown in step (3) and combined with the construction method described in step (2) to obtain the Her2 / CD3 / CD28-ABDCon multifunctional antibody. The specific method is as follows: (a) Anti-CD28-VHH was linked to the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a rigid linker PD Linker, and anti-CD28-VHH was linked to the anti-CD28-VHH region at the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a flexible linker 218 Linker. Then, anti-CD28-VHH was linked to the anti-CD28-VHH region at the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a flexible linker 212 Linker, and ABDCon was linked to the C-terminus of the light chain using a (G4S)3 Linker, labeled Her2 / CD3 / CD28-ABDCon1. (b) Anti-CD28-VHH was linked to the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a rigid linker PD Linker, and anti-CD28-VHH was tandemly linked to the anti-CD28-VHH region at the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a flexible linker 218 Linker. Then, anti-CD28-VHH was tandemly linked to the anti-CD28-VHH region at the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a flexible linker 212 Linker, and tandemly linked to the C-terminus of the light chain using a (G4S)3 Linker, labeled Her2 / CD3 / CD28-ABDCon2. (c) Anti-CD28-VHH was linked to the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a rigid linker PD Linker, and anti-CD28-VHH was tandemly linked to the anti-CD28-VHH region at the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a flexible linker 218 Linker. Then, anti-CD28-VHH was tandemly linked to the anti-CD28-VHH region at the N-terminus of the variable region (VL) domain of the SP34 Fab light chain using a flexible linker 212 Linker, and tandemly linked to the C-terminus of the light chain using a (G4S)3 Linker, labeled Her2 / CD3 / CD28-ABDCon3.

7. The method for constructing a multifunctional antibody molecule according to claim 2, characterized in that, The multifunctional antibody molecule is CD19 / CD22 / CD3-ABDCon, and the method for constructing the CD19 / CD22 / CD3-ABDCon multifunctional antibody molecule includes the following steps: (1) The anti-CD22-VHH is connected to the C-terminus of the SP34 Fab light chain constant region (CL) structure through (G4S)3 Linker, and ABDCon is connected to the C-terminus of the anti-CD22-VHH through 218 Linker. The structure formed from the N-terminus to the C-terminus is SP34 Fab light chain variable region—SP34 Fab light chain constant region—(G4S)3 Linker—anti-CD22-VHH—218 Linker—ABDCon. The heavy chain is expressed as SP34 Fab heavy chain variable region—SP34 Fab heavy chain constant region—(G4S)3 Linker—CD19-scFv, labeled as CD19 / CD22 / CD3-ABDCon LC; (2) The anti-CD19-scFv was linked to the C-terminus of the constant region (CH) domain of the SP34 Fab heavy chain via the (G4S)3 Linker, and then the ABDCon was linked to the C-terminus of the anti-CD19-scFv via the 218 Linker. The structure formed from the N-terminus to the C-terminus was SP34 Fab heavy chain variable region—SP34 Fab heavy chain constant region—(G4S)3 Linker—CD19-scFv—218 Linker-ABDCon. The light chain was expressed as SP34 Fab light chain variable region—SP34 Fab light chain constant region—(G4S)3 Linker—anti-CD22-VHH, and labeled as CD19 / CD22 / CD3-ABDCon HC.

8. The method for constructing a multifunctional antibody molecule according to claim 2, characterized in that, The multifunctional antibody molecule is Her2 / CD3 / 4-1BB-ABDCon, and the method for constructing the Her2 / CD3 / 4-1BB-ABDCon multifunctional antibody molecule includes the following steps: (1) Anti-4-1BB-VHH is linked to the C-terminus of the constant region (CL) domain of the SP34 Fab light chain via (G4S)3 Linker. The second anti-4-1BB-VHH is tandemly linked to the C-terminus of the first anti-4-1BB-VHH via 218 Linker. The third anti-4-1BB-VHH is then tandemly linked to the C-terminus of the second anti-4-1BB-VHH via 212 Linker. The albumin-binding domain is then linked to the C-terminus of anti-4-1BB-VHH via GS(G4S)3 Linker. From the N-terminus to the C-terminus, the structure is formed as follows: SP34 Fab light chain variable region—SP34 Fab light chain constant region—(G4S)3 Linker—anti-4-1BB-VHH—218 Linker—anti-4-1BB-VHH—212 Linker—anti-4-1BB-VHH—GS(G4S)3 Linker—ABDCon. The heavy chain is SP34. Fab heavy chain variable region—SP34 Fab heavy chain constant region—(G4S)3 Linker—expressed against Her2-VHH, labeled Her2 / CD3 / 4-1BB-ABDCon LC; (2) ABDCon is connected to the C-terminus of anti-Her2-VHH via GS(G4S)3 Linker, and anti-Her2-VHH is connected to the C-terminus of the SP34 Fab heavy chain constant region (CH) domain via (G4S)3 Linker. The structure formed from the N-terminus to the C-terminus is SP34 Fab heavy chain variable region—SP34 Fab heavy chain constant region—(G4S)3 Linker—anti-Her2-VHH—GS(G4S)3 Linker—ABDCon. The light chain is SP34 Fab light chain variable region—SP34 Fab light chain constant region—(G4S)3 Linker—anti-4-1BB-VHH—218 Linker—anti-4-1BB-VHH—212 Linker—anti-4-1BB-VHH, and is expressed as Her2 / CD3 / 4-1BB-ABDCon HC.

9. A method for preparing the multifunctional antibody according to any one of claims 1-8, characterized in that, Includes the following steps: (1) Obtain the fusion gene of the long-acting multifunctional antibody and construct the expression vector of the long-acting multifunctional antibody; (2) The above expression vector was transfected into host cells using genetic engineering methods; (3) The host cells described above are cultured under conditions that allow the production of the long-acting multifunctional antibody; (4) Separate and purify the resulting antibody protein; The expression vector in step (1) is a eukaryotic cell expression vector or a prokaryotic expression vector; the eukaryotic cell expression vector is selected from pFuse, pSeqtag, pCMV, pcDNA, pFastBac1, pPIC9K, pcAGGS, and the prokaryotic expression vector is selected from pET, pGEX, pMAL, pQE, pTrc, pBV, pTXB, with eukaryotic expression vectors being preferred; The host cells in step (2) are selected from Escherichia coli, Bacillus thuringiensis, Pichia pastoris, insect cells, 293 suspension cells or Chinese insect ovarian cells, preferably 293 suspension cells.

10. The use of the multifunctional antibody obtained by any one of the construction methods of claims 1-8 in the preparation of a medicament for treating cancer, said cancer including breast cancer, colorectal cancer, anal cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, head and neck cancer, nasopharyngeal cancer, skin cancer, melanoma, ovarian cancer, prostate cancer, urethral cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, brain tumor, glioma, neuroblastoma, esophageal cancer, gastric cancer, liver cancer, kidney cancer, bladder cancer, cervical cancer, endometrial cancer, thyroid cancer, eye cancer, sarcoma, bone cancer, leukemia, myeloma, or lymphoma.

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