Membrane surface protease response type C1s complement effect antibody-biomacromolecule conjugate based on HER2 targeting antibody

Abstract

The invention relates to an antibody-biomacromolecule conjugate. The antibody-biomacromolecule conjugate comprises an antibody targeting module, a complement effect module and a connexon, the antibody targeting module can be specifically combined with a cell membrane surface antigen, and the complement effect module is a key enzyme C1s in a classical complement pathway or a functional structural domain with complement splitting activity of the key enzyme C1s. The linker is used for connecting the antibody targeting module with the complement effect module, and is designed to generate cutting or conformational change on the outer side of a cell membrane or in an extracellular microenvironment, so that the complement effect module is converted from a limited state to a functional accessible state; to promote initiation of a classical complement pathway in the neighborhood of the cell membrane located by the antibody targeting module. The antibody-biomacromolecule conjugate provided by the invention can be used for engineering regulation and control of complement-related reactions.

Description

A membrane-surface protease-responsive C1s complement effector antibody-biomacromolecule conjugate based on a HER2-targeting antibody. Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, specifically relating to antibody engineering, the engineered application of complement system-related molecules, and the design and construction of biomolecular conjugates. More specifically, this invention relates to an antibody-biomolecular conjugate that uses an antibody targeting module to localize cell membrane surface antigens and uses an engineered connection method to guide effector modules related to the classical complement pathway to the localized region to participate in extracellular complement-related reaction processes. Background Technology

[0002] Aberrant expression of cell surface receptors is a key molecular characteristic of various pathological conditions. Human epidermal growth factor receptor 2 (HER2), as a transmembrane receptor protein, exhibits well-defined membrane localization characteristics in various cell types, making it a commonly used example of a cell membrane surface antigen in antibody engineering and targeted molecule construction. Various pathways have been developed for constructing antibody molecules around cell membrane surface receptors, including monoclonal antibodies, antibody-small molecule conjugates, and other antibody-related engineering forms. These techniques typically rely on the structural features of the antibody itself or its carried functional molecules to trigger corresponding molecular events upon cell membrane binding. However, existing antibody-related technologies largely focus on the antibody's intrinsic effects or intracellular mechanisms of action, with relatively limited engineering applications in extracellular immune effector systems.

[0003] The complement system is an important component of innate immunity, and its classical pathway amplifies signals through a series of cascade reactions. The initiation of the classical complement pathway depends on molecular events following antibody recognition of the target antigen, with the spatial localization and accessibility of complement components significantly influencing the occurrence of the cascade reactions. However, in existing technologies, activation of the classical complement pathway typically relies on the interaction between the antibody Fc domain and the complement initiation component, and the triggering efficiency of this approach varies considerably across different application scenarios.

[0004] Furthermore, complement-regulating molecules are expressed on the membrane surfaces of various cell types. These molecules can regulate the complement cascade at different stages, thus affecting the overall efficiency of complement-related reactions. Therefore, relying solely on the native antibody structure to trigger the complement reaction often makes it difficult to effectively engineer and regulate the initiation of the complement cascade. To improve the triggering efficiency of the classical complement pathway, existing technologies have attempted optimization through the engineering of antibody Fc domains or by jointly regulating the activity of complement-related molecules. However, these approaches typically focus on modifying existing antibody structures and do not directly introduce key complement effector components at the molecular conformation level, thus still resulting in limited efficiency in the initiation stage of the complement cascade.

[0005] In the classical complement pathway, the C1 complex consists of components such as C1q, C1r, and C1s. Among them, C1s, as a key enzyme performing complement cleavage, is responsible for the initial cleavage step of complement components and occupies a pivotal position in the complement cascade reaction. However, current technologies for utilizing C1s are mostly limited to their natural state, and there is a lack of mature solutions for introducing C1s as an independent functional module into antibody targeting systems through engineering. On the other hand, the cascade reaction of the complement system has significant amplification characteristics, and its range and intensity are closely related to the spatial distribution of complement-related components. Therefore, how to regulate the accessibility of key complement effector modules outside the cell membrane or in specific extracellular microenvironments is an urgent problem to be solved in complement-related engineering technologies.

[0006] In summary, existing antibody engineering and complement-related technologies still have shortcomings in the engineering utilization of the initial stage of the classical complement pathway. There is a lack of a technical solution that can combine key complement effector modules with antibody targeting through molecular configuration design and regulate complement-related responses in the cell membrane region. Summary of the Invention

[0007] Based on the aforementioned technical background, it is evident that existing antibody engineering and complement-related technologies still have shortcomings in the engineering utilization of the initiation stage of the classical complement pathway, and a molecular configuration scheme capable of effectively regulating the spatial accessibility of key complement effector modules has not yet been formed. Therefore, the overall objective of this invention is to provide a novel design approach for antibody-biomacromolecule conjugates, which introduces key effector enzyme modules from the classical complement pathway into antibody targeting systems through engineering methods, thereby regulating complement-related responses on the extracellular surface or in the extracellular microenvironment.

[0008] One of the objectives of this invention is to provide an engineered implementation path for complement classical pathway-related reactions that differs from the traditional Fc-dependent approach, thereby improving the limited initiation efficiency of existing antibody molecules when triggering complement-related reactions.

[0009] Another objective of this invention is to regulate the spatial distribution and accessibility of key complement effector modules in the vicinity of the cell membrane through molecular configuration design, thereby providing a structural basis for the occurrence of complement-related reactions in a specific space.

[0010] Furthermore, this invention aims to regulate the action site of complement effector modules at the molecular configuration level by introducing a linker structure that can be recognized by extracellular or cell membrane surface proteases, in order to meet the needs of spatial selectivity and range of action regulation in complement engineering applications.

[0011] Furthermore, the technical solution described in this invention is not limited to specific antigen types. By replacing the antibody targeting module, it can be extended to various cell membrane surface antigen-related engineering systems, thereby constructing a universal complement-related molecular engineering platform.

[0012] (I) Overview The technical solution of this invention revolves around the engineered combination of antibody targeting and complement-related effect modules. By structurally linking the key enzyme module in the classical complement pathway with the antibody targeting module, an antibody-biomacromolecule conjugate is constructed for regulating complement-related responses on the outer side of the cell membrane or in the extracellular microenvironment.

[0013] The overall design concept of this technical solution is as follows: without relying on endocytosis or lysosomal lysis, the antibody targeting module is used to locate the antigen on the cell membrane surface, and the complement effector module is made functionally accessible within the localization area, thereby providing an engineered implementation path for the occurrence of complement classical pathway-related reactions.

[0014] From an overall structural perspective, the antibody-biomacromolecule conjugate of the present invention comprises at least the following three basic modules: (1) an antibody targeting module; (2) an engineered linker module; and (3) a complement effector module. These modules are structurally interconnected and functionally complementary, together forming a molecular configuration capable of spatially regulating complement-related responses in the vicinity of the cell membrane. The antibody targeting module functions to recognize and bind to cell membrane surface antigens, thereby positioning the antibody-biomacromolecule conjugate on the membrane surface of the target cell. In some embodiments, the antibody targeting module may be selected from antibodies or antigen-binding fragments capable of recognizing specific cell membrane surface receptors, such as antibody structures that recognize HER2.

[0015] Engineered linker modules are used to achieve structural connections between antibody targeting modules and complement effector modules, and their designability allows for the regulation of the spatial location and conformational state of the complement effector module. In some embodiments, the linker is designed to be recognized by extracellular or cell membrane surface proteases, thereby transforming the complement effector module from a restricted state to a functionally accessible state in a specific spatial environment.

[0016] The complement effector module is a functional unit in this invention used to participate in reactions related to the classical complement pathway. In some embodiments, the complement effector module is a key enzyme C1s in the classical complement pathway or a functional domain with complement cleavage activity. The complement effector module can participate in complement-related cleavage reactions under appropriate spatial conditions, thereby providing initiation conditions for subsequent complement cascade reactions.

[0017] At the mechanism of action level, the antibody-biomacromolecule conjugate described in this invention, after application, can bind to cell membrane surface antigens through an antibody targeting module, thereby achieving localization to cell membrane regions. Subsequently, extracellular or membrane surface proteases act on the linker structure, enabling the complement effector module to gain functional access within the localized region, thus participating in complement-related reactions in the classical pathway.

[0018] It should be noted that, in some embodiments of the present invention, the complement effector module is not required to be completely dissociated from the antibody targeting module. As long as its spatial conformation allows the complement substrate to approach and maintains enzyme activity, it can participate in complement-related cleavage reactions while remaining connected.

[0019] Through the above-mentioned modular structural design and functional synergy, this invention provides an engineered molecular configuration for regulating complement-related responses in the cell membrane vicinity, offering a new technical approach for the engineering application of the classical complement pathway.

[0020] (II) Selection and Engineering Design of Antibody Targeting Module In the overall technical solution of this invention, the antibody targeting module, as an important component for realizing the spatial localization of the complement effector module, primarily functions to guide the complement effector module to specific cell membrane surface regions through antigen recognition capabilities, thereby providing a spatial basis for complement-related reactions to occur on the outer side of the cell membrane or in the extracellular microenvironment. Therefore, the target selection, structural form, and engineering design of the antibody targeting module are of great significance to the construction of the antibody-biomacromolecule conjugate described in this invention.

[0021] From the perspective of target selection, this invention uses human epidermal growth factor receptor 2 (HER2) as the recognition antigen for antibody targeting modules in some embodiments. HER2, as a transmembrane receptor protein, has its extracellular domain exposed on the outer side of the cell membrane, exhibiting well-defined membrane localization characteristics, making it suitable as an anchor point for antibody-mediated localization of biomacromolecule modules. Furthermore, HER2 can exhibit varying expression levels or spatial distribution characteristics in different cell types, thus providing diverse engineering application scenarios for antibody targeting.

[0022] It should be noted that this invention is not limited to HER2 under specific expression levels. Through antibody-mediated targeting and the amplification properties of the complement system cascade reaction, a structural basis can still be provided for the accessibility of the complement effector module in the cell membrane vicinity, even under relatively low antigen density. Therefore, the antibody targeting module described in this invention has engineering application potential under different antigen expression conditions.

[0023] From the perspective of antibody structure, the antibody targeting module of this invention can be a full-length immunoglobulin molecule, such as IgG1, IgG2, or IgG4 subtypes, or it can be an antigen-binding fragment of an antibody, including but not limited to Fab, F(ab′)2, single-chain variable fragments, single-domain antibodies, or combinations thereof. Different antibody structures can be selected according to specific engineering requirements to achieve a balance between targeting capability, molecular size, and structural stability.

[0024] In antibody engineering design, this invention allows for the engineering modification of variable or constant regions of antibodies to adapt to the connection requirements of complement effector modules or linker structures. For example, stable coupling sites can be provided for linkers or complement effector modules by introducing amino acid modifications at specific sites without affecting antigen binding capacity. Furthermore, the spatial distribution of antibody-biomacromolecule conjugates on the cell membrane surface can be optimized by adjusting the binding characteristics of the antibody to the target antigen.

[0025] In some embodiments, the antibody targeting module can be designed as a monospecific antibody structure. In other embodiments, bispecific or multispecific antibodies can also be used, enabling the antibody to simultaneously recognize two or more cell membrane surface antigens, thereby further enhancing the flexibility of targeting. The selection of the different antibody forms mentioned above are all optional engineering solutions of the present invention.

[0026] It is important to emphasize that the core function of the antibody targeting module in this invention is to achieve spatial localization, rather than to induce endocytosis. The antibody-biomacromolecule conjugate described in this invention does not rely on the endocytic behavior after antibody-antigen binding; its design focuses on the antibody's residence characteristics on the cell membrane surface, so as to facilitate the complement effector module's participation in complement-related reactions on the outer side of the cell membrane.

[0027] Finally, it should be noted that the antibody targeting module described in this invention is not based on the specific amino acid sequence of the antibody. The antibody targeting module described in this invention can be a known anti-HER2 antibody or a functionally equivalent variant thereof, such as anti-HER2 monoclonal antibodies reported in clinical trials or published literature.

[0028] This invention does not limit the specific variable region sequence, complementarity-determining region (CDR) sequence, or gene accession number of the antibody. As long as the antibody can specifically recognize the HER2 antigen and achieve targeting function, it can be used to implement the technical solution of this invention. Therefore, the specific sequence selection of the antibody targeting module is a conventional technical application by those skilled in the art based on existing antibody resources, and is not a necessary technical feature of this invention.

[0029] For known anti-HER2 antibodies, their preparation methods, sequence information, and biological characteristics have been disclosed in the prior art. Those skilled in the art can obtain the corresponding antibodies through commercial means or publicly available literature and apply them without any inventive effort.

[0030] In summary, through the systematic selection and engineering design of antibody targeting modules, this invention provides a reliable structural basis for the spatial positioning and functional accessibility of complement effector modules in the vicinity of the cell membrane.

[0031] (III) Selection and Engineering Design of the C1s Complement Effector Module In the overall technical solution of this invention, the complement effector module, as a functional unit participating in the relevant reactions of the classical complement pathway, plays a crucial role in the construction of antibody-biomacromolecule conjugates due to its selection criteria, structural form, and engineering design. This section describes the engineering implementation of the complement effector module, focusing on the key enzyme C1s in the classical complement pathway.

[0032] The cascade reactions of the classical complement pathway depend on the sequential action of multiple complement components. C1s, as a serine protease directly involved in complement cleavage, is responsible for the initial cleavage steps of complement components C4 and C2, thus providing the initiation conditions for subsequent complement cascade reactions. Therefore, compared to other components in the classical complement pathway, C1s plays a crucial role in complement-related reactions. Based on the aforementioned biological characteristics of complement, this invention selects C1s as a complement effector module for engineering design in some embodiments.

[0033] From the perspective of existing technology, the triggering of the classical complement pathway usually depends on the interaction between the antibody Fc domain and the complement initiation component. The triggering efficiency of this approach is easily affected by various factors in different application scenarios. In contrast, introducing C1s as an independent functional module into the antibody targeting system can provide an engineering implementation path for complement-related reactions at the molecular configuration level that differs from the traditional Fc-dependent approach.

[0034] In terms of structural form, the complement effector module of this invention can be a full-length C1s protein or a functional domain of C1s with complement cleavage activity. A full-length C1s protein structurally contains multiple functional segments, enabling it to maintain its enzymatic activity under appropriate spatial conditions. In other embodiments, selecting a functional domain of C1s is beneficial for regulating molecular size or structural properties. All of the above-mentioned different structural forms can be selected according to specific engineering requirements and do not constitute a limitation of the technical solution of this invention.

[0035] In terms of engineering design, this invention regulates the spatial accessibility of the complement effector module. The complement cascade reaction exhibits significant amplification characteristics, and its location is closely related to the spatial distribution of the complement effector module. Therefore, this invention introduces an engineered linker structure between the C1s complement effector module and the antibody targeting module, enabling the complement effector module to be in a restricted state when specific spatial conditions are not met, and to become functionally accessible after predetermined conditions are satisfied.

[0036] In some embodiments, the linker can be recognized by extracellular or cell membrane surface proteases, thereby modulating the conformation or spatial location of the complement effector module in the vicinity of the cell membrane. Furthermore, in some embodiments, the complement effector module can be designed as a precursor form or a low-basal-activity form to facilitate the linkage structure's regulation of its spatial accessibility.

[0037] It should be noted that this invention does not require the complement effector module to be completely dissociated from the antibody targeting module. As long as its spatial conformation allows the complement substrate to approach and maintains complement cleavage function, it can participate in complement-related reactions in the classical pathway while remaining connected.

[0038] From an engineering implementation perspective, the complement effector module can be connected to the antibody targeting module via gene fusion or chemical conjugation. In the gene fusion implementation, the complement effector module is expressed as part of the fusion protein; in the chemical conjugation implementation, the connection site can be controlled using site-specific conjugation technology. All of the above implementation methods are optional engineering solutions of this invention.

[0039] Finally, it should be noted that the complement effector module described in this invention is based on its functional characteristics in the classical complement pathway, rather than relying on a specific amino acid sequence itself. Complement enzyme C1s, as a known serine protease, has a full-length protein structure, functional domain divisions, and enzymatic properties that are well-known to those skilled in the art and documented in the literature.

[0040] The "functional domain with complement cleavage activity" described in this invention refers to a protein structural portion that, while maintaining the ability of C1s to cleave complement components C4 and / or C2, can participate in complement-related responses as a complement effector module. Those skilled in the art can obtain a full-length C1s protein or its functional domain that meets the above functional requirements using conventional protein engineering techniques based on existing publicly available information, without requiring inventive effort.

[0041] Furthermore, this invention does not limit the complement effector module to using a single, fixed amino acid sequence. As long as the complement effector module possesses complement cleavage functionality under engineering design conditions and can achieve spatial localization with the antibody targeting module through a linker structure, it can be used to implement the technical solution of this invention. Therefore, the selection and optimization of specific sequences are conventional technical adjustments by those skilled in the art and do not constitute essential technical features of this invention.

[0042] In summary, through the selection and engineering design of C1s complement effect modules, this invention provides a technical solution for spatially controlling key complement effect modules at the molecular configuration level, providing a structural basis for the engineering application of complement classical pathway-related reactions.

[0043] (iv) Types and Design Principles of Membrane Surface Protease-Cleavable Linkers In the overall technical solution of this invention, engineered linkers are used to realize the structural connection between the antibody targeting module and the complement effector module, and their designability allows for the regulation of the spatial accessibility of the complement effector module. Therefore, the selection of linker type and structural design is of great significance for the construction of antibody-biomacromolecule conjugates.

[0044] In some embodiments, the linker of the present invention is designed as a structure that can be recognized by proteases on the extracellular or cell membrane surface. Related studies have shown that various cell types and their extracellular environments contain proteases with membrane-binding or secretory properties, whose enzymatic activity is mainly distributed on the outer side of the cell membrane or in the extracellular space. Based on these characteristics, introducing protease recognition sequences into the linker structure can serve as an engineered means of regulating the spatial state of complement effector modules.

[0045] In some embodiments, the linker comprises a polypeptide sequence that can be recognized by matrix metalloproteinases or members of the detegrin metalloproteinase family. For example, a sequence sensitive to cleavage by MMP family proteases or ADAM family proteases can be selected. It should be noted that the present invention is not limited to a specific type of protease; any protease capable of acting on the linker structure outside the cell membrane or in the extracellular environment can be considered as an option in linker design.

[0046] From the perspective of the linker's structural form, the cleavable linker can be a single polypeptide sequence or a composite structure containing a cleavage sequence and a flexible spacer region. In some embodiments, the flexible spacer region is used to adjust the spatial distance or conformational degrees of freedom between the antibody targeting module and the complement effector module, thereby improving the accessibility of the complement effector module in different spatial states.

[0047] Furthermore, in some embodiments, the connector substructure may further include a shielding structure to restrict substrate accessibility of the complement effect module under specific spatial conditions. When the connector undergoes cutting or conformational changes, the shielding structure can be displaced or removed, thereby altering the spatial state of the complement effect module. This type of structural design provides more engineering implementation methods for the spatial control of the complement effect module.

[0048] It should be noted that the linker described in this invention does not require the cleavage reaction to be complete or occur in a single step. In some embodiments, even if the linker undergoes only partial cleavage or conformational change, as long as the spatial accessibility of the complement effect module can be altered, engineering design requirements can be met. Therefore, this invention does not impose an absolute limitation on the degree of linker cleavage.

[0049] From an engineering perspective, membrane surface protease-cleavable linkers can be designed and optimized using conventional protein engineering methods, including peptide sequence screening, in vitro protease activity verification, and structural stability assessment. These methods are well-known to those skilled in the art and can be used to construct and verify the linkers described in this invention.

[0050] Finally, it should be noted that the cleavable linker described in this invention does not require a specific amino acid sequence as an essential technical feature. The "linker that can be recognized by extracellular or cell membrane surface proteases" mentioned in this invention refers to a linker structure that can undergo cleavage or conformational changes under the action of corresponding proteases, thereby regulating the spatial accessibility of the complement effector module.

[0051] The recognition characteristics and commonly used cleavage sequences of matrix metalloproteinases (MMP family) and detegrin metalloproteinases (ADAM family) for their substrates have been disclosed in the art and are widely used in drug conjugate and prodrug design. Those skilled in the art can obtain cleavable linker sequences suitable for this invention based on existing publicly available information and through conventional sequence selection or screening methods, without requiring inventive effort.

[0052] This invention does not limit the linker to a single, fixed amino acid sequence. As long as the linker can adjust the spatial state of the complement effector module under predetermined spatial conditions, it can be used to implement the technical solution of this invention. Therefore, the selection, length, and amino acid composition of the specific linker sequence are conventional technical adjustments made by those skilled in the art based on specific engineering needs, and do not constitute essential technical features of this invention.

[0053] In summary, by introducing a linker structure that can be recognized by extracellular or cell membrane surface proteases, this invention provides an engineered connection method for regulating the spatial state of complement effector modules, offering a flexible and feasible technical means for the construction of antibody-biomacromolecule conjugates.

[0054] (V) Mechanism of Action and Activation Pathway of the Classical Complement Pathway The purpose of designing the antibody-biomacromolecule conjugate described in this invention is to provide an achievable spatial condition for complement-related reactions in the vicinity of the cell membrane through the engineered combination of antibody targeting and complement effector modules. The following content is used to illustrate the complement-related reaction pathways that may be involved at the molecular and structural levels in the technical solution described in this invention, to help understand its engineering design principles, rather than to limit specific biological results.

[0055] Under application conditions, the antibody-biomacromolecule conjugate can bind to cell membrane surface antigens via an antibody targeting module, thereby localizing the conjugate to the membrane surface of the target cell. This localization process helps confine the complement effector module to the vicinity of the cell membrane, providing local accessibility for complement-related responses.

[0056] In some implementations, extracellular or cell membrane surface proteases can act on linker structures, transforming the complement effector module from a spatially restricted state to a functionally accessible state. This process does not rely on endocytosis or lysosomal action, but rather achieves spatial regulation of the complement effector module through conformational changes or cleavage of the linker.

[0057] When the complement effector module is functionally accessible, it can participate in reactions related to the classical complement pathway. For example, when the complement effector module is C1s or a domain with complement cleavage activity, the module can act on complement components C4 and C2 under appropriate spatial conditions, thereby providing initiation conditions for subsequent complement cascade reactions.

[0058] As the complement cascade continues, complement components can sequentially cleave and deposit, forming the intermediate and terminal complexes known in the classical complement pathway. This process illustrates one possible pathway by which the complement effector module participates in the classical complement pathway and does not limit the specific extent or final outcome of the complement reaction.

[0059] It should be noted that the location of complement-related reactions described in this invention is mainly influenced by the localization of the antibody targeting module and the spatial regulation of the linker structure. Through the above-described engineering design, the effective range of the complement effector module can be limited at the molecular configuration level, thereby preventing complement-related reactions from occurring in non-target regions.

[0060] Through the above-described schematic diagram of the action pathway, this invention provides an engineered implementation of introducing a key complement effector module into the cell membrane region, providing a structural and mechanistic understanding basis for the engineering application of the classical complement pathway.

[0061] Compared with existing technologies, the present invention provides a new technical implementation method in the field of complement-related molecular engineering and antibody conjugation technology through the combined design of antibody targeting module, engineered linker module and complement effector module. Its beneficial effects are mainly reflected in the following aspects.

[0062] First, this invention provides a complement-related engineering scheme that differs from traditional intracellular pathways. Existing antibody-related technologies largely rely on antibody intrinsic effects or intracellular mechanisms of action. However, this invention, by introducing a key complement effector module into the cell membrane region, provides an engineered implementation path for classic complement pathway-related reactions, thereby expanding the mode of action of antibody-conjugated technologies.

[0063] Secondly, this invention has technical advantages in the engineered regulation of the initiation stage of the classical complement pathway. By structurally linking the key complement enzyme module with the antibody targeting module, this invention provides a different approach from the traditional Fc-dependent method for the occurrence of complement-related reactions in the initiation stage, which is beneficial to improving the controllability of complement-related reactions under specific spatial conditions.

[0064] Furthermore, this invention modulates the spatial accessibility of the complement effect module through the design of engineered linkers. This design allows the complement effect module to be in a restricted or accessible state under different spatial conditions, thereby restricting the location of complement-related reactions at the molecular configuration level and helping to prevent complement-related reactions from occurring in non-target regions.

[0065] Furthermore, the technical solution described in this invention does not rely on endocytosis following antibody-antigen binding. Compared to antibody-conjugation technologies that rely on intracellular transport and lysosomal processing, this invention achieves the engineering application of complement effector modules in the extracellular environment through localization and spatial regulation on the cell membrane surface, thereby reducing dependence on specific intracellular processes.

[0066] Furthermore, the antibody targeting module described in this invention can be replaced according to different engineering requirements, and the complement effector module and linker structure can also be adjusted according to specific application scenarios. Therefore, this invention has excellent modularity and platform characteristics. This feature is beneficial for expanding complement-related engineering schemes under different target antigen conditions.

[0067] Finally, the antibody-biomacromolecule conjugates described in this invention can be constructed and produced using existing protein engineering and biomacromolecule preparation technologies without the need to introduce highly toxic small molecule loads or complex intracellular release mechanisms, demonstrating good feasibility in engineering implementation and process scale-up.

[0068] In summary, this invention provides a new technical solution for the engineering application of the classical complement pathway through molecular configuration and modular design, which has positive technical effects in terms of structural design, spatial control and engineering implementation. Attached Figure Description

[0069] To further clarify the technical solution and mechanism of action of the present invention, the present invention will be described below in conjunction with the accompanying drawings. It should be noted that the accompanying drawings are only for illustrative purposes, illustrating the technical concept and implementation of the present invention, and do not constitute a limitation on the scope of protection of the present invention. Those skilled in the art can make various adjustments or modifications to the content of the drawings according to actual needs without departing from the technical concept of the present invention.

[0070] Figure 1 is a schematic diagram of the overall structure of the antibody-biomacromolecule conjugate described in this invention. This figure schematically illustrates the connection relationships between the antibody targeting module, the engineered linker module, and the complement effector module, serving to explain the modular design concept of this invention, rather than limiting the specific molecular configuration.

[0071] Figure 2 is a schematic diagram of the spatial positioning of the antibody-biomacromolecule conjugate described in this invention in the vicinity of the cell membrane. This figure is used to illustrate the relative spatial relationship of the complement effector module on the outer side of the cell membrane after the antibody targeting module binds to the cell membrane surface antigen.

[0072] Figure 3 is a schematic diagram of the linker cleavage process mediated by membrane surface proteases. This diagram illustrates how membrane-bound or secreted proteases, highly expressed in tumor cells and their microenvironment, cleave linkers, thereby transforming the complement effector module from a restricted state to a functionally accessible state. The cleavage process shown in the figure occurs in the extracellular environment and is independent of drug endocytosis or lysosomal lysis.

[0073] Figure 4 is a schematic diagram of the activation of the classical complement pathway on the tumor cell membrane surface. This figure illustrates how the complement effector module C1s cleaves complement components C4 and C2 on the tumor cell membrane surface, forming C3 convertase and triggering a complement cascade amplification reaction, ultimately guiding the assembly of the membrane attack complex on the tumor cell membrane. This figure is used to illustrate the activation of the classical complement pathway described in this invention. Detailed Implementation Methods: Implementation Method 1, Example

[0075] The technical solution of the present invention will be further described below with reference to specific embodiments. It should be noted that these embodiments are only used to illustrate the technical concept and implementation of the present invention and do not constitute a limitation on the scope of protection of the present invention. Without departing from the technical concept of the present invention, those skilled in the art can make various equivalent substitutions or improvements to the implementation methods, all of which should fall within the scope of protection of the present invention.

[0076] [Example 1: Construction of Anti-HER2 Antibody-C1s Conjugate Drug and Verification of its Mechanism of Action] This example provides an antibody-biomacromolecule conjugate drug with HER2 as the target antigen and complement enzyme C1s as the effector module, to illustrate the feasibility of the ABC drug of the present invention activating the classical complement pathway on the surface of tumor cell membranes.

[0077] (I) Selection and Preparation of Antibody Targeting Module In this embodiment, a clinically validated anti-HER2 monoclonal antibody was selected as the targeting module. The antibody can be a full-length IgG molecule or an antibody fragment with equivalent antigen-binding ability. The antibody was prepared using a conventional recombinant protein expression system and purified to obtain antibody raw materials that meet quality requirements. The antibody maintains its specific binding ability to the HER2 antigen during the preparation process.

[0078] (II) Selection and Processing of the Complement Effector Module In this embodiment, the complement effector module selected is the key enzyme C1s of the classical complement pathway or its functional domain with complement cleavage activity. C1s can be obtained through recombinant expression and, if necessary, engineered to maintain a low basal activity before activation. This process helps reduce the risk of non-specific complement activation in non-target tissues.

[0079] (III) Design and Connection of Membrane Surface Protease-Cleavage Linkers In this embodiment, the antibody targeting module and the complement effector module are connected via linkers that can be cleaved by tumor-associated membrane surface proteases. The linker contains a polypeptide sequence that can be recognized by MMP or ADAM family proteases and may further include a flexible spacer region to regulate spatial conformation. Antibodies and C1s can form stable conjugated drug molecules through gene fusion or site-specific coupling.

[0080] (iv) Description of the in vitro action of ABC drugs: In the in vitro experimental system, the ABC drugs constructed above were brought into contact with tumor cells expressing HER2. The antibody targeting module first binds to the HER2 antigen on the surface of the tumor cell membrane, allowing the ABC drugs to be located on the outer side of the cell membrane. Subsequently, under the action of proteases present in the tumor cells and their microenvironment, the linker is cleaved or conformationally relaxed, making the complement effector module functionally accessible.

[0081] In the presence of complement components, activated C1s cleave complement components C4 and C2 on the tumor cell membrane surface, initiating the classical complement pathway and forming C3 convertase. As the complement cascade proceeds, the membrane attack complex assembles on the tumor cell membrane, leading to lytic death of the tumor cells.

[0082] (V) Explanation of the Technical Effects of the Embodiments Through the above-described embodiments, the classical complement pathway can be effectively activated on the surface of tumor cell membranes, inducing complement-dependent cytotoxicity. This process does not depend on the endocytic capacity of tumor cells or the state of intracellular signaling pathways, thus retaining potential application value even in the context of multiple drug resistance. This embodiment demonstrates that the technical solution of the present invention is feasible in engineering and can achieve the expected complement-enhanced antitumor effect.

[0083] II. Example 2

[0084] The following embodiments, without altering the overall technical concept of the present invention, vary the structural form of the antibody targeting module and its connection method with the complement effector module to illustrate the diversity and flexibility of the technical solution of the present invention in terms of structural design and engineering implementation. These embodiments are merely illustrative of optional implementation methods and do not constitute a limitation on the scope of protection of the present invention.

[0085] [Example 2: Construction Example of HER2-Targeting-C1s Conjugate Drug Based on Antibody Fragment] (I) Composition of Antibody Targeting Module In this example, the antibody targeting module uses an antibody fragment structure with HER2 antigen-binding ability. The antibody fragment can be a single-chain variable fragment (scFv), a single-domain antibody (VHH), or other antibody-derived structures that can maintain HER2 specific recognition ability. Compared with full-length immunoglobulin molecules, antibody fragments usually have the characteristics of smaller molecular weight and relatively simpler structure, which is conducive to flexible engineering construction in different tissue environments. The antibody fragment can be prepared by conventional recombinant protein expression system and purified to obtain a targeting module with stable antigen-binding ability.

[0086] It should be noted that the present invention does not limit the specific sequence or source of the antibody fragment. As long as it can achieve specific recognition of the HER2 antigen, it can be used as the antibody targeting module in this embodiment.

[0087] (II) Configuration of the Complement Effector Module In this embodiment, the complement effector module still uses the key enzyme C1s in the classical complement pathway or its functional domain with complement cleavage activity. Compared with the full-length C1s protein, the functional domain form has certain advantages in terms of molecular size and structural complexity, which facilitates the overall engineering design with antibody fragments.

[0088] The C1s or their functional domains can be obtained through recombinant expression, while maintaining their complement-related enzymatic functions in the design. Before entering the targeting space, the complement effector module can be in a conformationally or spatially confined state to meet the engineering requirements of the overall molecule.

[0089] (III) Connection method between antibody fragment and complement effector module In this embodiment, the antibody fragment and complement effector module are connected by an engineered linker. The linker is preferably a structure that can be recognized by extracellular or cell membrane surface proteases, and may include a flexible spacer region as needed to adjust the spatial distance and conformational freedom between the antibody fragment and complement effector module.

[0090] Through this connection method, antibody fragments and complement effector modules can form structurally stable conjugate molecules. Under overall design conditions, the complement effector module remains confined until it reaches the target region, while in specific spatial environments, its functional domains can achieve greater spatial accessibility.

[0091] (iv) Technical Description of the Construction Example: By using antibody fragments instead of full-length antibody molecules, this embodiment illustrates that the antibody-biomacromolecule conjugate of the present invention does not depend on a specific antibody structural form. The core function of the antibody targeting module is to achieve the localization function of the HER2 antigen, which can be achieved by either a full-length antibody or an antibody fragment with equivalent antigen-binding ability.

[0092] Therefore, without changing the complement effector module and its spatial regulation design principles, by rationally selecting antibody fragment types and cooperating with engineered linker design, it is also possible to construct antibody-complement coupling structures that conform to the technical concept of this invention.

[0093] (V) Significance of the Embodiments This embodiment further illustrates that the technical solution of the present invention has good structural adaptability and engineering flexibility. The present invention does not limit the antibody targeting module to adopt a full-length immunoglobulin structure, but allows the use of various antibody-derived forms with antigen recognition capabilities. This embodiment is used to illustrate the constructability of the technical solution of the present invention under different structural combinations, providing supplementary explanation for those skilled in the art to understand and implement the present invention, and also supporting the limitation on the diversity of antibody targeting modules in the claims.

[0094] III. Alternative Implementation Methods

[0095] Without departing from the overall technical concept and core technical features of this invention, those skilled in the art can make various substitutions, modifications, or combinations to the foregoing embodiments. The following alternative embodiments are used to further illustrate the openness, stability, and applicability of the technical solution of this invention in terms of structural design and engineering implementation, and do not constitute a limitation on the scope of protection of this invention.

[0096] (I) Alternative Implementation Methods for Incomplete Release of the Complement Effector Module In some alternative implementation methods, the complement effector module does not need to be completely dissociated from the antibody targeting module after targeting. By rationally designing the length, flexibility, and spatial orientation of the linker, even if the complement effector module remains connected to the antibody module through the linker, it can participate in complement-related reactions on the target cell membrane surface as long as its spatial conformation allows access to the complement substrate. This alternative method reduces the dependence on complete cleavage events and improves the adaptability of the present invention under different engineering conditions.

[0097] (ii) Alternative Implementation Methods Involving Conformal Changes Rather Than Complete Cleavage: In another alternative implementation method, the linker does not undergo complete cleavage, but rather local conformational relaxation or spatial rearrangement occurs under the action of extracellular or cell membrane surface proteases. This conformational change can remove the spatial constraints on the active sites of the complement effector module, allowing the complement effector module to transition from a restricted state to a functionally accessible state. This alternative approach emphasizes functional release through spatial regulation, rather than relying on the complete cleavage of the linker.

[0098] (III) Alternative Implementation Methods Triggered by Different Tumor-Associated Proteases In addition to the aforementioned preferred MMP or ADAM family proteases, any protease with relatively specific activity on the outer side of the target cell membrane or in its adjacent microenvironment can be used to trigger linker cleavage or conformational changes. By selecting appropriate protease-responsive linkers for different tissue or pathological microenvironment characteristics, the technical solution of this invention can be adapted to different application scenarios.

[0099] (iv) Alternative Implementations of Complement Effect Modules in Different Active States In some alternative implementations, the complement effect module can be designed as a low-basic-activity or precursor form. This state remains stable in a cyclic environment, while under specific spatial conditions, higher functional accessibility is achieved through connector cleavage or conformational changes. This design helps to balance functional implementation with system stability requirements in the overall engineering design.

[0100] (v) Alternative Implementation Methods for Varying Antibody Targeting Module Forms: Without altering the fundamental principle of complement enhancement, antibody targeting modules can be antibodies or antibody fragments from different sources, with different structural forms, or different affinity ranges. By rationally selecting antibody characteristics, structural design and optimization can be performed for different antigen densities or tissue distribution characteristics. This alternative method further illustrates that the technical solution of the present invention does not depend on a single antibody form for implementation.

[0101] In summary, the above alternative embodiments demonstrate that the antibody-guided complement-related reaction technical solution of the present invention has good engineering flexibility and structural adaptability. Reasonable adjustments to the structural form, spatial regulation method, or triggering conditions, without departing from the core technical concept of the present invention, are all within the optional implementation scope of the technical solution of the present invention.

Claims

1. An antibody-biomacromolecule conjugate, characterized in that, include: The system comprises: an antibody-targeting module capable of specifically binding to cell membrane surface antigens; a complement effector module, which is a key enzyme C1s in the classical complement pathway or a functional domain with complement cleavage activity; and a linker connecting the antibody-targeting module to the complement effector module. The linker is designed to undergo cleavage or conformational changes on the outer surface of the cell membrane or in the extracellular microenvironment, thereby transforming the complement effector module from a restricted state to a functionally accessible state, enabling it to participate in and promote the initiation of the classical complement pathway in the cell membrane region where the antibody-targeting module is located.

2. The antibody-biomacromolecule conjugate according to claim 1, wherein, The antibody targeting module is a full-length immunoglobulin, Fab, F(ab′)2, single-chain variable fragment (scFv), or single-domain antibody.

3. The antibody-biomacromolecule conjugate according to claim 1 or 2, wherein, The antibody targeting module can specifically bind to receptors on the cell membrane surface.

4. The antibody-biomacromolecule conjugate according to claim 3, wherein, The cell membrane surface receptor is human epidermal growth factor receptor 2 (HER2).

5. The antibody-biomacromolecule conjugate according to claim 1, wherein, The complement effector module is a full-length C1s protein.

6. The antibody-biomacromolecule conjugate according to claim 1, wherein, The complement effect module is a functional domain of C1s with complement cleavage activity.

7. The antibody-biomacromolecule conjugate according to claim 1, wherein, The linker is a polypeptide linker that can be recognized by extracellular or cell membrane surface proteases.

8. The antibody-biomacromolecule conjugate according to claim 7, wherein, The extracellular or cell membrane surface proteases are selected from matrix metalloproteinases or ADAM family proteases.

9. The antibody-biomacromolecule conjugate according to claim 1, wherein, When the connector does not undergo cleavage or conformational change, it spatially restricts the active sites of the complement effector module.

10. The antibody-biomacromolecule conjugate according to claim 1, wherein, The complement effector module, while maintaining its connection with the antibody targeting module, can still mediate cleavage reactions related to the classical complement pathway.

11. A method for preparing the antibody-biomacromolecule conjugate of claim 1, characterized in that, include: Provide antibody targeting modules; A complement effector module is provided; the antibody targeting module is connected to the complement effector module via a connector.

12. The method according to claim 11, wherein, The connection is achieved through gene fusion or site-specific coupling.

13. The method according to claim 11, wherein, The linker contains a polypeptide sequence that can be recognized by extracellular or cell membrane surface proteases.

14. The antibody-biomacromolecule conjugate according to claim 1, wherein, The antibody-biomacromolecule conjugate promotes the initiation of the classical complement pathway on the outer side of the cell membrane, and this process does not depend on endocytosis.

15. The antibody-biomacromolecule conjugate according to claim 1, wherein, The connector further includes a flexible spacer region for adjusting the spatial configuration.

Images