Engineered immunoglobulin, and strategy and method for engineering same

Abstract

The present application relates to an engineered immunoglobulin or a fragment thereof, and a strategy and method for engineering same. Specifically, disclosed are an immunoglobulin monomer or fragment engineered to be suitable for forming a polymeric immunoglobulin (pIg), a polymeric protein complex formed therefrom, and a method for using same. Further disclosed is a method for treating or preventing a disease in a subject by using the engineered immunoglobulin, the fragment thereof, or the polymeric protein complex obtained therefrom.

Description

Engineered immunoglobulins and their engineering strategies and methods

[0001] This application claims priority to Chinese Invention Patent Application No. 202510088656.1, filed on January 20, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to engineered immunoglobulins and their engineering strategies and methods, particularly to immunoglobulin monomers engineered to be suitable for forming polymerized immunoglobulin (pIg), the resulting polymerized protein complexes, and methods of using them. Background Technology

[0003] Polymerized immunoglobulins (pIg) are an important component of the immune system in all jawed vertebrates. 1，2 Immunoglobulin M (IgM) is one of the oldest antibody classes, having survived the entire course of evolution, and is the prototype of polymerized immunoglobulins. Mammalian IgM and its linker chain (J chain). 3-5 After binding, they typically assemble into a pentamer. The J chain endows the IgM pentamer with unique functions and enhances its interactions with a variety of receptors and binding partners, such as pIgR, FcμR, and CD5L. 3，8-10 The C-terminus of the IgM heavy chain has an 18-residue tailpiece (tp) domain, which is crucial for polymerization and binding to the J chain. A similar tailpiece domain exists in IgA, which promotes its polymerization and interacts with the J chain. 11-13 In the absence of the J chain, mammalian IgM can form various polymeric structures, including hexamers, pentamers, and tetramers. 14-20 Interestingly, bony fish lack the J chain, but their IgM exhibits a unique tetrameric structure. 21-23 .

[0004] The naturally occurring multivalent nature of pIg endows it with high affinity, which is crucial for binding antigens with high affinity and triggering specific antibody effectors. For example, IgM is a highly efficient activator of the complement system, an important component of the innate immune system that helps clear microbial pathogens and damaged cells. 24，25The classical complement pathway is initiated by activation of the C1 complex, in which C1q binds to IgM or IgG already bound to the antigen. Notably, the IgM hexamer activates the complement system significantly more effectively than the pentamer, likely due to the hexamer structure of C1q as an adaptor protein.17,19,26-28 IgG can also activate the classical complement pathway; however, because IgG is a monomer in solution, multiple IgG molecules (ideally six) need to aggregate together to recruit C1q. 29-32 .

[0005] Engineering IgG to promote hexamer formation has become a key focus in antibody therapy development because these engineered molecules possess high complement-dependent cytotoxic (CDC) activity. 33 The strategy for producing IgM-like IgG involves adding 18 amino acids from the IgM tail domain (μtp). 34，35 Or C575S variant 36 It fuses with the heavy chain of IgG, thereby utilizing the polymerizing ability of μTP. Furthermore, an L309C mutation was introduced into IgG-Fc (Fcγ) to mimic Cys414 in IgM, promoting the formation of disulfide bonds between adjacent IgG molecules. 34，35，37 Further modifications include changing the C-terminal six amino acids of Fcγ (SLSPGK) to the IgM sequence preceding μtp (DKSTGK), and introducing two point mutations, V567I and A572G, into μtp to improve polymerization. (Hereinafter referred to as Fcγ) Innovent ) 38 Furthermore, IgG hexamer structures were found in the crystal lattices of several antibodies, including two anti-HIV-1 gp120 antibodies, b12 and 2G12. 39，40 Based on the reconstructed b12 hexamer structure using crystal symmetry, point mutations such as E345R, E430G, and S440Y were strategically designed to enhance Fcγ-Fcγ interactions and promote hexamer formation. 30，41 These innovations form the basis of the HexaBody antibody technology platform and mark a key advancement in antibody engineering. Similarly, H429Y or H429F (Stellabody) 42 and Q311R / M428E / N434W 43 Mutations in this substance can also lead to increased formation of IgG oligomers and / or increased complement activation.

[0006] The African clawed frog (Xenopus laevis) serves as an evolutionary link between bony fish and more complex vertebrates, and is an ideal model for studying early immunoglobulin differentiation events. Two types of pIg exist in the clawed frog: IgM and IgX. IgX is primarily found in mucus secretions and is considered a functional analogue of IgA. 6，44 However, it is structurally more similar to IgM, with four constant structural domains, while IgA only has three constant structural domains. 45 IgX forms a polymer. 7 However, unlike IgM and IgA, IgX does not appear to bind to the J chain. 6 This observation prompted us to further investigate its assembly mechanism. Summary of the Invention

[0007] This invention provides engineered immunoglobulins and their engineering strategies and methods, particularly relating to immunoglobulin monomers engineered to be suitable for forming polymerized immunoglobulin (pIg), the resulting polymerized protein complexes, and methods of using them.

[0008] In some aspects of the invention, an engineered immunoglobulin or fragment thereof is provided, wherein the C-terminus of the heavy chain of the immunoglobulin or fragment thereof is modified to promote the formation of a polymerized protein complex, preferably the fragment being an Fc region. In some embodiments, such modification of the engineered immunoglobulin or fragment thereof includes adding a tail flap domain to the C-terminus of the heavy chain, or substituting or deleting amino acid residues of the tail flap domain at the C-terminus of the heavy chain. In some specific embodiments, the tail flap domain is a tail flap domain derived from the Fc region of an IgX or IgM immunoglobulin. For example, such a tail flap domain is a tail flap domain of IgX from Xenopus laevis, for example, with the amino acid sequence SSLVNVNLVMS; or a tail flap domain of IgM from Xenopus laevis, for example, with the amino acid sequence PTNVNVSLVLSDTC; or a tail flap domain of IgM from human, for example, with the amino acid sequence PTLYNVSLVMSDTAGTCY.

[0009] In some embodiments, the engineered immunoglobulin or fragment thereof of the present invention is derived from IgG or IgM. In some embodiments, such engineered immunoglobulin or fragment thereof is derived from human.

[0010] In some embodiments, such engineered immunoglobulins or fragments thereof may be derived from human IgM, and the modification therein is a substitution of amino acid residues in the tail flap domain at the C-terminus of the heavy chain. In some specific embodiments, such a substitution may be replacing 18 amino acids at the C-terminus of the Fc domain of human IgM with, for example, SSLVNVNLVMS. In other embodiments, the modification may be a deletion of amino acid residues in the tail flap domain at the C-terminus of the heavy chain. In some specific embodiments, such a deletion is a deletion of one or more amino acids at the C-terminus of the Fc domain of human IgM. In some more specific embodiments, such a deletion is a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 amino acids at the C-terminus of the Fc domain of human IgM, for example, a deletion of 7 amino acids at the C-terminus of the Fc domain of human IgM.

[0011] In some embodiments, such engineered immunoglobulins or fragments thereof may be derived from human IgG, and the modification is the addition of a tail flap domain to the C-terminus of the heavy chain. In some specific embodiments, the added tail flap domain may be derived from the tail flap domain of Xenopus laevis IgX or the tail flap domain of human IgM. In some specific embodiments, the added tail flap domain may be derived from the tail flap domain of human IgM, and one or more amino acids may be deleted at the C-terminus of the tail flap domain of said IgM. In some more specific embodiments, the deletion may be a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 amino acids at the C-terminus of the tail flap domain of said IgM, for example, a deletion of 7 amino acids at the C-terminus of the Fc domain of human IgM. In some more specific embodiments, the amino acid sequence of the added tail flap domain may be, for example, PTLYNVSLVMS. In some embodiments, the added tail flap domain may be linked to the C-terminus of the Fc domain of said IgG heavy chain. In some more specific implementations, the six amino acids at the C-terminus of the Fc domain of the IgG heavy chain can be replaced with, for example, DKSTGK.

[0012] In some embodiments, such engineered immunoglobulins or fragments thereof may be in the form of IgM antibodies, IgG antibodies, or variants thereof. For example, such engineered immunoglobulins or fragments thereof may be Fc domains derived from IgM antibodies, IgG antibodies, or variants thereof. In some specific embodiments, the Fc domain may be linked to the heavy chain of an antigen-binding fragment. In some specific embodiments, the heavy chain of such an antigen-binding fragment may be derived from the heavy chain of any antibody, for example, it may be derived from an anti-CD20 antibody. In some more specific embodiments, such an anti-CD20 antibody may be rituximab.

[0013] In some aspects of the invention, a fusion molecule is provided comprising any engineered immunoglobulin or fragment thereof provided by the invention, said engineered immunoglobulin or fragment thereof being linked to a target molecule, preferably said fragment being an Fc region.

[0014] In some aspects of the invention, engineered multimeric protein complexes are provided that comprise any engineered immunoglobulin or fragment thereof provided by the invention. In some specific embodiments, such multimeric protein complexes are in tetrameric, pentamer, or hexamer form. In some more specific embodiments, such multimeric protein complexes may be in hexamer form.

[0015] In some embodiments, such a polymerized protein complex is in hexammeric form, and each monomer in the hexamer is an engineered immunoglobulin or a fragment thereof provided by the present invention. In some embodiments, the fragment is an Fc region. In some embodiments, the Fc region is linked to a target molecule. In some specific embodiments, such monomers form a hexamer through association with tail-plate domains. In some embodiments, each monomer contained in the polymerized protein complex is identical. In other embodiments, the monomers contained in the polymerized protein complex may be different.

[0016] In some embodiments, each monomer in the hexamer is the Fc region of the engineered immunoglobulin provided by the present invention, and the Fc region is linked to the target molecule. In some embodiments, the target molecule includes ACE2 or GCSFR. In some embodiments, the Fc region is linked to the target molecule to form a fusion molecule, for example, by linking the Fc region to the C-terminus of the target molecule. The fusion molecule thus obtained enables the target molecule to form a hexamer, thereby improving its affinity and specificity and significantly enhancing the corresponding therapeutic effect.

[0017] In some embodiments, the monomers comprising the polymerized protein complex of the present invention can form a hexamer through association with a tail-like domain, wherein the tail-like domain may be a tail-like domain of IgX from Xenopus laevis, for example, whose amino acid sequence may be SSLVNVNLVMS. In other embodiments, such a tail-like domain may be a tail-like domain of IgM from humans. In some specific embodiments, there is a deletion of one or more amino acids at the C-terminus of the tail-like domain of said IgM. In some more specific embodiments, such a deletion may be a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 amino acids at the C-terminus of the tail-like domain of said IgM. For example, such a deletion may be a deletion of 7 amino acids at the C-terminus of the tail-like domain of said IgM. In some more specific embodiments, the amino acid sequence of the tail-like domain may be, for example, PTLYNVSLVMS.

[0018] In some embodiments, the monomers comprising the polymerized protein complex of the present invention may be in the form of IgM antibodies, IgG antibodies, or variants thereof. For example, such monomers may be derived from the Fc domain of an IgM antibody, IgG antibody, or a variant thereof, optionally linked to a target molecule. In some specific embodiments, such Fc domains are linked to the heavy chain of an antigen-binding fragment. In some specific embodiments, the heavy chain of such an antigen-binding fragment may be derived from the heavy chain of any antibody, for example, from an anti-CD20 antibody. In some more specific embodiments, such an anti-CD20 antibody may be rituximab.

[0019] In some embodiments, the polymerized protein complex of the present invention may be, for example, a hexamer formed by an hFcμ-χtp chimera, such an hFcμ-χtp chimera may be obtained by replacing the tail-like domain at the C-terminus of the Fc domain of the human IgM heavy chain with SSLVNVNLVMS.

[0020] In some embodiments, the polymerized protein complex of the present invention can be a hexamer formed from a human Fcμ variant, which can be obtained by deleting one or more amino acids from the C-terminus of the tail region in the Fc domain of the human IgM heavy chain. In some specific embodiments, the deletion can be the deletion of 1, 2, 3, 4, 5, 6, 7, or 8 amino acids at the C-terminus of the tail region. In some more specific embodiments, the deletion can be the deletion of 7 amino acids at the C-terminus of the tail region. In some more specific embodiments, the amino acid sequence of the tail region of such a human Fcμ variant can be, for example, PTLYNVSLVMS.

[0021] In some embodiments, the polymerized protein complex of the present invention can be a hexamer formed from a human Fcγ variant, wherein the human Fcγ variant can be obtained by adding a tail flap domain to the C-terminus of the Fc domain of a human IgG heavy chain. In some specific embodiments, the added tail flap domain can be derived from the tail flap domain of human IgM, and there can be a deletion of one or more amino acids at the C-terminus of the tail flap domain. In some more specific embodiments, such a deletion can be a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 amino acids at the C-terminus of the tail flap domain, for example, a deletion of 7 amino acids at the C-terminus of the tail flap domain. In some more specific embodiments, the amino acid sequence of the tail flap domain of the human Fcγ variant is PTLYNVSLVMS. In some specific embodiments, the six amino acids at the C-terminus of the IgG heavy chain Fc domain can be replaced with DKSTGK, thereby linking it to the added tail flap domain.

[0022] In some embodiments, the monomers comprising the polymerized protein complex of the present invention may be in the form of IgM antibodies, IgG antibodies, or variants thereof, wherein the monomers may form hexamers through association with a tail-plate domain, and wherein the amino acid sequence of the tail-plate domain may be, for example, PTLYNVSLVMS. In some specific embodiments, the heavy chain of the antigen-binding fragment in the monomer is derived from any antibody, for example, from an anti-CD20 antibody. In some more specific embodiments, such an anti-CD20 antibody may be rituximab.

[0023] In some aspects of the invention, the use of engineered immunoglobulins or fragments thereof, or fusion molecules comprising said engineered immunoglobulins or fragments thereof, or corresponding polymerized protein complexes, is provided, for example, for use in the preparation of pharmaceuticals, in the treatment or prevention of diseases. In some specific embodiments, the resulting pharmaceuticals may have the activity of inducing complement-dependent cytotoxicity or may be able to enhance the neutralizing efficacy against viruses such as respiratory infectious viruses. In some specific embodiments, the engineered immunoglobulins or fragments thereof or corresponding polymerized protein complexes of the present invention, or pharmaceuticals prepared therefrom, may be used to treat patients with cancer. In some more specific embodiments, such patients may have hematologic malignancies, such as B-cell lymphoma. In other specific embodiments, the engineered immunoglobulins or fragments thereof or corresponding polymerized protein complexes of the present invention, or pharmaceuticals prepared therefrom, may be used to inhibit or block complement activation. Therefore, in some more specific embodiments, the engineered immunoglobulins or fragments thereof or corresponding polymerized protein complexes of the present invention, or pharmaceuticals prepared therefrom, may be used to treat diseases or conditions that can be improved or alleviated by inhibiting or blocking complement activation.

[0024] In some aspects of the invention, methods are provided for obtaining engineered immunoglobulins or fragments thereof, or fusion molecules or polymerized protein complexes comprising said engineered immunoglobulins or fragments thereof. In some embodiments, the fragment is an Fc region. In some embodiments, such methods include modifying the C-terminus of the heavy chain of an immunoglobulin or fragment thereof to promote the formation of polymerized protein complexes from said immunoglobulin or fragment thereof. In some specific embodiments, such modification includes adding a tail flap domain to the C-terminus of the heavy chain, or substituting or deleting amino acid residues of the tail flap domain at the C-terminus of the heavy chain. In some specific embodiments, the tail flap domain is a tail flap domain derived from the Fc region of an IgX or IgM immunoglobulin. For example, such a tail domain is the tail domain of IgX from Xenopus laevis, for example, with the amino acid sequence SSLVNVNLVMS; or the tail domain of IgM from Xenopus laevis, for example, with the amino acid sequence PTNVNVSLVLSDTC; or the tail domain of IgM from humans, for example, with the amino acid sequence PTLYNVSLVMSDTAGTCY.

[0025] In some embodiments, the engineered immunoglobulin or fragment thereof provided by the method of the present invention may be derived from IgA, IgG, or IgM. In some embodiments, such engineered immunoglobulin or fragment thereof may be derived from human.

[0026] In some embodiments, the engineered immunoglobulin or fragment thereof provided by the method of the present invention may be derived from human IgM, and the modification therein is a substitution of amino acid residues in the tail-fin domain at the C-terminus of the heavy chain. In some specific embodiments, such a substitution may be replacing 18 amino acids at the C-terminus of the Fc domain of human IgM with, for example, SSLVNVNLVMS. In other embodiments, the modification may be a deletion of amino acid residues in the tail-fin domain at the C-terminus of the heavy chain. In some specific embodiments, such a deletion is a deletion of one or more amino acids at the C-terminus of the Fc domain of human IgM. In some more specific embodiments, such a deletion is a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 amino acids at the C-terminus of the Fc domain of human IgM, for example, a deletion of 7 amino acids at the C-terminus of the Fc domain of human IgM.

[0027] In some embodiments, the engineered immunoglobulin or fragment thereof provided by the method of the present invention may be derived from human IgG, and the modification is the addition of a tail flap domain to the C-terminus of the heavy chain. In some specific embodiments, the added tail flap domain may be derived from the tail flap domain of Xenopus laevis IgX or the tail flap domain of human IgM. In some specific embodiments, the added tail flap domain may be derived from the tail flap domain of human IgM, and one or more amino acids may be deleted at the C-terminus of the tail flap domain of said IgM. In some more specific embodiments, the deletion may be a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 amino acids at the C-terminus of the tail flap domain of said IgM, for example, a deletion of 7 amino acids at the C-terminus of the Fc domain of human IgM. In some more specific embodiments, the amino acid sequence of the added tail flap domain may be, for example, PTLYNVSLVMS. In some embodiments, the added tail flap domain may be linked to the C-terminus of the Fc domain of said IgG heavy chain. In some more specific implementations, the six amino acids at the C-terminus of the Fc domain of the IgG heavy chain can be replaced with, for example, DKSTGK.

[0028] In some embodiments, the engineered immunoglobulin or fragment thereof provided by the method of the present invention may be in the form of an IgM antibody, an IgG antibody, or a variant thereof. For example, such an engineered immunoglobulin or fragment thereof may be an Fc domain derived from an IgM antibody, an IgG antibody, or a variant thereof. In some specific embodiments, the Fc domain may be linked to the heavy chain of an antigen-binding fragment. In some specific embodiments, the heavy chain of such an antigen-binding fragment may be derived from the heavy chain of any antibody, for example, from an anti-CD20 antibody. In some more specific embodiments, such an anti-CD20 antibody may be rituximab. Attached Figure Description

[0029] Figure 1. Xenopus laevis Fcχ can form stable hexamers. a. Cryo-electron microscopy two-dimensional classification analysis shows that both Fcχ and FcχCχ3-Cχ4-χtp can form hexamers. b. FcχCχ3-Cχ4-χtp hexamers in The 3D structural model reconstruction at high resolution is presented as a superimposed electronic density structural model from two angles. As shown by the dashed circle, the χtp region cannot be clearly resolved due to density blurring in this area. c. Side-by-side comparison of Fcχ hexamer (blue), human Fcμ-J pentamer (Fcμ is brown, J chain is orange), and bony fish Fcμ tetramer (cyan). d. Side-by-side comparison of Fc-Fc interactions in Fcχ hexamer, human Fcμ-J pentamer, and bony fish Fcμ tetramer. Interference regions of Cμ4 or Cχ4 domains, interchain disulfide bonds, and FG rings are marked in pink, yellow, and red, respectively.

[0030] Figure 2. hFcμ-χtp chimera forming a hexamer. a. Sequence alignment of the caudal domain regions of Xenopus xenograft IgX and IgM, bony fish IgM (from rainbow trout (Oncorhynchus mykiss)), and human IgM. The caudal domain regions are highlighted. b. hFcμ-μtp 18 SV-AUC analysis showed the presence of monomers, tetramers, pentamers, and hexamers in the solution. Cryo-electron microscopy two-dimensional classification analysis showed hFcμ-μtp 18 The polymer is composed of a mixture of tetramers, pentamers, and hexamers. d.SV-AUC analysis showed that, with hFcμ-μtp 18 Conversely, the hFcμ-χtp multimer is a homogeneous hexamer. e. Cryo-electron microscopy two-dimensional classification analysis of hFcμ-χtp chimeras. f. hFcμ-χtp hexamers in The 3D structural model was reconstructed at a resolution and displayed as a superimposed electronic density structural model from two angles.

[0031] Figure 3. Human Fcμ variants with shorter tail domains can form hexamers more efficiently. a. SV-AUC analysis shows that, in addition to monomers, hFcμ-μtp 11 a. Exists only in solution as a hexamer. b. Two-dimensional cryo-electron microscopy analysis of hFcμ-μtp variants with different tail domain lengths. c. Native-PAGE analysis of hFcμ variants. d. hFcμ-μtp 11 hexamer in Reconstruction of a frozen 3D structural model at high resolution, presented as a superimposed electron density structural model from two angles. e.hFcμ-μtp 11 A structural comparison of the hexamer and the hFcμ-J pentamer is conducted, with a focus on the Cμ4 region. hFcμ-μtp 11 The Fcμ molecules in the hexamer are shown in green, while the Fcμ molecules in the Fcμ-J pentamer are shown in white. The J chain is marked in orange.

[0032] Figure 4. hFcγ-μtp 11 Hexamers. a.hFcμ, hFcγ and engineered hFcγ-μtp 11 Sequence alignment of the C-terminal region of the chimera. b. SEC analysis showed hFcγ-μtp 11 It exists primarily in solution as a hexamer. hFcγ-μtp at resolution 11 Reconstruction of the three-dimensional structural model of the hexamer. d.hFcγ-μtp 11 Structural comparison of the hexamer and the IgG1-b12 hexamer in the crystal lattice. hFcγ-μtp11 The hexamer appears purple, while the IgG1-b12 hexamer appears pale blue. The Fc-Fc interaction within the e.Fcγ hexamer is shown. The interaction regions between the two Fcγ molecules are marked in orange and yellow, respectively.

[0033] Figure 5. With μtp 11 The IgM and IgG hexamers showed stronger CDC activity. a. CDC activity of engineered RTX-IgG and RTX-IgM antibodies was evaluated using OCI-Ly10 cells in the presence of human complement. Data are presented as mean ± 1 s.d. n = 3 biological replicates. Source data are provided in the source data file. b. CDC activity of engineered RTX-IgG and RTX-IgM antibodies against Daudi cells. For ease of detection, Daudi cells and Raji cells in c were first cultured for several days in RPMI-1640 medium supplemented with 10% heat-inactivated FBS. Data are presented as mean ± 1 s.d. n = 3 biological replicates. c. CDC activity data of engineered RTX-IgG and RTX-IgM antibodies against Raji cells are presented as mean ± 1 s.d. n = 3 biological replicates.

[0034] Figure 6. With μtp 11 Engineered IgG-Fc hexamers (instead of IgM-Fc hexamers) can block RTX-IgG-mediated complement activation and alleviate LPS-mediated acute lung injury. a. Containing μtp 11 The IgG-Fc hexamer can block RTX-IgG-mediated complement activation in Daudi cells. Containing μtp 11 No inhibitory effect was observed in IgM-Fc hexamer or IgG-Fc monomer. Data are presented as mean ± 1 s.d. n = 3 biological replicates. Source data can be found in the source data file. b. with μtp 11 The IgG-Fc hexamer blocked RTX-IgG-mediated complement activation in Raji cells. Data are presented as mean ± 1 s.d. n = 3 biological replicates. c. At the administered dose, Fcγ-μtp was administered via intraperitoneal injection (ip) of PBS. 11 LPS-mediated lung / body weight ratio determination in BALB / c mice after hexamer or hFcγ monomer administration. Data are presented as mean ± 1 s.d. Black dots represent individual data points of n = 14. Statistical analysis was performed using two-way ANOVA, and p-values ​​are indicated in the figures. d. At the administered dose, via intravenous injection (iv) PBS, Fcγ-μtp 11Total white blood cells (WBCs) in bronchoalveolar lavage fluid of BALB / c mice after hexamerization or Fcγ monomerization using LPS. Data are shown as mean ± 1 s.d. Black dots represent individual data points of n=14. e. Histological analysis of lung tissue. Data show the mean (±1 s.d.) histological score of lung tissue (0 = normal, 1 = mild, 2 = moderate, 3 = significant, 4 = severe). Data are shown as mean ± 1 s.d. Black dots represent individual data points of n=14. f. Representative H&E staining sections of lung tissue from BALB / c mice using LPS. The lungs of untreated (naive) mice are also shown in the figure. Histopathological changes include: mild to moderate thickening of alveolar walls (black arrows), rare alveolar inflammatory cell infiltration (blue arrows), rare alveolar erythrocyte infiltration (green arrows), and extensive interstitial vascular congestion (red arrows). g. Enzyme-linked immunosorbent assay (ELISA) was used to determine and compare the production of C4d in human serum. Background (BKG) levels of C4d in serum are represented by dashed lines. Ab: Antibody. Data are presented as mean ± 1 s.d. n = 3 biological replicates.

[0035] Figure 7. Protein purification. a. Purified Fcχ and Fcχ Δχtp Size exclusion chromatography (SEC) chromatogram. Fcχ is shown in blue. Δχtp a. Orange. b. SEC map of purified hFcμ-μtp variant. c. SDS-PADE analysis of purified hFcμ-μtp variant. d. SDS-PAGE analysis of purified engineered RTX-hFcγ and RTX-hFcμ antibodies. e. Yields (mg / L) of various immunoglobulins purified from HEK293F cells under our laboratory conditions.

[0036] Figure 8. Cryo-EM 3D reconstruction of FcχCχ3-Cχ4-χtp hexamer. a. Cryo-EM data processing flowchart of FcχCχ3-Cχ4-χtp. b. Resolution estimation of the overall FcχCχ3-Cχ4-χtp map. c. Resolution estimate of the local map around the Fcχ-Fcχ interface of FcχCχ3-Cχ4-χtp. d. FSC curve with predicted resolution of FcχCχ3-Cχ4-χtp. e. Angular distribution of FcχCχ3-Cχ4-χtp particles used for final 3D reconstruction. f. FSC curve and predicted resolution of local map. g. Angular distribution of FcχCχ3-Cχ4-χtp particles used in the final 3D reconstruction of the local map.

[0037] Figure 9. Structural comparison. a. Overlay of the structures of Fcχ hexamer and hFcμ-J pentamer. Fcχ is shown as white, while hFcμ is brown. The J chain in hFcμ-J is shown as orange. b. Overlay of the structures of Fcχ hexamer and bony fish tFcμ tetramer. Fcχ is shown as white, while tFcμ is cyan. c. Comparison of the structures of Fcχ hexamer and hFcμ-χtp hexamer. Fcχ is shown as white, while hFcμ is green. d. hFcμ-μtp 11 The hexamer and hFcμ-J pentamer are located in the Fcμ1-Cμ4 and Fcμ5-Cμ4 regions. hFcμ-μtp 11 The Fcμ molecules in the hexamer and Fcμ-J pentamer are shown in green and white, respectively. The J chain is represented in orange.

[0038] Figure 10. Sequence alignment of the CH3-CH4 regions of Xenopus IgX, Xenopus IgM, bony fish IgM, and human IgM. Secondary structures are depicted above and below the sequence blocks, with color coding corresponding to the molecular tags. Orange rectangles highlight Cys430 in the Cχ3 domain of IgX, as well as the corresponding Cys residues in Xenopus and human IgM. In contrast, similar Cys residues are significantly lacking in bony fish IgM.

[0039] Figure 11. Cryo-electron microscopy 3D reconstruction of hFcμ-χtp chimeras. a. Cryo-electron microscopy data processing flowchart for hFcμ-χtp. b. Resolution estimation of the overall hFcμ-χtp image. c. FSC curve with estimated hFcμ-χtp resolution. d. Angular distribution of hFcμ-χtp particles used for final 3D reconstruction.

[0040] Figure 12. hFcμ-μtp 11 Cryo-electron microscopy three-dimensional reconstruction of the hexamer. a.hFcμ-μtp 11 Flowchart of cryo-electron microscopy data processing. b.hFcμ-μtp 11 Overall image resolution estimation. c. With hFcμ-μtp 11 d. FSC curve for estimated resolution. hFcμ-μtp used for final 3D reconstruction. 11 Angular distribution of particles.

[0041] Figure 13. hFcγ-μtp 11 Cryo-electron microscopy three-dimensional reconstruction of the hexamer. a.hFcγ-μtp 11 Flowchart of cryogenic EM data processing. b.hFcγ-μtp 11 Overall spectrum resolution estimation. c. With hFcγ-μtp 11 FSC curve for estimated resolution. d. hFcγ-μtp used for final 3D reconstruction. 11Angular distribution of particles. e. Interaction between two adjacent hFcγ structural domains. f. Density map of Fcγ-Fcγ interface interactions.

[0042] Figure 14. Hexamers that xlFcμ can form. a. Size exclusion chromatography (SEC) chromatogram of purified xlFcμ. b. Cryo-electron microscopy two-dimensional classification results show that xlFcμ forms stable hexamers.

[0043] Figure 15. hACE2-hFcμ-μtp 11 It can form a hexamer. a. Purified hACE2-hFcμ-μtp 11 Size exclusion chromatography (SEC) chromatogram. b. Purified hACE2-hFcμ-μtp 11 SDS-PADE analysis. c. Cryo-electron microscopy two-dimensional classification results show that hACE2-hFcμ-μtp 11 It can form a stable hexamer.

[0044] Figure 16. GCSFR-hFcμ-μtp 11 It can form a hexamer. a. Purified Spytag003-hFcμ-μtp 11 Size exclusion chromatography (SEC) chromatogram. b. Purified Spytag003-hFcμ-μtp 11 c. Size exclusion chromatogram (SEC) of purified GCSFR-SpyCatcher003. d. SDS-PADE analysis of purified GCSFR-SpyCatcher003. e. GCSFR-hFcμ-μtp 11 Construction diagram. f.GCSFR-hFcμ-μtp 11 Size exclusion chromatography (SEC) chromatogram. g.GCSFR-hFcμ-μtp 11 SDS-PADE analysis. h. Cryo-electron microscopy two-dimensional classification results showed that GCSFR-hFcμ-μtp 11 It can form a stable hexamer.

[0045] Figure 17. Structure predictions using AlphaFold3. a. Comparison of Fcχ cryo-electron microscopy structures with the top five Fcχ hexamer structures predicted by AlphaFold3. b. “6+6” Fcχ assembly structure predicted by AlphaFold3. c. “5+7” Fcχ assembly structure predicted by AlphaFold3. Detailed Implementation

[0046] definition

[0047] Unless the context explicitly indicates otherwise, nouns without quantifiers used herein refer to one or more types. For example, the term "antigen" includes one or more antigens and may be considered equivalent to the phrase "at least one antigen." The term "comprising" as used herein means "including." Thus, "comprising antigen" means "including antigen" without excluding other elements. The phrase "and / or" means "and" or "or." It should also be understood that, unless otherwise indicated, any and all base sizes or amino acid sizes given for nucleic acids or polypeptides, as well as all molecular weights or molecular mass values, are approximate and provided for descriptive purposes. While many methods and materials similar to or equivalent to those described and used herein may be used, specific suitable methods and materials are described below. In case of conflict, this specification (including the explanation of terms) shall prevail. Furthermore, materials, methods, and examples are illustrative only and are not intended to be limiting. For the convenience of summarizing various embodiments, the following explanations of terms are provided:

[0048] The term “about” when referring to a measurable value (e.g., quantity, duration, etc.) means covering a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1%, because such variation is suitable for carrying out the disclosed method.

[0049] Unless otherwise noted, the technical terms used in this article are used according to their usual usage. Definitions of commonly used terms in molecular biology can be found in Benjamin Lewin, Genes VII, Oxford University Press, 1999; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc., 1995; and other similar references.

[0050] As used herein, the terms "protein" or "polypeptide" refer to a molecule composed of multiple monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). Therefore, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to one or more chains of two or more amino acids are included within the definition of "protein," and the term "polypeptide" may be used interchangeably with any of these terms. The terms "protein" or "polypeptide" also refer to the product of its post-expression modifications, including but not limited to glycosylation, acetylation, phosphorylation, amidation, and amino acid modifications derived from known protecting / blocking groups, protease cleavage, or non-naturally generated amino acids. Polypeptides may be derived from biological sources or generated by recombinant technologies, but are not necessarily translated from a specified nucleic acid sequence. They can be generated in any manner, including through chemical synthesis.

[0051] The size of the proteins disclosed herein can have about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids.

[0052] "Isolated" proteins, or their fragments, variants, or derivatives, refer to proteins that are not in their native environment. No specific purification level is prescribed. For example, isolated proteins may be removed from their natural or native environment. Recombinant proteins expressed in host cells and proteins considered isolated as disclosed herein, as well as native or recombinant proteins isolated, fractionated, or partially or substantially purified by any suitable technique, are also considered.

[0053] The term “non-naturally occurring protein” or any grammatical variation thereof, as used in this article, is a conditional qualifier that explicitly excludes, but only excludes, those protein forms that a judge or administrator or judicial authority may determine or interpret as “naturally occurring”.

[0054] Other proteins disclosed herein are fragments, derivatives, analogs, or variants of the aforementioned proteins, and any combination thereof. The terms "fragment," "variant," "derivative," and "analyte" as used herein include any form that retains at least some of the properties of the corresponding original antibody or protein (e.g., specific binding to an antigen). In addition to the specific antibody fragments discussed elsewhere herein, fragments of peptides also include, for example, protease-hydrolyzed fragments and deletion fragments. For example, variants of peptides include fragments as described above, as well as peptides having altered amino acid sequences due to amino acid substitutions, deletions, or insertions. In some aspects, variants may be non-naturally generated. Non-naturally generated variants can be generated using mutagenesis techniques known in the art. Variant peptides may contain conserved or non-conserved amino acid substitutions, deletions, or additions. Derivatives are peptides that have been altered to exhibit additional features not present on the original peptide. Examples include fusion proteins. Peptide variants may also be referred to herein as "peptide analogs." The term "derivative" as used herein may also refer to a target peptide having one or more amino acids derived chemically via a reaction of functional side groups. "Derivative" also includes those peptides containing one or more derived forms of the 20 standard amino acids. For example, 4-hydroxyproline can replace proline; 5-hydroxylysine can replace lysine; 3-methylhistidine can replace histidine; homoserine can replace serine; and ornithine can replace lysine.

[0055] "Conservative amino acid substitution" is a situation where one amino acid is replaced by another amino acid having a similar side chain. Families of amino acids with similar side chains have been defined in the art, including basic side chains (such as lysine, arginine, histidine), acidic side chains (such as aspartic acid, glutamic acid), uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (such as threonine, valine, isoleucine), and aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). For example, phenylalanine replacing tyrosine is a conservative substitution. In some embodiments, the conservative substitution in the sequence of the polypeptides and antibodies of the present invention does not invalidate the binding of the polypeptide or antibody containing the amino acid sequence to the antigen bound to the binding molecule. Methods for identifying conserved substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).

[0056] The term "polynucleotide" is intended to include both single and multiple nucleic acids, referring to isolated nucleic acid molecules or constructs such as messenger RNA (mRNA), cDNA, or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or unconventional bonds (such as amide bonds, as found in peptide nucleic acids (PNA)). The term "nucleic acid" or "nucleic acid sequence" refers to any one or more segments of nucleic acids present in a polynucleotide, such as fragments of DNA or RNA.

[0057] "Isolated" nucleic acids or polynucleotides are any form of nucleic acids or polynucleotides isolated from their original environment. For example, gel-purified polynucleotides or recombinant polynucleotides encoding polypeptides contained in a vector will be considered "isolated." Furthermore, polynucleotide segments engineered to have restriction sites for cloning, such as PCR products, are considered "isolated." Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or recombinant polynucleotides purified (partially or substantially) in non-original solutions (e.g., buffers or saline). Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides, wherein the transcripts are not naturally occurring transcripts. Isolated polynucleotides or nucleic acids also include molecules produced synthetically. Furthermore, polynucleotides or nucleic acids can be or may include regulatory elements such as promoters, ribosome binding sites, or transcription terminators.

[0058] The term “non-naturally occurring polynucleotide” or any grammatical variation thereof as used in this article is a conditional qualifier that explicitly excludes, but only excludes, those nucleic acid or polynucleotide forms that the judged body or regulatory authority or judicial body may determine or interpret as “naturally occurring”.

[0059] As used herein, the term "coding region" refers to the portion of a nucleic acid consisting of codons that translate into amino acids. While "stop codons" (TAG, TGA, or TAA) do not translate into amino acids, they can be considered part of a coding region, and any side sequences such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of a coding region. Two or more coding regions may exist in a single polynucleotide construct, such as on a single vector, or in separate polynucleotide constructs, such as on separate (different) vectors. Furthermore, any vector can contain a single coding region, or may contain two or more coding regions; for example, a single vector may separately encode the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region. In addition, vectors, polynucleotides, or nucleic acids may include heterologous coding regions that are fused to or not fused to another coding region. Heterologous coding regions include, but are not limited to, those encoding proprietary elements or motifs (e.g., secretory signal peptides or heterologous functional domains).

[0060] In some embodiments, the polynucleotide or nucleic acid is DNA. For DNA, a polynucleotide containing a polypeptide encoding a nucleic acid typically contains a promoter and / or other transcriptional or translational control elements that are operatively linked to one or more coding regions. An operative link means that when the coding region of a gene product, such as a polypeptide, is linked to one or more regulatory sequences in this manner, the expression of that gene product is placed under the influence or control of those regulatory sequences. Two DNA segments (such as a polypeptide coding region and its associated promoter) are "operatively linked" if the induction of promoter function leads to the transcription of mRNA encoding the desired gene product, and if the properties of the link between the two DNA segments do not interfere with the ability of the expression regulatory sequence to direct the expression of the gene product or to transcribe the DNA template. Therefore, if a promoter can influence the transcription of a nucleic acid, the promoter region is operatively linked to the polypeptide-encoded nucleic acid. The promoter can be a cell-specific promoter that directs the transcription of the DNA substance in a predetermined cell. Other transcriptional control elements besides promoters, such as enhancers, operons, repressors, and transcription termination signals, can be operatively linked to polynucleotides to direct cell-specific transcription.

[0061] Various transcriptional control regions are known to those skilled in the art. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (immediate early promoter, used in conjunction with intron-A), simian virus 40 (early promoter), and retroviruses (such as Raoul's sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes, such as actin, heat shock proteins, bovine growth hormone, and rabbit β-globulin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcriptional control regions include tissue-specific promoters and enhancers, and lymphokine-inducible promoters (e.g., promoters induced by interferon or interleukin).

[0062] This document discloses certain binding molecules, or antigen-binding fragments, variants, or derivatives thereof. Unless specifically referred to as a full-size antibody, the term "binding molecule" encompasses full-size antibodies and antigen-binding subunits, fragments, variants, analogs, or derivatives of said antibodies, such as engineered antibody molecules or fragments that bind antigens in a manner similar to antibody molecules but employ a different scaffold.

[0063] As used herein, the term "binding molecule" in its broadest sense refers to a molecule that specifically binds to a target or molecular determinant (e.g., an epitope or antigenic determinant). As further described herein, a binding molecule may comprise one or more "antigen-binding domains" as described herein. A non-limiting example of a binding molecule is an antibody or a fragment thereof that maintains antigen-specific binding.

[0064] As used herein, the term "binding domain" or "antigen-binding domain" refers to a region of a binding molecule sufficient to specifically bind to an epitope. For example, "Fv," such as the variable heavy chain and variable light chain of an antibody, as two separate polypeptide subunits or as a single chain, is considered a "binding domain." Other binding domains include, but are not limited to, the variable heavy chain (VHH) of antibodies derived from camelid species, or the six immunoglobulin complementarity-determining regions (CDRs) expressed on a fibronectin scaffold. The term "binding molecule" as used herein may include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more "antigen-binding domains."

[0065] The terms “antibody” and “immunoglobulin” are used interchangeably herein. An antibody (or a fragment, variant, or derivative thereof, as described herein) comprises: at least a variable region of the heavy chain (for camelid species) or at least a variable region of both the heavy and light chains. The basic immunoglobulin structures in vertebrate systems are relatively well understood. See, for example, Harlow et al., *Antibodies: A Laboratory Manual* (Cold Spring Harbor Laboratory Press, 2nd ed., 1988). Unless otherwise stated, the term “antibody” encompasses any substance ranging from small antigen-binding fragments to full-size antibodies, such as IgG antibodies comprising two complete heavy chains and two complete light chains, IgA antibodies comprising four complete heavy chains and four complete light chains and may include a J chain and / or a secretory component, or IgM antibodies comprising ten or twelve complete heavy chains and ten or twelve complete light chains and may include a J chain.

[0066] As discussed in more detail below, the term "immunoglobulin" encompasses a variety of types of polypeptides distinguishable by their biochemical properties. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with further subclasses (such as γ1-γ4 or α1-α2). The characteristics of this chain determine whether an antibody "isotype" is IgG, IgM, IgA, IgG, or IgE. Immunoglobulin subclasses (subtypes) such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 have been well characterized and are known to confer functional specialization. Based on the disclosure herein, those skilled in the art can readily distinguish the respective modified forms of these immunoglobulins, and thus, they are covered within the scope of this invention.

[0067] Light chains are classified as kappa or lambda (κ, λ). Each heavy chain class can bind to either a κ or λ light chain. Typically, light and heavy chains are covalently linked, and when immunoglobulins are expressed by host cells such as hybridomas, B cells, or genetically engineered cells, the “tail” portions of the two heavy chains are linked together via covalent disulfide bonds or non-covalent connections. In the heavy chain, amino acids extend from the N-terminus at the bifurcation end of the Y-configuration to the C-terminus at the base of each chain. The basic structure of some antibodies (e.g., IgG antibodies) comprises two heavy chain subunits and two light chain subunits covalently linked by disulfide bonds to form a “Y” structure, also referred to herein as the “H2L2” structure, or “binding unit.”

[0068] As used herein, the term "binding unit" refers to a portion of a binding molecule, such as an antibody or its antigen-binding fragment, corresponding to a standard immunoglobulin structure, such as two heavy chains or fragments thereof and two light chains or fragments thereof, or two heavy chains or fragments thereof, such as antibodies derived from camelids or cartilaginous fish. In some aspects, such as where the binding molecule is a divalent IgG antibody or its antigen-binding fragment, the terms "binding molecule" and "binding unit" are equivalent. In other aspects, such as where the binding molecule is an IgG hexamer, IgM pentamer, or IgM hexamer, the binding molecule comprises two or more "binding units." A binding unit need not include the full-length antibody heavy and light chains, but will generally be divalent, i.e., will include two "binding domains," as defined below. Some binding molecules provided herein are dimers, tetramers, pentamers, or hexamers, and comprise two, four, five, or six divalent binding units. As used herein, a binding molecule comprising two or more binding units, for example, two, four, five, or six binding units, may be referred to as a "polymer."

[0069] The term “J chain” as used in this article refers to the J chain of a sequence antibody of any animal species, including the mature human J chain.

[0070] The terms “valence,” “bivalent,” “multivalent,” and their grammatical equivalents refer to the number of antigen-binding domains in a given binding molecule or binding unit. Thus, referring to a given binding molecule, such as an IgM antibody or a fragment thereof, the terms “bivalent,” “tetravalent,” and “hexavalent” indicate the presence of two, four, and six antigen-binding domains, respectively. Bivalent or multivalent binding molecules can be monospecific, meaning all antigen-binding domains are identical, or they can be bispecific or multispecific, meaning two or more antigen-binding domains are different, for example, binding to different epitopes on the same antigen, or binding to completely different antigens.

[0071] The term "epitaph" includes any molecular determinant capable of specifically binding to an antibody. In some respects, an epitope may include a chemically active surface group of a molecule (e.g., an amino acid, sugar side chain, phosphoryl group, or sulfonyl group), while in other respects, an epitope may have certain three-dimensional structural features and / or specific charge characteristics. An epitope is a region of the target that an antibody binds to.

[0072] "Multispecific binding molecules or antibodies" or "bispecific binding molecules or antibodies" refer to binding molecules, antibodies, or their antigen-binding fragments that have the ability to specifically bind to two or more different epitopes on the same or different targets. "Monospecific" refers to the ability to bind to only one type of epitope.

[0073] The term "target" is used in its broadest sense to include substances that can be bound by binding molecules. Targets can be, for example, peptides, nucleic acids, carbohydrates, lipids, or other molecules. Furthermore, for example, a "target" can be a cell, organ, or organism containing an epitope that can be bound by binding molecules.

[0074] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used from a functional perspective. In this regard, it should be understood that the variable regions of the variable light (VL) chain or variable heavy (VH) chain portion determine antigen recognition and specificity. Conversely, the constant regions (CL) of the light chain and the constant regions of the heavy chain (e.g., CH1, CH2, CH3, or CH4) provide biological properties such as secretion, transplacental movement, Fc receptor binding, complement binding, etc. By convention, the numbering of constant region domains increases with distance from the antigen-binding site or the amino terminus of the antibody. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 (or CH4 in the case of IgM) and CL domains are actually the carboxyl terms of the heavy and light chains, respectively.

[0075] The terms "Fc region" and "Fc domain" are used interchangeably in this document, referring to crystallizable fragments, which are functional regions of immunoglobulins produced after hydrolysis by papain. These fragments do not possess antigen-binding capabilities, but mediate immune responses such as complement activation, antibody-dependent cytotoxicity (ADCC), and regulation of phagocytosis by binding to Fc receptors on the surface of immune effector cells. For example, in IgG, the Fc region corresponds to the CH2 and CH3 domains of IgG and is the site of interaction between IgG and effector molecules or cells. The Fab region contains the complete variable region and the CH1 region of the constant region; the Fc region refers only to the CH2 and CH3 regions of the IgG constant region, equivalent to the lower part of the Y-shaped structure. Similarly, in IgM, the Fc region corresponds to the CH2, CH3, and CH4 domains of IgM and is the site of interaction between IgM and effector molecules or cells. The Fab region contains the complete variable region and the CH1 region of the constant region; the Fc region refers only to the CH2, CH3, and CH4 regions of the IgM constant region, equivalent to the lower part of the Y-shaped structure.

[0076] Unless explicitly stated otherwise, where a term used herein has two or more definitions, the definition of the term should include all of those meanings. A specific example is the use of the term “complementarity-determining region” (“CDR”) to describe non-contiguous antigen-binding sites found within the variable regions of heavy and light chain polypeptides. These specific regions have been described, for example, by Kabat et al., U.S. Department of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983), and by Chothia et al., J. Mol. Biol. 196: 901-917 (1987). The definitions by Kabat and Chothia include overlaps or subsets of amino acids when compared to each other. The exact amino acid numbering covering a specific CDR will vary depending on the sequence and size of the CDR. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which amino acids contain a specific CDR. Kabat et al. have also defined a numbering system applicable to the variable region sequence of any antibody. Those skilled in the art can readily apply the “Kabat numbering system” to any variable region sequence without relying on any experimental data outside the sequence itself.

[0077] Binding molecules, such as antibodies or their antigen-binding fragments, variants or derivatives, including but not limited to polyclonal, monoclonal, human, humanized or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments containing VL or VH domains, and fragments generated by Fab expression libraries.

[0078] "Specific binding" generally refers to a binding molecule, such as an antibody or a fragment, variant, or derivative thereof, binding to an epitope via its antigen-binding domain, and said binding requires some complementarity between the antigen-binding domain and the epitope. According to this definition, a binding molecule is said to "specifically bind" to an epitope when it binds to the epitope more readily through its antigen-binding domain than to a randomly unrelated epitope. In this paper, the term "specificity" is used to qualitatively analyze the relative affinity of an antibody for a given epitope. For example, binding molecule "A" may be considered to have higher specificity for a given epitope than binding molecule "B," or it may be said that binding molecule "A" binds to epitope "C" with higher specificity or affinity than to the associated epitope "D."

[0079] If a binding molecule, such as an antibody or a fragment, variant, or derivative thereof, preferentially binds to the epitope to a degree that blocks the binding of a reference antibody or antigen-binding fragment to that epitope, then it can be said that the binding molecule, such as an antibody or a fragment, variant, or derivative thereof, competitively inhibits the binding of the reference antibody or antigen-binding fragment to the given epitope. Competitive inhibition can be determined by any method known in the art, for example, a competitive ELISA assay. It can be said that the binding molecule competitively inhibits at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% of the binding of the reference antibody or antigen-binding fragment to the given epitope.

[0080] As used herein, the term "affinity" refers to a measure of the strength of the binding of an individual epitope to one or more binding domains, such as those of an immunoglobulin molecule. The term "avidity" as used herein refers to the overall stability of the complex between a population of antigen-binding domains and the antigen. Affinity relates both to the affinity of an individual antigen-binding domain within a population to a specific epitope and to the valence state of the immunoglobulin molecule and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen, such as a multimer, with a highly repetitive epitope structure is an example of high affinity. The interaction between a bivalent monoclonal antibody and a receptor present at high density on the cell surface will also exhibit high affinity.

[0081] The binding molecules or their antigen-binding fragments, variants, or derivatives disclosed herein may also be described or illustrated in terms of their cross-reactivity. As used herein, the term "cross-reactivity" refers to the ability of a binding molecule (e.g., an antibody or its fragments, variants, or derivatives) to be specific to one antigen and react with a second antigen; it is a measure of the affinity between two different antigenic substances. Therefore, a binding molecule is cross-reactive if it binds to an epitope other than the one that induces its formation. Cross-reactive epitopes typically contain many of the same complementary structural features as the inducing epitope, and in some cases even match it more closely than the original epitope.

[0082] Antibody fragments, including single-chain antibodies or other binding domains, may exist alone or in combination of one or more of the following: a hinge region, a CH1, CH2, CH3, or CH4 domain, a J chain, or a secretory component. Antigen-binding fragments may also be included, which may comprise any combination of one or more variable regions with one or more of the hinge region, CH1, CH2, CH3, or CH4 domain, J chain, or secretory component. Binding molecules, such as antibodies or their antigen-binding fragments, may be derived from any animal source, including fish, amphibians, birds, and mammals. "Human" antibodies as used herein include antibodies having the amino acid sequence of human immunoglobulins, and include antibodies isolated from human immunoglobulin libraries or derived from animal transgenes of one or more human immunoglobulins, and which may express endogenous immunoglobulins in some examples, but not in others.

[0083] As used herein, the term "heavy chain subunit" includes an amino acid sequence derived from the immunoglobulin heavy chain. Binding molecules, such as antibodies containing heavy chain subunits, may include at least one of the following: a VH domain, a CH1 domain, a hinge domain (e.g., upper, middle, and / or lower hinge regions), a CH2 domain, a CH3 domain, a CH4 domain, or variants or fragments thereof. For example, binding molecules, such as antibodies or fragments, variants, or derivatives thereof, may include, in addition to the VH domain, including but not limited to: a CH1 domain; a CH1 domain, a hinge, and a CH2 domain; a CH1 domain and a CH3 domain; a CH1 domain, a hinge, and a CH3 domain; or a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. In some aspects, binding molecules, such as antibodies or fragments, variants, or derivatives thereof, may also include, in addition to the VH domain, a CH3 domain and a CH4 domain; or a CH3 domain, a CH4 domain, and a J chain. Furthermore, the binding molecules used in this invention may lack certain constant region portions, such as all or part of the CH2 domain. Those skilled in the art will understand that these domains (e.g., heavy chain subunits) can be modified so that their amino acid sequence differs from the original immunoglobulin molecule.

[0084] As used herein, the term "light chain subunit" includes an amino acid sequence derived from the immunoglobulin light chain. A light chain subunit includes at least a VL domain and may also include a CL (e.g., Cκ or Cλ) domain.

[0085] A binding molecule, such as an antibody or its antigen-binding fragment, variant, or derivative, may be described or characterized in relation to the epitope or antigenic moiety that it recognizes or specifically binds to. The portion of the target antigen that specifically interacts with the antigen-binding domain of the antibody is called an "epitaph" or "antigenic determinant." A target antigen may contain a single epitope, or at least two epitopes, and may include any number of epitopes depending on the size, conformation, and type of the antigen.

[0086] As used in this article, the term "disulfide bond" refers to a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge a second thiol group.

[0087] As used in this article, the term "chimera" refers to an antibody in which the immunoreactive region or site is derived from or derived from a first species, while the constant region (which may be complete, partial, or modified) is derived from a second species.

[0088] As used herein, the term "engineered" includes manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g., by recombinant technology, in vitro peptide synthesis, by enzyme or chemically coupled peptides, or some combination of these techniques).

[0089] The terms “connected,” “fused,” or “fusion,” or other grammatical equivalents used herein are used interchangeably. These terms refer to the connection of two or more elements or components together by means including chemical coupling or recombination.

[0090] The term "target molecule" as used in this article can refer to any molecule with therapeutic effects, such as any molecule used to treat acute or chronic diseases (including but not limited to acute infectious diseases, chronic infectious diseases, cancer, diabetes, obesity, dry eye syndrome, etc.). Target molecules include antibodies, antigens, receptors, ligands, cytokines, and small molecule active substances (e.g., activators or inhibitors).

[0091] As used herein, the term "fusion molecule" refers to a fusion comprising any engineered immunoglobulin or fragment thereof provided by the present invention and a target molecule, wherein the engineered immunoglobulin or fragment thereof (e.g., the Fc region) is linked to the target molecule.

[0092] As used herein, the term "expression" refers to the process by which a gene produces a biochemical substance such as a polypeptide. This process includes any manifestation of gene function within the cell, including but not limited to gene knockout, transient expression, and stable expression. It includes, but is not limited to, gene transcription into RNA, such as messenger RNA (mRNA), and the translation of such mRNA into a polypeptide. If the desired end product is a biochemical substance, expression includes the production of that biochemical substance and any precursors. Gene expression produces a "gene product." As used herein, a gene product can be a nucleic acid, such as messenger RNA produced through gene transcription, or a polypeptide translated from a transcript. Gene products described herein also include nucleic acids with post-transcriptional modifications, such as polyadenylation, or polypeptides with post-translational modifications, such as methylation, glycosylation, addition of lipids, attachment to other protein subunits, or cleavage by proteases.

[0093] Terms such as “treating,” “treatment,” “to treat,” “alleviating,” or “to alleviate” refer to therapeutic measures that cure, alleviate, or reduce the symptoms of an existing, diagnosed pathological condition or disorder, and / or halt or slow the progression of an existing, diagnosed pathological condition or disorder. Terms such as “prevent,” “prevention,” “avoid,” or “deterrence” refer to preventive or preventative measures that prevent the progression of an undiagnosed target pathological condition or disorder. Therefore, those who need treatment may include those who already have the disease; those who are susceptible to the disease; and those who need to prevent the disease.

[0094] The term "object," "individual," "animal," "patient," or "mammal" refers to any object requiring diagnosis, prevention, or treatment, particularly mammalian objects. Mammal objects include humans, domestic animals, livestock and zoo animals, sports animals, or pets such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, etc.

[0095] Phrases used herein, such as “subjects that will benefit from treatment” and “animals in need of treatment,” include subjects, such as mammals, that will benefit from the administration of binding molecules, such as antibodies containing one or more antigen-binding domains. These binding molecules, such as antibodies, can be used, for example, in diagnostic procedures and / or for the treatment or prevention of disease.

[0096] Polymerized immunoglobulins (pIg) are an important component of the immune system in jawed vertebrates. 1，2In mammals, immunoglobulin M (IgM) typically forms a pentamer linked by a joining chain (J chain). 3-5 In contrast, the IgX of Xenopus laevis uniquely assembles into a multimer independent of the J chain. 6，7 Here, we present the cryo-electron microscopy (cryo-EM) structure of IgX, revealing its hexameric conformation. By adding an 11-residue IgX tailpiece domain to human IgM, we successfully promoted the formation of IgM hexamers. Notably, we found that the length of the tailpiece domain affects the hexamer formation efficiency, as truncating the 18 amino acids of the native IgM tailpiece domain to 11–16 residues enhances hexamer assembly. Importantly, introducing a shortened IgM tailpiece domain into IgG efficiently forms IgG hexamers. The cryo-electron microscopy structures of these engineered proteins demonstrate their assembly mechanisms. We further confirm that these engineered CD20-targeting IgM and IgG hexamers exhibit potent complement-dependent cytotoxicity (CDC) against various B lymphoma cells. Furthermore, this engineered IgG-Fc hexamer effectively acts as a decoy, mitigating CDC. By linking engineered Fc molecules with target molecules (e.g., ACE2, GCSFR) to form fusion molecules, the target molecules can form hexamers, thereby improving their affinity and specificity and significantly enhancing the corresponding therapeutic effects. These findings deepen our understanding of pIg evolution and provide new strategies for developing therapeutic biologics based on IgM, IgG, or the engineered Fc molecules of this invention.

[0097] Example

[0098] Example 1. Method

[0099] Cell culture

[0100] HEK293F cells (Thermo Fisher, 11625019) were cultured in SMM 293-TI medium (Sino Biological) at 37°C, 5% CO2, and 55% humidity in a humidified shaker. OCI-Ly10 cells (RRID: CVCL_8795), Raji cells, and Daudi cells were originally purchased from the American Type Culture Collection and cultured in RPMI-1640 medium (Gibco) supplemented with 10% FBS (PAN Seratech) and 1% penicillin-streptomycin (Gibco) in a humidified incubator at 37°C and 5% CO2.

[0101] animal

[0102] Male BALB / c mice (6-8 weeks old) were obtained from Sford (Beijing) Biotechnology Co., Ltd., catalog number: 110324241105187741. Mice were isolated in an isolation chamber for at least one week, during which time they were identified using ear tags, sex classification, and cage cards. Mice were housed in an SPF (specifically pathogen-free) environment with a temperature maintained between 20.0 and 26.0°C, humidity between 40% and 60%, and a 12-hour light / dark cycle. Bedding consisted of sterile wood shavings, and each cage contained three animals, labeled with their corresponding animal number and sex. Throughout the isolation and experimental period, mice had free access to water and food, and all food and water were free of contaminants that could affect the research results.

[0103] Protein expression and purification

[0104] Codon-optimized DNA fragments encoding Fcχ (residues 240-584, UniProtKB: Q6INK3) or Fcχcχ3-cχ4-χtp (residues 360-584) were cloned into a modified pcDNA vector, with an N-terminal IL-2 signal peptide added and an 8×His tag attached to the tail. The constructs were transiently transfected into HEK293F cells using polyethyleneimine (Polysciences) and cultured for 4 days. Proteins were extracted from the conditioned medium using Ni-NTA affinity resin (Smart Lifesciences) and further purified using a Superose 6 incremental column (GE Healthcare) in a final buffer containing 20 mM HEPES (pH 7.2) and 150 mM NaCl.

[0105] For hFcμ with different tail domains, the DNA fragment encoding hFcμ (amino acid residues 229-576, UniProtKB: P0DOX6) and its variants with χtp or μtp of different lengths were cloned into modified pcDNA and IL-2 signal peptide was attached to the N-terminus and a twin-strep tag was attached to the tail. The protein was expressed under similar conditions, first purified with Strep-Tactin resin (Smart Lifesciences), and then further purified using a Superose 6 chromatography column.

[0106] To express hFcγ-μtp 11 Chimeric proteins, truncated μTP 11The fragment introduces the C-terminus of hFcγ (amino acid residues 218-449, UniProtKB: P0DOX5) and replaces the C-terminal region (SLSPGK) of hFcγ with the corresponding region (DKSTGK) of hFcμ. This will encode hFcγ-μtp. 11 The DNA fragment was cloned into a modified pcDNA vector with an N-terminal IL-2 signal peptide and a Flag tag. The expression hFcγ-μtp was then... 11 The construct was transfected into HEK293F cells. Four days later, conditioned medium was incubated with an anti-Flag M2 affinity gel (A2220, Sigma-Aldrich) and eluted with binding buffer containing 200 μg / mL 3×Flag peptide (NJP50002, NJPeptide). The protein was then purified using a Superose 6 column in buffer containing 20 mM HEPES (pH 7.2) and 150 mM NaCl. The purified protein was detected by reducing SDS-PAGE or Native-PAGE and Coomassie Brilliant Blue staining.

[0107] For xlFcμ, a codon-optimized DNA fragment encoding xlFcμ residues (residues 257–605, UniProtKB: Q6GN83) was cloned into a modified pcDNA vector with an N-terminal IL-2 signal peptide and a flag tag. The xlFcμ-expressing construct was transfected into HEK293F cells. After 4 days, conditioned medium was incubated with an anti-Flag M2 affinity gel (A2220, Sigma-Aldrich) and eluted with 200 μg / mL of 3×Flag peptide (NJP50002, NJPeptide) binding buffer. Further purification was then achieved using a Superose 6Increase column in a buffer containing 20 mM HEPES (pH 7.2) and 150 mM NaCl.

[0108] To express hACE2-hFcμ-μtp 11 The protein will encode hACE2 (residues 18-740, UniProtKB: Q9BYF1) and hFcμ-μtp. 11 The DNA fragment (residues 229-569, UniProtKB: P0DOX6) was cloned into a modified pcDNA vector, and an IL-2 signal peptide and a Flag tag were ligated to the N-terminus. The expression of hACE2-hFcμ-μtp was then visualized using polyethyleneimine (Polysciences). 11The construct was transiently transfected into HEK293F cells and cultured for 4 days. Conditioned medium was incubated with an anti-Flag M2 affinity gel (A2220, Sigma-Aldrich) and eluted with binding buffer containing 200 μg / mL 3×Flag peptide (NJP50002, NJPeptide). The protein was then further purified using a Superose 6-Increase column in buffer containing 20 mM HEPES (pH 7.2) and 150 mM NaCl. The purified protein was detected by reducing SDS-PAGE and Coomassie Brilliant Blue staining.

[0109] To express Spytag003-hFcμ-μtp 11 The protein will encode Spytag003 (residues 1-16) and hFcμ-μtp. 11 The DNA fragment (residues 229-569, UniProtKB: P0DOX6) was cloned into a modified pcDNA vector, in which Spytag003 was joined at both ends with GGGGS and an IL-2 signal peptide, an 8×His tag, and a Flag tag were attached to the N-terminus. The expression Spytag-hFcμ-μtp was then visualized using polyethyleneimine (Polysciences). 11The construct was transiently transfected into HEK293F cells and cultured for 4 days. Conditioned medium was incubated with Ni-NTA affinity resin (Smart Lifesciences) and eluted with binding buffer containing 500 mM imidazole. The protein was then further purified using a Superose 6-Increase column in buffer containing 20 mM HEPES (pH 7.2) and 150 mM NaCl. The purified protein was detected by reducing SDS-PAGE and Coomassie Brilliant Blue staining. To express the GCSFR-SpyCatcher003 protein, DNA fragments encoding GCSFR (residues 25-333, UniProtKB: Q99062) and SpyCatcher003 (residues 26-113) were cloned into a modified pcDNA vector, with SpyCatcher003 linked at both ends by GGGGS, an IL-2 signal peptide linked at the N-terminus, and an 8×His tag and a TwinStrep tag linked at the C-terminus. The GCSFR-SpyCatcher003 construct was transiently transfected into HEK293F cells using polyethyleneimine (Polysciences) and cultured for 4 days. Conditioned medium was incubated with Ni-NTA affinity resin (Smart Lifesciences) and eluted with binding buffer containing 500 mM imidazole. The protein was then further purified using a Superose 6-Increase column in buffer containing 20 mM HEPES (pH 7.2) and 150 mM NaCl. The purified protein was detected by reducing SDS-PAGE and Coomassie Brilliant Blue staining.

[0110] In order to construct GCSFR-hFcμ-μtp 11 Protein (Figure 16e), Spytag003-hFcμ-μtp 11 The protein and GCSFR-SpyCatcher003 protein were incubated overnight at 4°C at a molar ratio of 1:30. The protein was then further separated and purified using a Superose 6-Increase column in a buffer containing 20 mM HEPES (pH 7.2) and 150 mM NaCl, and detected by reducing SDS-PAGE and Coomassie Brilliant Blue staining.

[0111] Sedimentation velocity analysis using ultracentrifugation (SV-AUC)

[0112] SV-AUC experiments were performed in an Optima AUC analytical ultracentrifuge (Beckman Coulter) (absorbance measured at 16°C, 280 nm), using a 12 mm carbon-filled Epon central element (Beckman, 392778) and a four-hole An60Ti rotor at 50,000 rpm. The buffer solution consisted of 25 mM Tris-HCl (pH 7.4) and 150 mM NaCl. Data were analyzed using SEDFIT software to determine the sedimentation coefficient distribution c(s).

[0113] Cryo-EM Sample Preparation and Data Collection

[0114] Protein samples were concentrated to 1.2–1.5 mg / mL and then treated with 0.05% glutaraldehyde (Sigma) for 10 minutes. 4 μL of cross-linked sample was aliquoted onto glow discharge porous carbon-gold meshes (Quantifoil, R1.2 / 1.3) using a Vitrobot Mark IV at 4°C and 100% humidity. The meshes were then immersed in liquid ethane. The meshes were screened using a 200 kV Talos Arctica microscope equipped with a Ceta camera. Data acquisition was performed using a 300 kV Titan Krios G3 electron microscope with a K3 Summit direct detection camera or a 300 kV Titan Krios G4 electron microscope with a Falcon 4 camera. Data were collected using EPU software (E Pluribus Unum, Thermo Fisher) with a defocus range set to -1.0 to -1.4 μm.

[0115] Frozen EM data processing and model building

[0116] Using MotionCor2 (v1.4.4) 51 Motion correction is performed on the original video frames. Gctf (v1.06) is used. 52 Estimate the contrast transfer function (CTF) parameters. Then use cryoSPARC (v3.2). 53 Data processing is performed. Images are manually sifted and summed, and particles are selected using a blob picker. A template is generated through 2D classification, and the particles picked by the template undergo multiple rounds of 2D classification to eliminate inaccurate particles. Further in-situ reconstruction and heterogeneous refinement are then performed. Using the particles retained by heterogeneous refinement, the final 3D reconstruction is generated through homogeneous and non-homogeneous refinement. To improve the density of the tp region, FcχCχ3–Cχ4–χtp and Fcμ-μtp are... 11The particles are input into RELION (v5.0), where C6 symmetry 3D refinement is performed, followed by C6 symmetry expansion to generate all symmetry-related views. Subsequently, 3D classification is performed without alignment using local masks targeting the tp regions. Local resolution maps are generated using ResMap. 54 Analysis was performed using UCSF ChimeraX. 55 Display using Coot. 56 Adjust the structural model and use Phenix 57 The real space refinement function in the software refines the structural model.

[0117] complement-dependent cytotoxicity assay

[0118] To generate engineered anti-CD20 IgM and IgG variants, the heavy chain DNA of the rituximab (RTX) antigen-binding fragment was installed in the pcDNA vector as hFcμ and hFcμ-μtp. 11 and hFcγ-μtp 11 The resulting plasmid, along with the corresponding light chain, was transfected into HEK293F cells, with the J-chain expression plasmid transfected or not transfected at a ratio of 1:1:2 or 1:1. Proteins were then isolated from the conditioned medium using Ni-NTA or anti-Flag-M2 affinity gels, followed by separation using a Superose 6 column as described above. All proteins were analyzed by reducing SDS-PAGE or Native-PAGE and Coomassie Brilliant Blue staining.

[0119] Complement-dependent cytotoxicity assays used multiple B-lymphoma cell lines, including OCI-Ly10, Daudi, and Raji. OCI-Ly10 cells were directly used for assays after culture in standard FBS; while Daudi and Raji cells were cultured for several days in RPMI-1640 medium supplemented with 10% heat-inactivated FBS before assays. RTX-hFcμ or RTX-hFcγ protein was sequentially incubated with an equal volume of cell culture (approximately 20,000 cells) and 12% normal human serum complement (Quidel), then transferred to 96-well microplates. After incubation at 37°C for 8 hours, 50 μl of CellTiter-Glo reagent (Promega, G7572) was added to each well, and the plates were incubated at room temperature for 10 minutes. Luminescence was measured using a Cytation 5 cell imaging multimodal reader (BioTek). A 4-parameter curve fitting method was used in GraphPad Prism to plot the luminescence units versus protein concentration, and the data were analyzed.

[0120] To verify the engineered hFcγ-μtp 11hexamer, hFcμ-μtp 11 The role of hexamer and in RTX-hFcγ-mediated classical complement activation was investigated. RTX-hFcγ monomeric protein (1.2 μg / mL) was mixed with serially diluted hFcγ or hFcμ protein. The resulting sample was incubated sequentially with an equal volume of B lymphoma cell culture (approximately 20,000 cells) and 12% normal human serum complement (Quidel), and then transferred to 96-well microplates. After incubation at 37°C for 8 hours, the samples were measured and analyzed as described above.

[0121] Liquid-phase complement activation assay

[0122] Liquid-phase complement activation was assessed by measuring the concentration of C4d (a marker of the classical complement pathway). The specific assay involved placing 100 μg / ml of antibody in 90% normal human serum complement and incubating at 37°C for 1 hour. Subsequently, the concentration of C4d was determined using the Micro Vue C4d EIA kit (Quidel) according to the manufacturer's instructions.

[0123] LPS-induced acute lung injury

[0124] To induce lung injury, mice were administered 2 mg / kg LPS (Solarbio, L8880) via nebulization once daily for three days; the control group received an equal volume of physiological saline. The LPS solution was prepared at a concentration of 0.8 mg / mL, and 50 μL of the solution was drawn into a nebulizer needle (Beijing Huironghe Technology Co., Ltd., HRH-MAG4). After anesthetizing the mice with isoflurane gas, the needle was carefully inserted into the trachea under laryngoscope-assisted guidance, and 50 μL of the solution was injected. Mice were observed for 30 minutes after injection to ensure there were no signs of asphyxiation.

[0125] Following the third LPS injection, mice were randomly assigned to receive either a specific antibody (50 mg / kg) or phosphate-buffered saline (PBS, Solarbio, P1033) via tail vein injection. Treatments were formulated and numbered by independent researchers to ensure double-blind administration of the injections and subsequent analyses. All mice were euthanized 12 hours after antibody or PBS injection. Gross organ observations were then recorded, and lung tissue weight was calculated to determine the lung / body weight ratio. Half of the lung tissue was used for bronchoalveolar lavage, and the other half was preserved for pathological examination. Leukocyte counts were analyzed in the bronchoalveolar lavage fluid, and the remaining fluid was retained. Lung tissue was resampled, dehydrated, embedded, sectioned, stained with hematoxylin and eosin (H&E), and analyzed under a microscope. Histopathological changes in the lung tissue were observed, including rare alveolar wall granulocytic infiltration, alveolar wall thickening leading to alveolar size changes, rare eosinophils in the bronchioles, microbleeds, alveolar erythrocyte accumulation, and rare interstitial vascular congestion. The degree of pathological changes in lung tissue was assessed by blinded scoring by personnel who were unaware of the grouping: 0 = normal, 1 = mild, 2 = moderate, 3 = significant, 4 = severe.

[0126] Example 2. Xenopus laevis IgX forms hexamer

[0127] The Fc region of the IgX heavy chain (Fcχ) comprises three constant domains and a tail domain with 11 amino acid residues (Cχ2, Cχ3, Cχ4, and χtp). We recombinantly expressed Fcχ using HEK293F cells. The polymerized form was subsequently separated by size exclusion chromatography (SEC) (Fig. 7a). Cryo-EM 2D classification showed that the Fcχ multimer stably formed a hexamer (Fig. 1a). A fragment composed of Cχ3, Cχ4, and χtp of Fcχ (FcχCχ3-Cχ4-χtp) was also purified and found to form a stable hexamer. This fragment showed better quality on an electron microscopy grid and underwent detailed cryo-EM analysis, achieving [further details needed]. 3D reconstruction at resolution (Fig. 1b, Fig. 8, Table 1).

[0128] The FcχCχ3-Cχ4-χtp structure is characterized by six Cχ3-Cχ4 units arranged in a hexagonal symmetry on the same plane (Fig. 1c). In contrast, the human IgM pentamer core structure (hFcμ-J) exhibits five hFcμ molecules arranged in a pseudo-hexagonal symmetry pattern, with the J chain filling the gap between the first and fifth hFcμ molecules (Fig. 1c, Fig. 9a). On the other hand, the IgM-Fc(tFcμ) of bony fish exhibits a different configuration, forming a looser tetramer arrangement that lacks the hexagonal symmetry observed in the FcχCχ3-Cχ4-χtp and hFcμ-J structures (Fig. 1c, Fig. 9b).

[0129] Further local refinement of the interfaces between adjacent Fcχ units was performed to enhance the visualization of the interface amino acid residues (Fig. 1d). Similar to the hFcμ-hFcμ interface in the human IgM structure, both Cχ3 and Cχ4 domains are involved in Fcχ-Fcχ interactions. The Cχ4-Cχ4 interfaces between adjacent Fcχ units cover approximately [missing information - likely a specific area or region] of each Cχ4 domain. The surface area is comparable to the interface between the two Cμ4 domains in hFcμ-J. Cys430 in the Cχ3 domain participates in the formation of interchain disulfide bonds, similar to Cys414 in the hFcμCμ3 domain. In contrast, there is no similar Cys in bony fish IgM (Fig. 10), and the interface between adjacent tFcμ units in the tetramer is almost entirely provided by the Cμ4 domain (Fig. 1d).

[0130] In the EM density map refined using C1 symmetry, the χtp region was not clearly resolved (Fig. 1b). Since the unique hexagonal symmetry of the Cχ3–Cχ4 region may play a dominant role in particle alignment compared to the asymmetric χtp region, the FcχCχ3–Cχ4–χtp particles were extended with C6 symmetry (Fig. 8a). In the resulting C6 symmetry extension category (category 6), the secondary structure features of χtp became clearly discernible. Although a precise model could not be constructed due to resolution limitations, it is evident that the χtp region exhibits typical β-fold conformational features.

[0131] The β-chain regions of the caudal domains in χtp and hFcμ and tFcμ show high sequence homology (Fig. 2a), forming β-sandwich assemblies in human IgM pentamers and bony fish IgM tetramers, respectively, despite different configurations (Fig. 1c). It is likely that each χtp retains the ability to encode the β-chain due to the sequence similarity of each χtp to that of human and bony fish μtp. However, the twelve chains may combine in different arrangements to form β-sandwich structures, with chains from different Fcχ chains exchanging positions in these β assemblies. This leads to an obfuscated density map of the EM in this region after single-particle averaging, posing a challenge to the accurate interpretation of structural features. The pronounced hexagonal symmetry of the Cχ3-Cχ4 region may also complicate the precise registration of particles in this asymmetric central region. We also used AlphaFold3 to predict the structure of the Fcχ hexamer. While the Cχ3–Cχ4 hexamer was reasonably predicted, the central tail assembly exhibited a variety of patterns (Fig. 17a), which were inconsistent with the cryo-electron microscopy reconstructions obtained through C6 symmetry extension (Fig. 8a). When only the χtp region was predicted, AlphaFold3 showed that the 12 chains could assemble into two forms (“6+6” or “5+7”) (Figs. 17b and c). It remains unclear whether these predicted structures represent the true χtp assembly forms. In any case, removing χtp significantly reduced the formation of Fcχ hexamers, thus highlighting the crucial role of χtp in the IgX hexamerization process (Fig. 7a).

[0132] Table 1. Statistics on cryo-electron microscopy data collection, refinement and validation.

[0133] Example 3. hFcμ-χtp chimeras form homogeneous hexamers.

[0134] Mammalian IgM molecules form stable pentamers in the presence of the J chain; however, in the absence of the J chain, the stability of IgM decreases, and it may exhibit various polymeric forms, including hexamers, pentamers, and even tetramers. Consistent with this, recombinant Fcμ (denoted as Fcμ-μtp) with a complete 18-amino acid tail plate... 18 The compound exhibited heterogeneity in solution. SV-AUC results showed the formation of a mixture of tetramers, pentamers, and hexamers (Fig. 2b). These polymeric forms were enriched by SEC (Fig. 7b) and analyzed using cryo-EM. Consistent with the SV-AUC results, Fcμ-μtp 18 The tetramer, pentamer and hexamer forms were clearly identified in the 2D classification (Fig. 2c).

[0135] To obtain a homogeneous Fcμ hexamer, we first replaced the 18-amino acid tail of Fcμ with an 11-amino acid χtp, constructing an Fcμ-χtp chimera. This chimera was recombinantly expressed and purified by SEC (Figures 7b and 7c). SV-AUC analysis showed that the chimera consistently exhibited a homogeneous hexamer structure (Figure 2d). Further cryo-electron microscopy analysis of the Fcμ-χtp polymer confirmed the presence of only a homogeneous hexamer, with no other polymeric forms detected (Figure 2e). Subsequently, the cryo-electron microscopy structure of the Fcμ-χtp hexamer was resolved to... The resolution is shown in Figures 2f, 11, and Table 1. Similar to the Fcχ hexamer, the six Fcμ units in this structure are arranged in a perfect hexagonal geometry (Figure 9c).

[0136] The 18 amino acid tail domains (μtp) of hFcμ 18 In contrast, χtp contains only 11 amino acids (Figure 2a). This difference leads us to speculate that the length of the tail domain affects the assembly of the hFcμ hexamer. To test this hypothesis, we first truncated the last 7 amino acids at the C-terminus of μtp to create hFcμ-μtp. 11 Variant. This variant was recombinantly purified using the same method as the Fcχ and hFcμ-χtp chimeras (Figures 7b and 7c). SV-AUC analysis showed that hFcμ-μtp 11 The polymer is a homogeneous hexamer (Fig. 3a), which is also confirmed by cryo-electron microscopy analysis (Fig. 3b).

[0137] To further investigate the effect of tail domain length on hFcμ hexamerization, we systematically truncated μtp by removing C-terminal amino acids one by one (Fig. 7c). The multimeric components of each variant collected after SEC were analyzed using a two-dimensional classification method in cryo-electron microscopy (Fig. 3b). Analysis showed that hFcμ hexamers could be formed when the μtp length was between 11 and 16 amino acids. Notably, hFcμ-μtp... 10 The variant exhibits both hexamer and tetramer, while hFcμ-μtp 17 It exhibits a mixture of hexamers and pentamers. Native-PAGE analysis further confirmed the oligomeric state of these polymers: hFcμ-μtp 10 hFcμ-μtp 17 and hFcμ-μtp 18 Heterogeneous polymers are formed in solution (Fig. 3c). In contrast, hFcμ-χtp and hFcμ-μtp... 11 to hFcμ-μtp 16It appears in a uniform hexamer form. These results indicate that the tail domain containing 11–16 residues optimally supports hFcμ hexamer formation.

[0138] exist Based on the overall resolution, hFcμ-μtp 11 The three-dimensional structure of the hexamer was constructed and analyzed, revealing six hFcμ molecules, with the Cμ4 domain exhibiting the highest density (Fig. 3d, Fig. 12). This structure, together with the hFcμ-χtp structure, represents the first visualization of the human Fcμ hexamer. All six hFcμ units are aligned in the same plane, with the sixth hFcμ molecule occupying the position of the J chain in the hFcμ-J pentamer (Fig. 3e), consistent with previous predictions. When the hFcμ-J pentamer core, including five pairs of Cμ4 and J chains, is superimposed on the hFcμ hexamer, hFcμ1 and hFcμ5 in the pentamer exhibit slight rotation due to their direct connection to the J chain (Fig. 3e, Fig. 9d). This subtle asymmetry introduced by the J chain influences the interaction between the IgM pentamer and its specific receptor FcμR. 8 Similar to what was observed in the hexamer structures of FcχCχ3-Cχ4-χtp and hFcμ-χtp, and unlike the pentamer structure of hFcμ-J, although μtp 11 Spanning the entire length of the μtpβ chain, μtp 11 The segment remains unclear (Fig. 2a). We speculate that, without co-folding with the J chain, the twelve μtp chains may form β-sandwiches in different arrangements, similar to χtp; this therefore leads to an unclear EM density map of the region.

[0139] Example 4. Containing μtp 11 Engineered IgG is assembled into hexamer

[0140] To further study μtp 11 Whether it can also lead to the formation of IgG hexamer, we will investigate μtp. 11 The segment was introduced into the C-terminus of hIgG1-Fc (hFcγ), forming hFcγ-μtp. 11 Chimera (Fig. 4a). Furthermore, affected by hFcγ Innovent design 38 Inspired by this, we replaced the six C-terminal amino acids of hFcγ (SLSPGK) with the linker sequence (DKSTGK) between hCμ4 and hμtp. This substitution removed the rigid Pro445 in hFcγ, effectively enhancing the interaction between hFcγ and μtp. 11 The flexibility of the joint area between them.

[0141] As shown in the SEC analysis (Figure 4b), hFcγ-μtp 11 The assembly of the polymers was highly efficient. Subsequently, hFcγ-μtp was determined. 11 The cryo-electron microscopy structure of the hexamer, with a resolution of [resolution missing]. (Fig. 4c, Fig. 13). In this structure, six hFcγ molecules aggregate into a hexagon, highly similar to the b12 hexamer reconstructed based on crystal symmetry (Fig. 4d). Both the Cγ2 and Cγ3 domains contribute to the interactions between adjacent hFcγ molecules, with each molecule having approximately [missing information]. The surface area of ​​the surface covers the interface (Fig. 4e). Notably, the Glu380 and Glu382 residues in the C chain, the FG loop of the Cγ3 domain, and the His433-Gln438 segment in the G chain play crucial roles in mediating Fcγ-Fcγ interactions (Fig. 13e, f). Despite the high overall resolution, μtp could not be resolved. 11 This fragment is related to hFcμ-μtp 11 The results for hexamer are similar.

[0142] Example 5. Containing μtp 11 Enhanced CDC activity of IgM and IgG hexamers

[0143] To evaluate the potential of engineered hIgM and hIgG hexamers to enhance complement activation, we performed complement-dependent cytotoxicity (CDC) assays. We engineered the heavy chain region of the anti-CD20 IgG rituximab (RTX, hereinafter referred to as RTX-hFcγ) antigen-binding fragment to hFcμ and hFcμ-μtp. 11 and hFcγ-μtp 11 Constructs. These constructs were then co-expressed with the light chain of RTX and the J chain of the IgM pentamer to obtain recombinant anti-CD20 IgM pentamer (RTX-hFcμ-J) and IgM hexamer (RTX-hFcμ-μtp). 11 ) and IgG hexamer (RTX-hFcγ-μtp 11 (Figure 7d). For comparison, we also prepared recombinant RTX-hFcγ E430G and RTX-hFcγ Innovent .

[0144] First, OCI-Ly10 B lymphoma cells were used in human complement. 46The CDC activity was evaluated in the presence of 11 . As expected, among these molecules, the monomeric RTX-hFcγ had the lowest complement activation activity, with an IC50 of approximately 0.50 μg / mL (Figure 5a). In contrast, the engineered RTX-hFcμ-J pentamer had an IC50 of approximately 0.23 μg / mL, indicating a two-fold increase in its CDC activity. Compared to the RTX-hFcμ-J pentamer, the RTX-hFcμ-μtp 11 hexamer had a further two-fold increase in CDC activity, with an IC50 of 0.12 μg / mL. Importantly, RTX-hFcγ E430G and RTX-hFcγ Innovent both showed strong complement activation ability, with IC50 values of approximately 0.06 μg / mL, which was 9-fold that of the original RTX-hFcγ (Figure 5a); the CDC activity of our RTX-hFcγ-μtp 11 was comparable to that of RTX-hFcγ Innovent and RTX-hFcγ E430G .

[0145] We also determined the CDC activity of these engineered molecules in other B lymphoma cell lines, including Daudi and Raji. Different from OCI-Ly10 cells cultured with standard fetal bovine serum (FBS) and directly used for CDC assays, Daudi and Raji cells were first cultured with heat-inactivated FBS before the assay. This step seemed to increase their sensitivity to CDC-mediated killing. Nevertheless, the engineered molecules showed a generally consistent activity pattern in Daudi cells, with their potencies ranked in the following order: RTX-hFcγ monomer < RTX-hFcμ-J pentamer < RTX-hFcμ-μtp 11 hexamer < RTX-hFcγ E430G ≈ RTX-hFcγ-μtp 11 ≈ RTX-hFcγ Innovent . Notably, compared to the RTX-hFcμ-μtp 11 hexamer, the RTX-hFcμ-J pentamer showed a less pronounced concentration-dependent response in these cells. The different potencies of hexameric and pentameric IgM for complement activation are affected by antigen density 28 , which may account for the different patterns observed for the RTX-hFcμ-J pentamer and the RTX-hFcμ-μtp hexamer in OCI-Ly10 and Daudi cells. In Raji cells, these molecules showed activity comparable to that observed in Daudi cells, but notably, RTX-hFcγ Innovent and RTX-hFcγ-μtp 11Hexamer ratio RTX-hFcγ E430G It exhibits stronger CDC activity. In summary, these results indicate that using hμtp... 11 The complement activation ability of IgM and IgG hexamers was greatly enhanced by the strategy design.

[0146] Example 6. hFcγ-μtp 11 Instead of hFcμ-μtp 11 Block complement activation

[0147] Complement overactivation can lead to a variety of diseases, and recombinant Fc hexamer can serve as a "decoy" therapy to reduce complement-mediated tissue damage. For example, a recent study showed that recombinant hFcγ-μtp 18 The fusion protein CSL777 (with the L309C mutation) 37 It has a protective effect in a mouse model of xenoantibody-mediated acute lung injury. 47 To study hFcγ-μtp 11 and hFcμ-μtp 11 To investigate the effect of hexamer on complement activation, we evaluated CDC activity of RTX-hFcγ in Daudi and Raji cells where these proteins were present. Our results indicate that, unlike Fcγ monomers, hFcγ-μtp... 11 The hexamer effectively inhibits RTX-hFcγ-mediated complement activation (Figure 6). In contrast, Fcμ-μtp... 11 The hexamer has no inhibitory effect.

[0148] We also assessed the level of complement activation in the liquid phase by measuring the production of C4d in the serum of normal individuals. The results showed that Fcγ-μtp 11 Moderate liquid-phase complement activation was induced, while Fcμ-μtp 11 Then there is none (Figure 6g). It is noteworthy that under our experimental conditions, stable RTX hexamers (including RTX-Fcγ) Innovent RTX-Fcγ-μtp 11 and RTX-Fcμ-μtp 11 The liquid-phase complement activation triggered by γ-μtp was stronger than that of Fcγ-μtp alone. 11 Among them, RTX-Fcγ Innovent and RTX-Fcγ-μtp 11 The effect is the most potent. Its underlying mechanism is currently unclear, but the presence of the Fab fragment may promote the formation of more stable C1 binding sites on the Fcγ or Fcμ domains.

[0149] In summary, these results indicate that Fcγ-μtp 11Fcγ-μtp inhibits CDC by competitively binding to the C1 complex with IgG, thereby shifting complement activation from the target site to the liquid phase. 11 With Fcμ-μtp 11 The difference in activity of hexamers in inhibiting CDC, and RTX-Fcγ Innovent / RTX-Fcγ-μtp 11 With RTX-Fcμ-μtp 11 Differences in efficacy during liquid-phase activation.

[0150] These results are consistent with previous structural analyses, which indicate that IgG hexamers readily interact with the complement C1 complex in solution, while IgM pentamers or hexamers require significant conformational changes to expose their C1 binding sites. 27，31 .

[0151] We further evaluated Fcγ-μtp in a lipopolysaccharide (LPS)-induced acute lung injury mouse model. 11 The therapeutic efficacy of hexamer. In this study, wild-type BALB / c mice were first exposed to nebulized LPS for three consecutive days. Subsequently, these mice received a single intravenous injection of phosphate-buffered saline (PBS), Fcγ (50 mg / kg), or Fcγ-μtp. 11 (50 mg / kg). Results showed that the PBS group exhibited severe pulmonary edema, manifested as a significantly increased lung-to-body weight ratio (Figure 6c); while the Fcγ or Fcγ-μtp group... 11 This phenomenon was alleviated after treatment. Furthermore, the leukocyte level in the bronchoalveolar lavage fluid of the PBS group was significantly elevated, indicating lung inflammation (Figure 6d). Fcγ and Fcγ-μtp 11 Treatments reduced lung-to-body weight ratio and white blood cell count. High-dose human Fcγ may mimic the effects of intravenously administered immunoglobulin (IVIg), which reduces inflammation in mouse models of immune thrombocytopenic purpura, antibody-mediated lung injury, and human autoimmune diseases. Notably, Fcγ-μtp... 11 The treatment showed stronger efficacy, indicating that complement inhibition contributed to the therapeutic effect, similar to the results observed in CSL777 in an antibody-mediated lung injury model. Consistent with these findings, histopathological analysis of lung tissue revealed hemorrhage, alveolar wall thickening, and inflammatory cell infiltration in the PBS group (Fig. 6e, f). In contrast, lung tissue damage was alleviated in both treatment groups 12 hours after treatment, and Fcγ-μtp... 11 It exhibits superior therapeutic efficacy. Overall, these results indicate that Fcγ-μtp... 11 It has potential benefits in the prevention and treatment of complement-dependent inflammation and organ damage.

[0152] Example 7.xlFcμ can also form hexamers.

[0153] In its natural state, Xenopus laevis IgM forms a pentameric structure together with the J chain. Since its caudate domain is 14 amino acids long, and based on the influence of caudate domain length on hexamer formation in human IgM, we hypothesized that Xenopus laevis IgM would also form a hexamer in the absence of the J chain. To this end, we expressed xlFcμ and observed its assembly in the absence of the J chain. Size exclusion chromatography showed that only xlFcμ hexamers formed, in addition to the monomer (Fig. 14a). Cryo-electron microscopy two-dimensional classification results also showed that xlFcμ formed a hexamer (Fig. 14b). This further demonstrates that the length of the caudate domain is crucial for hexamer formation.

[0154] Example 8. hACE2-hFcμ-μtp 11 Able to form hexamer

[0155] Angiotensin-converting enzyme 2 (ACE2) is primarily the receptor for severe acute respiratory syndrome coronavirus (SARS-CoV-2). 58 We found that fusing ACE2 into hFcμ-μtp 11 The above-mentioned material can form a hexamer, thereby increasing its neutralizing efficacy against respiratory infectious viruses by increasing affinity. We expressed hACE2-hFcμ-μtp 11 The protein was analyzed, and its assembly was observed. Size exclusion chromatography and Coomassie brilliant blue staining showed that hACE2-hFcμ-μtp was formed. 11 Hexamers and monomers (Fig. 15a-b). Cryo-electron microscopy two-dimensional classification results also show hACE2-hFcμ-μtp. 11 A hexamer was formed (Figure 15c).

[0156] Example 9. GCSFR-hFcμ-μtp 11 Able to form hexamer

[0157] Granulocyte colony-stimulating factor (G-CSF), also known as colony-stimulating factor 3 (CSF3), mainly exerts its effects by stimulating the survival, proliferation, differentiation, and function of neutrophil progenitor cells and mature neutrophils. It is primarily used to treat granulocytopenia and to mobilize hematopoietic stem cells. 59-61 Granulocyte colony-stimulating factor receptor (GCSFR) belongs to the hematopoietic factor receptor family. G-CSF binds to GCSFR, inducing receptor dimerization and subsequently activating downstream signaling. 62-63 Furthermore, the binding of G-CSF to GCSFR can promote tumor cell proliferation, inhibit tumor cell apoptosis, or induce angiogenesis. 64We found that fusing GCSFR into hFcμ-μtp 11 The SpyTag-SpyCatcher method can form a hexamer, thereby inhibiting the occurrence and development of tumor cells. 65-67 GCSFR-hFcμ-μtp was expressed 11 Proteins were analyzed, and their assembly was observed. Size exclusion chromatography and Coomassie brilliant blue staining revealed Spytag003-hFcμ-μtp 11 It can form hexamers and monomers (Fig. 16a-b), GCSFR-SpyCatcher003 is in monomer form (Fig. 16c-d), while Spytag003-hFcμ-μtp 11 The GCSFR-SpyCatcher003 protein can covalently bind to SpyTag-SpyCatcher to form GCSFR-hFcμ-μtp. 11 Complex (Fig. 16e-g). Cryo-electron microscopy two-dimensional classification results show GCSFR-hFcμ-μtp 11 It can form a hexamer (Fig. 16h).

[0158] discuss

[0159] In this study, we demonstrated that Xenopus laevis IgX can form hexamers independently of the J chain. We further demonstrated that engineering an 11-amino acid tail fissure domain of IgX into human IgM, or truncating the 18 amino acids of the native tail fissure domain of IgM to 11–16 residues, can promote IgM hexamer formation. Mechanistically, tail fissure domains of fewer than 11 residues may compromise the integrity of the tail fissure domain β chain, potentially disrupting the stability of the IgM hexamer. Conversely, tail fissure domains of more than 16 residues may hinder hexamer formation due to steric hindrance. Specifically, the penultimate Cys575 participates in the formation of disulfide bonds between adjacent Fcμ units, while the terminal Tyr576 has a bulky side chain. These factors combined may pose a challenge to accommodating twelve complete tail fissure domains within the narrow central cavity of the hFcμ hexamer. Consistent with this conclusion, the 14-amino acid Xenopus laevis tail fissure domain also promotes the formation of stable hexamer structures.

[0160] IgM antibodies have been shown to have potential for therapeutic applications. 48 Under natural conditions, IgM antibodies typically exhibit low affinity and specificity. However, transposing variable domains of mature-affinity IgG to an IgM scaffold can significantly improve binding activity and potency. For example, converting neutralizing SARS-CoV-2 IgG to its IgM form can significantly improve antibody titer and broaden its binding range. 49This engineered IgM can also be administered intranasally, making it a promising candidate drug for the prevention and treatment of SARS-CoV-2 and other respiratory pathogens. Other biologics utilizing IgM scaffolds, such as IgM-like ACE2, have also been developed. 50 It can broadly neutralize SARS-CoV-2 variants and can be administered via aerosol inhalation. Our study provides a direct strategy for the engineered design of IgM hexamers, utilizing the shortened tail-plate domain of IgM itself or the tail-plate domain of IgX, which can improve the efficacy and usability of IgM-dependent biologics. Notably, although μtp 11 It has the same length as the 11-amino acid χtp, but their sequences are different. In particular, μtp... 11 It contains a more rigid Pro559 (instead of Ser in χtp) and an Asn563-Val564-Ser565 motif that leads to glycosylation at Asn563, unlike the Asn-Val-Asn sequence in χtp which does not support this modification (Fig. 2a). These differences do not appear to affect μtp. 11 The ability to assist in the formation of IgM hexamers; however, χtp and μtp 11 The impact on hexamer yield requires further investigation.

[0161] Importantly, by fusing a shortened IgM tail domain to IgG, we also assembled IgG into a hexamer. This IgG hexamer possesses potent complement-activating properties, potentially enhancing its therapeutic applications. 33 With the fusion of 18 amino acid μTP tail domain 34，35 Or the C575S and V567I / A572G variants with the same length. 36，38 Compared to the previous strategy, our strategy more effectively promotes hexamer formation. As a proof-of-concept, we designed RTX-hFcγ-μtp... 11 It possesses strong complement activation properties (Figure 5b), while hFcγ-μtp 11 The fragment effectively blocked the CDC activity of RTX IgG in a competitive manner (Figure 6). Furthermore, Fcγ-μtp 11 Hexamers effectively alleviate lung inflammation in mouse models. Furthermore, by linking the engineered Fc of this invention to the target molecule (e.g., linking the Fc to the C-terminus of the target molecule to form a fusion molecule, such as the fusion proteins of Examples 8 and 9), the target molecule can form a hexamer, thereby improving its affinity and specificity and significantly enhancing the corresponding therapeutic effect. In summary, these findings lay the foundation for alternative strategies for developing IgG or IgG-Fc hexamers for therapeutic purposes, or for developing hexamers of target molecules.

[0162] Every application and patent cited herein, and every reference cited in each of those applications and patents (including during the examination of each granted patent; “Application References”), and every PCT and foreign application or patent corresponding to and / or claiming priority to any of these applications and patents, and every reference cited or referenced in each Application References, is expressly incorporated herein by reference and may be used to practice this invention. More generally, several references are cited herein, whether in the reference list preceding the claims or in the text itself; and each of these references (“References Cited herein”) and every reference cited in each References herein (including any manufacturer’s specification, guide, etc.) is expressly incorporated herein by reference.

[0163] The foregoing description of some specific embodiments provides sufficient information to enable others to readily modify or adapt such specific embodiments for different applications by applying present knowledge without departing from the general concept, and therefore such modifications and alterations should and are intended to be included within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the phrases or terms used herein are for descriptive purposes and not for limitation. Exemplary embodiments have been disclosed in the drawings and specification, and although specific terms may have been used, they are used in a general descriptive sense only, and not for limiting purposes, unless otherwise stated, and the scope of the claims is therefore not limited thereto. Furthermore, those skilled in the art will understand that certain steps of the methods discussed herein can be ordered in an alternative order or the steps can be combined. Therefore, this means that the appended claims are not limited to the specific embodiments disclosed herein. Those skilled in the art will recognize or be able to determine many equivalents of the embodiments of the invention described herein using only conventional experimentation. Such equivalents are covered in the following claims.

[0164] References

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Claims

1. An engineered immunoglobulin or a fragment thereof, wherein the heavy chain C-terminus of the immunoglobulin or the fragment thereof is modified to promote the formation of a polymerized protein complex, preferably the fragment being an Fc region.

2. The engineered immunoglobulin or fragment thereof of claim 1, wherein the engineered immunoglobulin or fragment thereof is derived from IgG or IgM.

3. The engineered immunoglobulin or a fragment thereof according to claim 1 or 2, wherein the modification comprises: Add a tail flap domain to the C-terminus of the IgG heavy chain, or replace or delete amino acid residues in the tail flap domain at the C-terminus of the IgM heavy chain.

4. The engineered immunoglobulin or fragment thereof of claim 3, comprising a tail flap domain derived from the Fc region of IgX or IgM immunoglobulin.

5. The engineered immunoglobulin or fragment thereof as described in claim 3 or 4, comprising a tail flap domain of IgX from the African clawed frog (Xenopus laevis).

6. The engineered immunoglobulin or fragment thereof as claimed in claim 3 or 4, comprising a tail flap domain of IgM derived from Xenopus laevis.

7. The engineered immunoglobulin or fragment thereof as described in claim 3 or 4, comprising a tail flap domain derived from human IgM.

8. The engineered immunoglobulin or fragment thereof according to any one of claims 1 to 7, wherein the engineered immunoglobulin or fragment thereof is derived from a human, or wherein the engineered immunoglobulin is a human antibody or a humanized antibody.

9. The engineered immunoglobulin or fragment thereof according to any one of claims 1 to 8, wherein the engineered immunoglobulin or fragment thereof is derived from human IgM, and wherein the modification is a substitution or deletion of amino acid residues in the tail flap domain at the C-terminus of the heavy chain.

10. The engineered immunoglobulin or fragment thereof of claim 9, wherein the substitution is replacing the tail domain of the C-terminal 18 amino acids of the Fc domain of human IgM with the tail domain of IgX from Xenopus laevis or the tail domain of IgM from Xenopus laevis.

11. The engineered immunoglobulin or a fragment thereof of claim 10, wherein the first 1, 2, or 3 amino acids of the C-terminus of the IgM derived from Xenopus laevis are deleted.

12. The engineered immunoglobulin or a fragment thereof of claim 9, wherein the modification is the deletion of two or more amino acid residues of the tail flap domain at the C-terminus of the heavy chain.

13. The engineered immunoglobulin or a fragment thereof of claim 12, wherein the deletion is a deletion of the first 2, 3, 4, 5, 6 or 7 amino acids at the C-terminus of the Fc domain of human IgM.

14. The engineered immunoglobulin or fragment thereof according to any one of claims 9 to 13, wherein the tail domain comprises 11, 12, 13, 14, 15 or 16 amino acids in length.

15. The engineered immunoglobulin or fragment thereof according to any one of claims 11 to 14, wherein the tail domain comprises the first 11, 12, 13, 14, 15 or 16 amino acids of the N-terminus of the tail domain of natural human IgM.

16. The engineered immunoglobulin or fragment thereof of any one of claims 1 to 8, wherein the engineered immunoglobulin or fragment thereof is derived from human IgG, and wherein the modification is the addition of a tail flap domain to the C-terminus of the heavy chain.

17. The engineered immunoglobulin or a fragment thereof of claim 16, wherein the added caudal domain is derived from the caudal domain of Xenopus laevis IgX, the caudal domain of Xenopus laevis IgM, or the caudal domain of human IgM.

18. The engineered immunoglobulin or a fragment thereof according to claim 16 or 17, wherein the added tail flap domain is derived from the tail flap domain of human IgM, and the first 2, 3, 4, 5, 6 or 7 amino acids at the C-terminus of the tail flap domain of said human IgM are deleted.

19. The engineered immunoglobulin or a fragment thereof as claimed in claim 16 or 17, wherein the added tail domain is derived from the tail domain of Xenopus laevis IgM, and optionally the first 1, 2, or 3 amino acids at the C-terminus of the tail domain of Xenopus laevis IgM are deleted.

20. The engineered immunoglobulin or fragment thereof of any one of claims 16 to 19, wherein the length of the added tail domain is 11, 12, 13, 14, 15 or 16 amino acids.

21. The engineered immunoglobulin or fragment thereof of any one of claims 16 to 20, wherein the added tail flap domain is linked to the C-terminus of the IgG heavy chain Fc domain.

22. The engineered immunoglobulin or a fragment thereof of claim 21, wherein the first six amino acids of the C-terminus of the Fc domain of the IgG heavy chain are replaced with DKSTGK.

23. The engineered immunoglobulin or fragment thereof as claimed in claim 1, wherein it is in the form of an IgM antibody, an IgG antibody or a variant thereof.

24. The engineered immunoglobulin or fragment thereof of claim 1, comprising an Fc domain derived from an IgM antibody, an IgG antibody, or a variant thereof.

25. The engineered immunoglobulin or a fragment thereof as claimed in claim 23 or 24, wherein the Fc domain is linked to the heavy chain of the antigen-binding fragment.

26. The engineered immunoglobulin or a fragment thereof of claim 25, wherein the heavy chain of the antigen-binding fragment is derived from an anti-CD20 antibody.

27. The engineered immunoglobulin or a fragment thereof of claim 26, wherein the anti-CD20 antibody is rituximab.

28. A hexameric protein complex comprising any one of the engineered immunoglobulins or fragments thereof as claimed in any one of claims 1 to 27.

29. The hexameric protein complex of claim 28, wherein each monomer in the hexamer is an engineered immunoglobulin or a fragment thereof as described in any one of claims 1 to 27, preferably the fragment being an Fc region, optionally the Fc region being linked to a target molecule.

30. The hexameric protein complex of claim 28 or 29, wherein each engineered immunoglobulin or fragment thereof of any one of claims 1 to 26 has the same or different antigen specificity.

31. The hexameric protein complex of any one of claims 28 to 30, wherein the monomer forms the hexamer through association of tail domains.

32. The hexameric protein complex of claim 31, wherein the tail flap domain is the tail flap domain of IgX from Xenopus laevis.

33. The hexameric protein complex of claim 31, wherein the tail flap domain is a tail flap domain derived from human IgM.

34. The hexameric protein complex of claim 33, wherein the first 2, 3, 4, 5, 6, or 7 amino acids of the C-terminus of the tail-plate domain of the human IgM are deleted.

35. The hexameric protein complex of claim 31, wherein the tail flap domain is the tail flap domain of IgM from Xenopus laevis.

36. The hexameric protein complex of claim 35, wherein the first 1, 2, or 3 amino acids of the C-terminus of the tail domain of the Xenopus laevis IgM are deleted.

37. The hexameric protein complex of any one of claims 28 to 36, wherein the tail domain of each engineered immunoglobulin or fragment thereof of any one of claims 1 to 26 is 11, 12, 13, 14, 15 or 16 amino acids in length.

38. The hexameric protein complex of any one of claims 28 to 37, wherein the hexameric protein complex is an antibody hexamer.

39. The hexameric protein complex of any one of claims 28 to 38, wherein the hexameric protein complex is an IgM hexamer.

40. The hexameric protein complex of any one of claims 28 to 39, wherein the hexameric protein complex is a hexamer of IgG.

41. The hexameric protein complex of any one of claims 28 to 40, wherein each of the monomers is in the form of an IgM antibody, an IgG antibody, or a variant thereof.

42. The hexameric protein complex of any one of claims 28 to 40, wherein each of the monomers comprises an Fc domain derived from an IgM antibody, an IgG antibody, or a variant thereof, optionally said Fc domain being linked to a target molecule.

43. The hexameric protein complex of any one of claims 28 to 42, wherein the Fc domain is linked to the heavy chain of the antigen-binding fragment.

44. The hexameric protein complex of any one of claims 28 to 43, wherein the antigen-binding fragment is derived from an anti-CD20 antibody.

45. The hexameric protein complex of claim 44, wherein the anti-CD20 antibody is rituximab.

46. ​​Use of the engineered immunoglobulin or fragment thereof, or a fusion molecule comprising said engineered immunoglobulin or fragment thereof, or the hexameric protein complex of any one of claims 28 to 45, in the preparation of a medicament.

47. The use of claim 46, wherein the drug is used to induce complement-dependent cytotoxicity or to enhance neutralizing efficacy against viruses such as respiratory infectious viruses.

48. The use of claim 46, wherein the drug is used to treat a patient suffering from cancer such as B-cell lymphoma.

49. The use of claim 46, wherein the drug is used to inhibit or block complement activation.

50. A method for obtaining an engineered immunoglobulin or a fragment thereof capable of forming a hexamer, or a fusion molecule comprising said engineered immunoglobulin or a fragment thereof, or a hexamer protein complex formed from said engineered immunoglobulin or a fragment thereof, or a fusion molecule comprising said engineered immunoglobulin or a fragment thereof, preferably said fragment being an Fc region, said method comprising modifying the C-terminus of the heavy chain of the immunoglobulin or the fragment thereof.

51. The method of claim 50, wherein the engineered immunoglobulin or a fragment thereof is derived from IgG or IgM.

52. The method of claim 50 or 51, wherein the modification comprises: Add a tail flap domain to the C-terminus of the IgG heavy chain, or replace or delete amino acid residues in the tail flap domain at the C-terminus of the IgM heavy chain.

53. The method of any one of claims 50 to 52, wherein the engineered immunoglobulin or a fragment thereof comprises a tail flap domain of IgX from Xenopus laevis.

54. The method of any one of claims 50 to 52, wherein the engineered immunoglobulin or a fragment thereof comprises a tail flap domain of IgM from Xenopus laevis.

55. The method of any one of claims 50 to 52, wherein the engineered immunoglobulin or a fragment thereof comprises a tail flap domain derived from human IgM.

56. The method of any one of claims 50 to 55, wherein the engineered immunoglobulin or a fragment thereof is derived from a human, or wherein the engineered immunoglobulin is a human antibody or a humanized antibody.

57. The method of any one of claims 50 to 56, wherein the engineered immunoglobulin or a fragment thereof is derived from human IgM, and wherein the modification is a substitution or deletion of amino acid residues in the tail flap domain at the C-terminus of the heavy chain.

58. The method of claim 57, wherein the substitution is replacing the tail domain of the C-terminal 18 amino acids of the Fc domain of human IgM with the tail domain of IgX from Xenopus laevis or the tail domain of IgM from Xenopus laevis.

59. The method of claim 58, wherein the first 1, 2, or 3 amino acids of the C-terminus of the tail flap domain of the IgM from the African clawed frog are deleted.

60. The method of claim 57, wherein the deletion is a deletion of two or more amino acids at the C-terminus of the Fc domain of human IgM.

61. The method of claim 60, wherein the deletion is a deletion of 2, 3, 4, 5, 6 or 7 amino acids before the C-terminus of the Fc domain of human IgM.

62. The method according to any one of claims 57 to 61, wherein the length of the tail structure domain comprising 11, 12, 13, 14, 15 or 16 amino acids.

63. The method of any one of claims 60 to 62, wherein the tail domain comprises the first 11, 12, 13, 14, 15 or 16 amino acids of the N-terminus of the tail domain of natural human IgM.

64. The method of any one of claims 52 to 56, wherein the engineered immunoglobulin or a fragment thereof is derived from human IgG, and wherein the modification is the addition of a tail flap domain to the C-terminus of the heavy chain.

65. The method of claim 64, wherein the added caudal domain is derived from the caudal domain of IgX from Xenopus laevis, the caudal domain of IgM from Xenopus laevis, or the caudal domain of IgM from human.

66. The method of claim 64 or 65, wherein the added tail domain is derived from the tail domain of human IgM, and the first 2, 3, 4, 5, 6, or 7 amino acids at the C-terminus of the tail domain of said human IgM are deleted.

67. The method of claim 64 or 65, wherein the added tail domain is derived from the tail domain of Xenopus laevis IgM, and optionally the first 1, 2, or 3 amino acids at the C-terminus of the tail domain of Xenopus laevis IgM are deleted.

68. The method of any one of claims 64 to 67, wherein the length of the added tail domain is 11, 12, 13, 14, 15, or 16 amino acids.

69. The method of any one of claims 64 to 68, wherein the added tail flap domain is connected to the C-terminus of the IgG heavy chain Fc domain.

70. The method of claim 69, wherein the first six amino acids at the C-terminus of the Fc domain of the IgG heavy chain are replaced with DKSTGK.

71. The method of claim 50, wherein the engineered immunoglobulin or a fragment thereof is in the form of an IgM antibody, an IgG antibody, or a variant thereof.

72. The method of claim 50, wherein the engineered immunoglobulin or a fragment thereof comprises an Fc domain derived from an IgM antibody, an IgG antibody, or a variant thereof.

73. The method of claim 71 or 72, wherein the Fc domain is linked to the heavy chain of the antigen-binding fragment.

74. The method of claim 73, wherein the heavy chain of the antigen-binding fragment is derived from the anti-CD20 antibody.

75. The method of claim 74, wherein the anti-CD20 antibody is rituximab.

76. A fusion molecule comprising an engineered immunoglobulin or a fragment thereof as described in any one of claims 1 to 27, wherein the engineered immunoglobulin or the fragment thereof is linked to a target molecule, preferably the fragment being an Fc region.

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