Engineered IgG and IgA chimeric "four-leaf clover X antibodies"

Abstract

The present invention relates to a crossmab antibody molecule comprising antibody variable region sequences against various target antigens, having both an IgG antibody Fc region and an IgA antibody Fc region, retaining the binding capacity of the IgG Fc region and the IgA Fc region to their respective receptors and displaying combined effector functions of IgG molecules and IgA molecules. The present invention further relates to pharmaceutical compositions comprising said crossmab antibody molecule, methods of treatment and use in the treatment of diseases.

Description

Technical Field

[0001] This invention relates to the field of antibody engineering, specifically to a cloverleaf X-configuration antibody molecule compatible with variable region sequences against various target antigens, which simultaneously possesses both IgG and IgA antibody Fc regions, retains the binding ability of the IgG and IgA Fc regions to their respective receptors, and exhibits the combined effector functions of IgG and IgA molecules. This invention also relates to pharmaceutical compositions comprising said X-configuration antibody molecule, and their use in disease treatment. Background Technology

[0002] Therapeutic monoclonal antibodies targeting tumor antigens have become an effective approach for clinical cancer treatment. Currently, most clinically used anticancer monoclonal antibodies are IgG antibodies. These antibodies bind to the Fcγ receptor (FcγR) through their Fc region, recruiting corresponding effector cells and killing antigen-carrying tumor cells through antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), induction of cancer cell apoptosis, and complement-dependent cytotoxicity (CDC), thereby achieving therapeutic goals. However, in actual clinical applications, many problems still exist, such as subjects not responding to IgG antibodies, low response, and developing antibody resistance. Therefore, it is still necessary to develop antibodies that function through other mechanisms of action to provide alternative treatment options for cancer patients who cannot benefit from current immunotherapy.

[0003] Neutrophils are the most abundant cytotoxic immune cells in the human bloodstream, accounting for 50%-70% of blood lymphocytes. Neutrophils possess an IgA-Fc receptor, FcαRI (CD89), on their surface, which can be recruited through mutual recognition with IgA antibodies, killing antibody-promoted cancer cells via a unique mechanism called trogoptosis. IgA can also activate macrophages, the most abundant cells in the tumor microenvironment, killing tumor cells through phagocytosis. IgA, by binding to the receptor FcαRI (CD89) on neutrophils and macrophages, can effectively kill tumor cells in vitro and in various animal models. Furthermore, it has been reported that FcαRI-dependent activation of neutrophils or ungraded leukocytes via IgA antibodies is more effective in killing tumor cells than activation mediated by FcγR via IgG antibodies.

[0004] However, the development of IgA antibodies as an effective immunotherapy is limited by certain characteristics of IgA itself: (1) IgA cannot activate NK cells because NK cells do not express the receptor FcαRI; (2) IgA cannot induce CDC because it does not bind to C1q; (3) IgA has a short half-life because it cannot bind to FcRn.

[0005] To overcome the problems associated with IgG and IgA antibodies, many attempts have been made in existing technologies. For example, William Kelton et al. developed an IgGA molecule containing a chimeric Fc region, which includes the CH2 domain of the IgG antibody and the CH3 domain of the IgA antibody. However, the IgGA molecule does not bind FcgRIIIa, nor does it bind FcRn in a pH-dependent manner. Further modifications to the IgGA molecule are needed to retain the full spectrum of IgG Fc binding activity required for therapeutic applications. Niels Heemskerk et al. constructed a bispecific molecule called TrisomAb, in which one arm of the bispecific antibody targets FcαRI and the other arm targets tumor-associated antigens. Although TrisomAb can effectively recruit NK cells and macrophages, its antigen-binding titer is only 1, lower than the divalent antigen-binding titer of conventional antibodies. Therefore, the lower antigen-binding titer of TrisomAb offsets the IgA-mediated tumor-killing ability.

[0006] Therefore, in tumor immunotherapy, there remains an urgent need for new antibody molecules that can overcome the limitations of current IgG and IgA antibodies and provide highly effective treatment for cancer patients. The X-configuration antibody provided in this application meets this need. Invention Overview

[0007] This invention constructs a cloverleaf X-configuration antibody molecule compatible with variable region sequences against various target antigens. It simultaneously possesses both IgG and IgA antibody Fc regions, retaining the binding ability of the IgG and IgA Fc regions to their respective receptors and exhibiting combined effector functions of IgG and IgA molecules. This allows for the recruitment of a wider variety and greater number of effector cells, thereby maximizing biological function. Since the X-configuration antibody molecule has no restrictions on antigen recognition, it is possible to construct antibodies targeting any antigen known in the art or to be obtained in the future. Figure 1 The antibody molecule with configuration X shown in B.

[0008] It should be noted that the design of the X-configuration antibody chimeric light chain in this invention is not a simple imitation of the natural antibody heavy chain; that is, it is not simply a matter of linking the antibody light chain to the Fc via the natural antibody hinge region. Subsequent embodiments demonstrate that a chimeric light chain formed by simply linking the antibody light chain to the Fc via the natural antibody hinge region cannot be assembled with the heavy chain to form an X-configuration antibody molecule. The selection and design of the connection sequence (including the linker and hinge region) between the antibody Fab light chain and the Fc plays a decisive role in whether the X-configuration antibody can be formed and whether each structural part of the X-configuration antibody can function properly.

[0009] In one aspect, the present invention provides a novel IgG and IgA chimeric "cloverleaf X antibody" (referred to as X-body antibody or X-body) molecule, said molecule comprising: (1) two antibody Fabs; (2) an IgA-type antibody Fc domain; and (3) an IgG-type antibody Fc domain, which are assembled to form a cloverleaf X-body antibody by interaction between two light chains containing the IgA Fc domain and two heavy chains containing the IgG Fc domain, or by interaction between two light chains containing the IgG Fc domain and two heavy chains containing the IgA Fc domain. Figure 1 The X-configuration antibody molecule shown in B retains the affinity for a specific antigen target, the binding ability of the Fc domain of the IgA antibody to its receptor, and the binding ability of the Fc domain of the IgG antibody to its receptor. Therefore, while recognizing the target antigen, the X-configuration antibody molecule can simultaneously recruit more effector cells such as NK cells, macrophages, and neutrophils, thereby activating the corresponding effector cells more efficiently and exerting a killing effect.

[0010] In one embodiment, this application provides an X-configuration antibody molecule comprising two identical antibody (chimeric) heavy chains and two identical antibody (chimeric) light chains, wherein the antibody heavy chains and light chains of the X-configuration molecule sequentially comprise the following structures:

[0011] (1) The antibody heavy chain, from N-terminus to C-terminus, sequentially contains the antibody heavy chain variable region (VH)-CH1-linker sequence-IgG Fc sequence; the antibody light chain, from N-terminus to C-terminus, sequentially contains the antibody light chain variable region (VL)-light chain constant region (CL)-linker sequence-IgA Fc sequence; or

[0012] (2) The antibody heavy chain contains, from the N-terminus to the C-terminus, the antibody heavy chain variable region (VH)-CH1-linker sequence-IgA Fc sequence, and the antibody light chain contains, from the N-terminus to the C-terminus, the antibody light chain variable region (VL)-light chain constant region (CL)-linker sequence-IgG Fc sequence.

[0013] Among them, VH and VL recognize the same target antigen, and VH-CH1 and VL-CL assemble to form the corresponding Fab. Thus, the X-configuration antibody molecule forms a total of 2 Fabs with the same antigen binding site.

[0014] Two identical IgG Fc sequences dimerize to form an IgG Fc region; two identical IgA Fc sequences dimerize to form an IgA Fc region.

[0015] In one embodiment, the variable regions (VH and VL) of the antibody are derived from any antibody molecule that recognizes an antigen. In another embodiment, the antibody variable regions are derived from antibodies from different species that recognize any antigen, said species being mammals, such as mammals commonly used to produce antibodies, such as rodents, dogs, camels, horses, cattle, sheep, etc. In yet another embodiment, the antibody variable regions are derived from animal antibodies, humanized antibodies, chimeric antibodies, or human antibodies.

[0016] In one embodiment, the VH and VL originate from the same antibody molecule. In one embodiment, the antibody variable region recognizes a specific antigen or epitope expressed by tumor cells. In one embodiment, the antibody variable region recognizes cytokines that are abnormally expressed compared to normal cells. In one embodiment, the antibody variable region recognizes, for example, tumor-specific molecules such as CD20, CD19, and Her2.

[0017] In one embodiment, the linker sequence includes a connector. In another embodiment, the linker sequence includes a hinge region. In yet another embodiment, the linker sequence includes both a connector and a hinge region. In one embodiment, the hinge region and the Fc polypeptide sequence linked thereto are homologous or heterologous.

[0018] In one embodiment, the IgG Fc sequence may be derived from an antibody providing the heavy chain variable region, or may be selected from the Fc sequence of any IgG class antibody known in the art, such as concordant or germline IgG Fc sequences. In one embodiment, the IgG Fc sequence may be an Fc sequence from different IgG subclasses, such as Fc sequences from IgG1, IgG2, IgG3, and IgG4. In one embodiment, the IgG Fc sequence may contain mutations / modifications, such as those known in the art that enhance ADCC, ADCP, and CDC effector functions. In another embodiment, the IgG Fc sequence may be formed by chimerating Fc sequences from different IgG molecules, for example, CH2 and CH3 are derived from different IgG Fc sequences, or a portion of the amino acid sequence in the Fc sequence is heterologous to the remaining sequences. In one embodiment, the IgG Fc sequence comprises the Fc sequence of IgG1. In one embodiment, the IgG Fc sequence comprises the Fc sequence of IgG1 containing a C-terminal lysine. In another embodiment, the IgG Fc sequence comprises the Fc sequence of IgG2. In one embodiment, the IgG Fc sequence comprises the Fc sequence of IgG2 containing a C-terminal lysine. In another embodiment, the IgG Fc sequence comprises the Fc sequence of IgG3. In one embodiment, the IgG Fc sequence comprises the Fc sequence of IgG3 containing a C-terminal lysine. In one embodiment, the IgG Fc sequence comprises the Fc sequence of IgG4. In one embodiment, the IgG Fc sequence comprises the Fc sequence of IgG4 containing a C-terminal lysine. In yet another embodiment, the IgG Fc sequence comprises a sequence formed by chimerism of one or more sequences from the IgG1 Fc sequence, the IgG2 Fc sequence, the IgG3 Fc sequence, and the IgG4 Fc sequence. In yet another embodiment, the IgG Fc comprises a mutation that increases affinity for FcRn, such as the YTE mutation.

[0019] In one embodiment, the IgG Fc sequence comprises the Fc sequence shown in SEQ ID NO:21, or comprises an Fc sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the Fc sequence shown in SEQ ID NO:21.

[0020] In one embodiment, the IgA Fc sequence may be derived from an antibody providing the heavy chain variable region, or selected from the Fc sequence of any IgA class antibody known in the art, such as concordant or germline IgA Fc sequences. In one embodiment, the IgA Fc may be an Fc sequence of the IgA1 subclass or an Fc sequence of the IgA2 subclass. In one embodiment, the IgA Fc sequence may contain mutations / modifications, such as those known in the art that enhance ADCC, ADCP, and CDC effector functions. In another embodiment, the IgA Fc sequence may be formed by chimerism of Fc sequences from different IgA molecules, for example, CH2 and CH3 are derived from different IgA Fc sequences, or a portion of the amino acid sequence in the Fc sequence is heterologous to the remaining sequences. In one embodiment, the IgA Fc sequence comprises the IgA1 Fc sequence. In another embodiment, the IgA Fc sequence comprises the IgA2 Fc sequence. In yet another embodiment, the IgA Fc sequence comprises a chimerism of the IgA1 Fc sequence and the IgA2 Fc sequence.

[0021] In one embodiment, the IgA Fc sequence comprises the Fc sequence shown in SEQ ID NO:22, or comprises an Fc sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the Fc sequence shown in SEQ ID NO:22.

[0022] In one embodiment, CH1 and / or CL in the X-configuration antibody may be homologous or heterologous to the Fc sequence linked thereto. In another embodiment, CH1 and / or CL in the X-configuration antibody may be homologous or heterologous to the VH and / or VL linked thereto. In one embodiment, CH1 and / or CL in the X-configuration antibody may be derived from commonly used CH1 and CL known in the art, such as universal CH1 and CL.

[0023] In one embodiment, the present invention provides an X-configuration antibody molecule comprising two identical antibody (chimeric) heavy chains and two identical antibody (chimeric) light chains. The antibody heavy chains, from the N-terminus to the C-terminus, sequentially comprise a VH-CH1-IgG hinge region-IgG1 Fc sequence, and the antibody light chains, from the N-terminus to the C-terminus, sequentially comprise a VL-CL-IgA1 / IgA2 hinge region-IgA1 / IgA2 Fc sequence. VH and VL form two Fabs, the IgG1 Fc sequence dimerizes to form an IgG1 Fc region, and the IgA1 / IgA2 Fc sequence dimerizes to form an IgA1 / IgA2 Fc region.

[0024] In one embodiment, the IgG hinge region of the antibody heavy chain and the IgG Fc sequence linked thereto originate from the same IgG subclass. In another embodiment, the IgG hinge region of the antibody heavy chain and the IgG Fc sequence linked thereto originate from immunoglobulins of different IgG subclasses; for example, when the Fc sequence originates from IgG1, the hinge region may originate from IgG2, IgG3, or IgG4. In one embodiment, the hinge region of the antibody light chain and the Fc sequence linked thereto originate from the same IgA subclass. In another embodiment, the hinge region of the antibody light chain and the Fc sequence linked thereto originate from different IgA subclasses; for example, when the hinge region originates from IgA1, the Fc region originates from IgA2, or vice versa.

[0025] In a further embodiment, the antibody light chain and / or heavy chain may include a linker.

[0026] In another embodiment, the antibody light chain comprises, from the N-terminus to the C-terminus, a VL-CL-connector-IgA1 hinge region-IgA1 Fc sequence, a VL-CL-connector-IgA2 hinge region-IgA2 Fc sequence, a VL-CL-connector-IgA2 hinge region-IgA1 Fc sequence, or a VL-CL-connector-IgA1 hinge region-IgA2 Fc sequence.

[0027] In one embodiment, CH1 and / or CL in the X-configuration antibody may be homologous or heterologous to the Fc sequence linked thereto. In another embodiment, CH1 and / or CL in the X-configuration antibody may be homologous or heterologous to the VH and / or VL linked thereto. In one embodiment, CH1 and / or CL in the X-configuration antibody may be derived from commonly used CH1 and CL known in the art, such as universal CH1 and CL.

[0028] In one embodiment, the present invention provides an X-configuration antibody molecule comprising two identical antibody (chimeric) heavy chains and two identical antibody (chimeric) light chains. The antibody heavy chains, from the N-terminus to the C-terminus, sequentially comprise a VH-CH1-IgG hinge region-IgG2Fc sequence, and the antibody light chains, from the N-terminus to the C-terminus, sequentially comprise a VL-CL-IgA1 / IgA2 hinge region-IgA1 / IgA2 Fc sequence. VH and VL form two Fabs, the IgG1 Fc sequence dimerizes to form an IgG1 Fc region, and the IgA1 / IgA2 Fc sequence dimerizes to form an IgA1 / IgA2 Fc region.

[0029] In one embodiment, the IgG hinge region of the antibody heavy chain and the IgG Fc sequence linked thereto originate from the same IgG subclass. In another embodiment, the IgG hinge region of the antibody heavy chain and the IgG Fc sequence linked thereto originate from immunoglobulins of different IgG subclasses; for example, when the Fc sequence originates from IgG2, the hinge region may originate from IgG1, IgG3, or IgG4. In one embodiment, the hinge region of the antibody light chain and the Fc sequence linked thereto originate from the same IgA subclass. In another embodiment, the hinge region of the antibody light chain and the Fc sequence linked thereto originate from different IgA subclasses; for example, when the hinge region originates from IgA1, the Fc region originates from IgA2, or when the hinge region originates from IgA2, the Fc region originates from IgA1.

[0030] In a further embodiment, the antibody light chain and / or heavy chain may include a linker.

[0031] In another embodiment, the antibody light chain comprises, from the N-terminus to the C-terminus, a VL-CL-connector-IgA1 hinge region-IgA1 Fc sequence, a VL-CL-connector-IgA2 hinge region-IgA2 Fc sequence, a VL-CL-connector-IgA2 hinge region-IgA1 Fc sequence, or a VL-CL-connector-IgA1 hinge region-IgA2 Fc sequence.

[0032] In one embodiment, CH1 and / or CL in the X-configuration antibody may be homologous or heterologous to the Fc sequence linked thereto. In another embodiment, CH1 and / or CL in the X-configuration antibody may be homologous or heterologous to the VH and / or VL linked thereto. In one embodiment, CH1 and / or CL in the X-configuration antibody may be derived from commonly used CH1 and CL known in the art, such as universal CH1 and CL.

[0033] In one embodiment, the present invention provides an X-configuration antibody molecule comprising two identical antibody (chimeric) heavy chains and two identical antibody (chimeric) light chains. The antibody heavy chains, from the N-terminus to the C-terminus, sequentially comprise a VH-CH1-IgG hinge region-IgG3Fc sequence, and the antibody light chains, from the N-terminus to the C-terminus, sequentially comprise a VL-CL-IgA1 / IgA2 hinge region-IgA1 / IgA2 Fc sequence. VH and VL form two Fabs, the IgG3 Fc sequence dimerizes to form an IgG3 Fc region, and the IgA1 / IgA2 Fc sequence dimerizes to form an IgA1 / IgA2 Fc region.

[0034] In one embodiment, the IgG hinge region of the antibody heavy chain and the IgG Fc sequence linked thereto originate from the same IgG subclass. In another embodiment, the IgG hinge region of the antibody heavy chain and the IgG Fc sequence linked thereto originate from immunoglobulins of different IgG subclasses; for example, when the Fc sequence originates from IgG3, the hinge region may originate from IgG1, IgG2, or IgG4. In one embodiment, the hinge region of the antibody light chain and the Fc sequence linked thereto originate from the same IgA subclass. In another embodiment, the hinge region of the antibody light chain and the Fc sequence linked thereto originate from different IgA subclasses; for example, when the hinge region originates from IgA1, the Fc region originates from IgA2, or when the hinge region originates from IgA2, the Fc region originates from IgA1.

[0035] In a further embodiment, the antibody light chain and / or heavy chain may include a linker.

[0036] In another embodiment, the antibody light chain comprises, from the N-terminus to the C-terminus, a VL-CL-connector-IgA1 hinge region-IgA1 Fc sequence, a VL-CL-connector-IgA2 hinge region-IgA2 Fc sequence, a VL-CL-connector-IgA2 hinge region-IgA1 Fc sequence, or a VL-CL-connector-IgA1 hinge region-IgA2 Fc sequence.

[0037] In one embodiment, CH1 and / or CL in the X-configuration antibody may be homologous or heterologous to the Fc sequence linked thereto. In another embodiment, CH1 and / or CL in the X-configuration antibody may be homologous or heterologous to the VH and / or VL linked thereto. In one embodiment, CH1 and / or CL in the X-configuration antibody may be derived from commonly used CH1 and CL known in the art, such as universal CH1 and CL.

[0038] In one embodiment, the present invention provides an X-configuration antibody molecule comprising two identical antibody (chimeric) heavy chains and two identical antibody (chimeric) light chains. The antibody heavy chains, from the N-terminus to the C-terminus, sequentially comprise a VH-CH1-IgG hinge region-IgG4Fc sequence, and the antibody light chains, from the N-terminus to the C-terminus, sequentially comprise a VL-CL-IgA1 / IgA2 hinge region-IgA1 / IgA2 Fc sequence. VH and VL form two Fabs, the IgG4 Fc sequence dimerizes to form an IgG4 Fc region, and the IgA1 / IgA2 Fc sequence dimerizes to form an IgA1 / IgA2 Fc region.

[0039] In one embodiment, the IgG hinge region of the antibody heavy chain and the IgG Fc sequence linked thereto originate from the same IgG subclass. In another embodiment, the IgG hinge region of the antibody heavy chain and the IgG Fc sequence linked thereto originate from immunoglobulins of different IgG subclasses; for example, when the Fc sequence originates from IgG4, the hinge region may originate from IgG1, IgG3, or IgG2. In one embodiment, the hinge region of the antibody light chain and the Fc sequence linked thereto originate from the same IgA subclass. In another embodiment, the hinge region of the antibody light chain and the Fc sequence linked thereto originate from different IgA subclasses; for example, when the hinge region originates from IgA1, the Fc region originates from IgA2, or when the hinge region originates from IgA2, the Fc region originates from IgA1.

[0040] In a further embodiment, the antibody light chain and / or heavy chain may include a linker.

[0041] In another embodiment, the antibody light chain comprises, from the N-terminus to the C-terminus, a VL-CL-connector-IgA1 hinge region-IgA1 Fc sequence, a VL-CL-connector-IgA2 hinge region-IgA2 Fc sequence, a VL-CL-connector-IgA2 hinge region-IgA1 Fc sequence, or a VL-CL-connector-IgA1 hinge region-IgA2 Fc sequence.

[0042] In one embodiment, CH1 and / or CL in the X-configuration antibody may be homologous or heterologous to the Fc sequence linked thereto. In another embodiment, CH1 and / or CL in the X-configuration antibody may be homologous or heterologous to the VH and / or VL linked thereto. In one embodiment, CH1 and / or CL in the X-configuration antibody may be derived from commonly used CH1 and CL known in the art, such as universal CH1 and CL.

[0043] In one specific embodiment, the present invention provides an X-configuration antibody molecule comprising two identical antibody (chimeric) heavy chains and two identical antibody (chimeric) light chains. The antibody heavy chains, from the N-terminus to the C-terminus, sequentially comprise a VH-CH1-IgG1 hinge region-IgG1 Fc sequence, and the antibody light chains, from the N-terminus to the C-terminus, sequentially comprise a VL-CL-linker-IgA2 hinge region-IgA1 Fc sequence. VH and VL form two Fabs, the IgG1 Fc sequence dimerizes to form an IgG1 Fc region, and the IgA1 Fc sequence dimerizes to form an IgA1 Fc region.

[0044] In one embodiment, the present invention provides an X-configuration antibody molecule targeting CD20, having the molecular structure described in any of the above embodiments.

[0045] In one specific embodiment, the present invention provides an X-configuration antibody molecule targeting CD20, wherein the heavy chain comprises the Fc sequence shown in SEQ ID NO:21, or comprises an Fc sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the Fc sequence shown in SEQ ID NO:21; the light chain comprises the Fc sequence shown in SEQ ID NO:22, or comprises an Fc sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the Fc sequence shown in SEQ ID NO:22.

[0046] In one specific embodiment, the present invention provides an X-configuration antibody molecule targeting CD20, wherein the heavy chain of the molecule includes the hinge region shown in SEQ ID NO:19 or 20, and the light chain includes the hinge region shown in SEQ ID NO:17 or 18.

[0047] In one specific embodiment, the present invention provides an X-configuration antibody molecule targeting CD20, wherein the molecular light chain comprises a adapter sequence selected from G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)6, (G4S)8, or 218 adapters.

[0048] In one specific embodiment, the present invention provides an X-configuration antibody molecule targeting CD20, wherein the molecule comprises two identical antibody (chimeric) heavy chains and two identical antibody (chimeric) light chains, the antibody heavy chains comprising the sequence shown in SEQ ID NO:1, and the antibody light chains comprising the sequence shown in SEQ ID NO:2.

[0049] In one embodiment, the present invention provides an X-configuration antibody molecule targeting CD19, having the molecular structure described in any of the above embodiments.

[0050] In one specific embodiment, the present invention provides an X-configuration antibody molecule targeting CD19, wherein the heavy chain comprises the Fc sequence shown in SEQ ID NO:21, or comprises an Fc sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the Fc sequence shown in SEQ ID NO:21; the light chain comprises the Fc sequence shown in SEQ ID NO:22, or comprises an Fc sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the Fc sequence shown in SEQ ID NO:22.

[0051] In one specific embodiment, the present invention provides an X-configuration antibody molecule targeting CD19, wherein the heavy chain of the molecule includes the hinge region shown in SEQ ID NO:19 or 20, and the light chain includes the hinge region shown in SEQ ID NO:17 or 18.

[0052] In one specific embodiment, the present invention provides an X-configuration antibody molecule targeting CD19, wherein the molecular light chain comprises a adapter sequence selected from G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)6, (G4S)8, or 218 adapters.

[0053] In one specific embodiment, the present invention provides an X-configuration antibody molecule targeting CD19, wherein the molecule comprises two identical antibody (chimeric) heavy chains and two identical antibody (chimeric) light chains, the antibody heavy chains comprising the sequence shown in SEQ ID NO:3, and the antibody light chains comprising the sequence shown in SEQ ID NO:4.

[0054] In one embodiment, the present invention provides an X-configuration antibody molecule targeting Her2, having the molecular structure described in any of the above embodiments.

[0055] In one specific embodiment, the present invention provides an X-configuration antibody molecule targeting Her2, wherein the heavy chain of the molecule comprises the Fc sequence shown in SEQ ID NO:21, or comprises an Fc sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the Fc sequence shown in SEQ ID NO:21; the light chain comprises the Fc sequence shown in SEQ ID NO:22, or comprises an Fc sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the Fc sequence shown in SEQ ID NO:22.

[0056] In one specific embodiment, the present invention provides an X-configuration antibody molecule targeting Her2, wherein the heavy chain of the molecule includes the hinge region shown in SEQ ID NO:19 or 20, and the light chain includes the hinge region shown in SEQ ID NO:17 or 18.

[0057] In one specific embodiment, the present invention provides an X-configuration antibody molecule targeting Her2, wherein the molecular light chain comprises a adapter sequence selected from G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)6, (G4S)8, or 218 adapters.

[0058] In one specific embodiment, the present invention provides an X-configuration antibody molecule targeting Her2, wherein the molecule comprises two identical antibody (chimeric) heavy chains and two identical antibody (chimeric) light chains, the antibody heavy chains comprising the sequence shown in SEQ ID NO:5, and the antibody light chains comprising the sequence shown in SEQ ID NO:6.

[0059] In one embodiment, the present invention provides an X-configuration antibody molecule comprising two identical antibody (chimeric) heavy chains and two identical antibody (chimeric) light chains. The antibody heavy chains, from the N-terminus to the C-terminus, sequentially comprise a VH-CH1-IgA1 / IgA2 hinge region-IgA1 / IgA2 Fc sequence, and the antibody light chains, from the N-terminus to the C-terminus, sequentially comprise a VL-CL-IgG hinge region-IgG1 Fc sequence. VH and VL form two Fabs, the IgG1 Fc sequence dimerizes to form an IgG1 Fc region, and the IgA1 / IgA2 Fc sequence dimerizes to form an IgA1 / IgA2 Fc region.

[0060] In one embodiment, the IgG hinge region of the antibody light chain and the IgG Fc sequence linked thereto originate from the same IgG subclass. In another embodiment, the IgG hinge region of the antibody light chain and the IgG Fc sequence linked thereto originate from immunoglobulins of different IgG subclasses; for example, when the Fc sequence originates from IgG1, the hinge region may originate from IgG4, IgG3, or IgG2. In one embodiment, the hinge region of the antibody heavy chain and the Fc sequence linked thereto originate from the same IgA subclass. In another embodiment, the hinge region of the antibody heavy chain and the Fc sequence linked thereto originate from different IgA subclasses; for example, when the hinge region originates from IgA1, the Fc region originates from IgA2, or when the hinge region originates from IgA2, the Fc region originates from IgA1.

[0061] In a further embodiment, the antibody light chain and / or heavy chain may include a linker.

[0062] In another embodiment, the antibody light chain comprises, from the N-terminus to the C-terminus, a VL-CL-linker-IgG1 hinge region-IgG1 Fc sequence. In one embodiment, the IgG hinge region of the antibody light chain and the IgG Fc sequence linked thereto are derived from different IgG subclasses of immunoglobulins; for example, when the Fc sequence is derived from IgG1, the hinge region may be derived from IgG4, IgG3, or IgG2.

[0063] In one embodiment, CH1 and / or CL in the X-configuration antibody may be homologous or heterologous to the Fc sequence linked thereto. In another embodiment, CH1 and / or CL in the X-configuration antibody may be homologous or heterologous to the VH and / or VL linked thereto. In one embodiment, CH1 and / or CL in the X-configuration antibody may be derived from commonly used CH1 and CL known in the art, such as universal CH1 and CL.

[0064] In one embodiment, the present invention provides an X-configuration antibody molecule, wherein the molecule comprises two identical antibody (chimeric) heavy chains and two identical antibody (chimeric) light chains, wherein the antibody heavy chains, from the N-terminus to the C-terminus, sequentially comprise a VH-CH1-IgA1 / IgA2 hinge region-IgA1 / IgA2 Fc sequence, and the antibody light chains, from the N-terminus to the C-terminus, sequentially comprise a VL-CL-IgG hinge region-IgG2 Fc sequence, wherein VH and VL form two Fabs, the IgG2 Fc sequence dimerizes to form an IgG2 Fc region, and the IgA1 / IgA2 Fc sequence dimerizes to form an IgA1 / IgA2 Fc region.

[0065] In one embodiment, the IgG hinge region of the antibody light chain and the IgG Fc sequence linked thereto originate from the same IgG subclass. In another embodiment, the IgG hinge region of the antibody light chain and the IgG Fc sequence linked thereto originate from immunoglobulins of different IgG subclasses; for example, when the Fc sequence originates from IgG2, the hinge region may originate from IgG4, IgG3, or IgG1. In one embodiment, the hinge region of the antibody heavy chain and the Fc sequence linked thereto originate from the same IgA subclass. In another embodiment, the hinge region of the antibody heavy chain and the Fc sequence linked thereto originate from different IgA subclasses; for example, when the hinge region originates from IgA1, the Fc region originates from IgA2, or when the hinge region originates from IgA2, the Fc region originates from IgA1.

[0066] In a further embodiment, the antibody light chain and / or heavy chain may include a linker.

[0067] In another embodiment, the antibody light chain comprises, from the N-terminus to the C-terminus, a VL-CL-linker-IgG2 hinge region-IgG2 Fc sequence. In one embodiment, the IgG hinge region of the antibody light chain and the IgG Fc sequence linked thereto are derived from different IgG subclasses of immunoglobulins; for example, when the Fc sequence is derived from IgG2, the hinge region may be derived from IgG4, IgG3, or IgG1.

[0068] In one embodiment, CH1 and / or CL in the X-configuration antibody may be homologous or heterologous to the Fc sequence linked thereto. In another embodiment, CH1 and / or CL in the X-configuration antibody may be homologous or heterologous to the VH and / or VL linked thereto. In one embodiment, CH1 and / or CL in the X-configuration antibody may be derived from commonly used CH1 and CL known in the art, such as universal CH1 and CL.

[0069] In one embodiment, the present invention provides an X-configuration antibody molecule comprising two identical antibody (chimeric) heavy chains and two identical antibody (chimeric) light chains. The antibody heavy chains, from the N-terminus to the C-terminus, sequentially comprise a VH-CH1-IgA1 / IgA2 hinge region-IgA1 / IgA2 Fc sequence, and the antibody light chains, from the N-terminus to the C-terminus, sequentially comprise a VL-CL-IgG hinge region-IgG3 Fc sequence. VH and VL form two Fabs, the IgG3 Fc sequence dimerizes to form an IgG3 Fc region, and the IgA1 / IgA2 Fc sequence dimerizes to form an IgA1 / IgA2 Fc region.

[0070] In one embodiment, the IgG hinge region of the antibody light chain and the IgG Fc sequence linked thereto originate from the same IgG subclass. In another embodiment, the IgG hinge region of the antibody light chain and the IgG Fc sequence linked thereto originate from immunoglobulins of different IgG subclasses; for example, when the Fc sequence originates from IgG3, the hinge region may originate from IgG4, IgG2, or IgG1. In one embodiment, the hinge region of the antibody heavy chain and the Fc sequence linked thereto originate from the same IgA subclass. In another embodiment, the hinge region of the antibody heavy chain and the Fc sequence linked thereto originate from different IgA subclasses; for example, when the hinge region originates from IgA1, the Fc region originates from IgA2, or when the hinge region originates from IgA2, the Fc region originates from IgA1.

[0071] In a further embodiment, the antibody light chain and / or heavy chain may include a linker.

[0072] In another embodiment, the antibody light chain comprises, from the N-terminus to the C-terminus, a VL-CL-linker-IgG3 hinge region-IgG3 Fc sequence. In one embodiment, the IgG hinge region of the antibody light chain and the IgG Fc sequence linked thereto are derived from different IgG subclasses of immunoglobulins; for example, when the Fc sequence is derived from IgG3, the hinge region may be derived from IgG4, IgG2, or IgG1.

[0073] In one embodiment, CH1 and / or CL in the X-configuration antibody may be homologous or heterologous to the Fc sequence linked thereto. In another embodiment, CH1 and / or CL in the X-configuration antibody may be homologous or heterologous to the VH and / or VL linked thereto. In one embodiment, CH1 and / or CL in the X-configuration antibody may be derived from commonly used CH1 and CL known in the art, such as universal CH1 and CL.

[0074] In one embodiment, the present invention provides an X-configuration antibody molecule comprising two identical antibody (chimeric) heavy chains and two identical antibody (chimeric) light chains. The antibody heavy chains, from the N-terminus to the C-terminus, sequentially comprise a VH-CH1-IgA1 / IgA2 hinge region-IgA1 / IgA2 Fc sequence, and the antibody light chains, from the N-terminus to the C-terminus, sequentially comprise a VL-CL-IgG hinge region-IgG4 Fc sequence. VH and VL form two Fabs, the IgG4 Fc sequence dimerizes to form an IgG4 Fc region, and the IgA1 / IgA2 Fc sequence dimerizes to form an IgA1 / IgA2 Fc region.

[0075] In one embodiment, the IgG hinge region of the antibody light chain and the IgG Fc sequence linked thereto originate from the same IgG subclass. In another embodiment, the IgG hinge region of the antibody light chain and the IgG Fc sequence linked thereto originate from immunoglobulins of different IgG subclasses; for example, when the Fc sequence originates from IgG4, the hinge region may originate from IgG2, IgG3, or IgG1. In one embodiment, the hinge region of the antibody heavy chain and the Fc sequence linked thereto originate from the same IgA subclass. In another embodiment, the hinge region of the antibody heavy chain and the Fc sequence linked thereto originate from different IgA subclasses; for example, when the hinge region originates from IgA1, the Fc region originates from IgA2, or when the hinge region originates from IgA2, the Fc region originates from IgA1.

[0076] In a further embodiment, the antibody light chain and / or heavy chain may include a linker.

[0077] In another embodiment, the antibody light chain comprises, from the N-terminus to the C-terminus, a VL-CL-linker-IgG4 hinge region-IgG4 Fc sequence. In one embodiment, the IgG hinge region of the antibody light chain and the IgG Fc sequence linked thereto are derived from different IgG subclasses of immunoglobulins; for example, when the Fc sequence is derived from IgG4, the hinge region may be derived from IgG2, IgG3, or IgG1.

[0078] In one embodiment, CH1 and / or CL in the X-configuration antibody may be homologous or heterologous to the Fc sequence linked thereto. In another embodiment, CH1 and / or CL in the X-configuration antibody may be homologous or heterologous to the VH and / or VL linked thereto. In one embodiment, CH1 and / or CL in the X-configuration antibody may be derived from commonly used CH1 and CL known in the art, such as universal CH1 and CL.

[0079] In one specific embodiment, the present invention provides an X-configuration antibody molecule comprising two identical antibody (chimeric) heavy chains and two identical antibody (chimeric) light chains. The antibody heavy chains, from the N-terminus to the C-terminus, sequentially comprise a VH-CH1-IgA1 hinge region-IgA1 Fc sequence, and the antibody light chains, from the N-terminus to the C-terminus, sequentially comprise a VL-CL-linker-IgG1 hinge region-IgG1 Fc sequence. VH and VL form two Fabs, the IgG1 Fc sequence dimerizes to form an IgG1 Fc region, and the IgA1 Fc sequence dimerizes to form an IgA1 Fc region.

[0080] In one embodiment, CH1 and / or CL in the X-configuration antibody may be homologous or heterologous to the Fc sequence linked thereto. In another embodiment, CH1 and / or CL in the X-configuration antibody may be homologous or heterologous to the VH and / or VL linked thereto. In one embodiment, CH1 and / or CL in the X-configuration antibody may be derived from commonly used CH1 and CL known in the art, such as universal CH1 and CL.

[0081] In one specific embodiment, the present invention provides an X-configuration antibody molecule targeting Her2, wherein the heavy chain of the molecule comprises the Fc sequence shown in SEQ ID NO:22, or comprises an Fc sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the Fc sequence shown in SEQ ID NO:22; the light chain comprises the Fc sequence shown in SEQ ID NO:21, or comprises an Fc sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the Fc sequence shown in SEQ ID NO:21.

[0082] In one specific embodiment, the present invention provides an X-configuration antibody molecule targeting Her2, wherein the heavy chain of the molecule includes the hinge region shown in SEQ ID NO:17 or 18, and the light chain includes the hinge region shown in SEQ ID NO:19 or 20. In a more specific embodiment, the present invention provides an X-configuration antibody molecule targeting Her2, comprising two identical antibody (chimeric) heavy chains and two identical antibody (chimeric) light chains, wherein the antibody heavy chains include the sequence shown in SEQ ID NO:7, and the antibody light chains include the sequence shown in SEQ ID NO:8.

[0083] In any of the foregoing embodiments, the connector is selected from (G4S)n, where n is an integer equal to or greater than 1, for example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9; 218 connector; GGGSG; GGSGG; GSGGG; SGGGG; GGTGS; GTSPGG; GNGGGS; G4S-GGSGG-G4S-SGGGG; GGG; DGGGS; TGEKP; GGRR; EGKSSGSGSESKVD; KESGSVSSEQLAQFRSLD; GGRRGGGS; LRQRDGERP; LRQKDGGGSERP; and GSTGSGSGKPGSGEGSTKG, etc., which can provide flexible connectors.

[0084] In one implementation, the connector is selected from the (G4S)2, (G4S)3, (G4S)4, (G4S)6, (G4S)8, or 218 connector sequence.

[0085] In one embodiment, the hinge region is derived from various classes of immunoglobulins, from a common hinge region of immunoglobulins, or from a hinge region of a germline antibody molecule. In one embodiment, the hinge region is derived from an IgG antibody or its subclass, or an IgA antibody or its subclass. In one embodiment, the hinge region is derived from the hinge region contained in, for example, IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 molecules. In a specific embodiment, the hinge region is an IgG hinge region sequence containing "CPPC" (e.g., the amino acid sequence "EPKSCDKTHTCPPCP" or "EPKSSDKTHTCPPCP"); or an IgA hinge region sequence VPSTPPTPSPSTPPTPSPS, VPPPPP. In one embodiment, the hinge region contains one or more amino acid mutations that do not affect its flexibility. In one embodiment, the hinge region and the Fc polypeptide sequence linked thereto are homologous or heterologous.

[0086] In one embodiment, the hinge region is derived from IgG1, such as the hinge region contained in SEQ ID NO:1. In another embodiment, the hinge region is derived from IgA1, such as the hinge region containing the sequence shown in SEQ ID NO:17. In one embodiment, the hinge region is derived from IgA2, such as the hinge region containing the sequence shown in SEQ ID NO:18.

[0087] In any of the foregoing embodiments, the CH1 domain may be homologous or heterologous to the heavy chain variable region and / or the Fc sequence.

[0088] Secondly, the present invention provides a method for preparing an X-configuration antibody molecule that recognizes a specific target, wherein the X-configuration antibody molecule comprises two identical antibody heavy chains and two identical antibody light chains, and the antibody heavy chains and light chains of the X-configuration molecule sequentially comprise the following structures:

[0089] (1) The antibody heavy chain, from N-terminus to C-terminus, sequentially contains the antibody heavy chain variable region (VH)-CH1-linker sequence-IgG Fc sequence; the antibody light chain, from N-terminus to C-terminus, sequentially contains the antibody light chain variable region (VL)-light chain constant region (CL)-linker sequence-IgA Fc sequence; or

[0090] (2) The antibody heavy chain contains, from the N-terminus to the C-terminus, the antibody heavy chain variable region (VH)-CH1-linker sequence-IgA Fc sequence, and the antibody light chain contains, from the N-terminus to the C-terminus, the antibody light chain variable region (VL)-light chain constant region (CL)-linker sequence-IgG Fc sequence.

[0091] Among them, VH and VL recognize the same target antigen, and VH-CH1 and VL-CL assemble to form the corresponding Fab. Thus, the X-configuration antibody molecule forms a total of 2 Fabs with the same antigen binding site.

[0092] Two identical IgG Fc sequences dimerize to form an IgG Fc region; two identical IgA Fc sequences dimerize to form an IgA Fc region.

[0093] In one embodiment, the present invention provides a method for preparing an X-configuration antibody molecule that recognizes a specific target, comprising:

[0094] 1. Obtain the coding sequences or amino acid sequences of the heavy chain variable regions and light chain variable regions of antibodies that recognize specific targets;

[0095] 2. Construct an X-configuration antibody heavy chain sequence with the structure VH-CH1-linker sequence-IgG Fc and an X-configuration antibody light chain sequence with the structure VL-CL-linker sequence-IgA Fc; or construct an X-configuration antibody heavy chain sequence with the structure VH-CH1-linker sequence-IgA Fc and an X-configuration antibody light chain sequence with the structure VL-CL-linker sequence-IgG Fc.

[0096] 3. Under suitable conditions, express the antibody heavy chain and light chain sequences obtained in step 2, wherein two heavy chain polypeptides and two light chain polypeptides assemble into an X-configuration antibody in the form of tetramers. VH-CH1 and VL-CL assemble to form the corresponding Fab, two identical IgG Fc sequences dimerize to form the IgG Fc region, and two identical IgA Fc sequences dimerize to form the IgAFc region, thereby obtaining the corresponding X-configuration antibody molecule.

[0097] 4. Optionally, purify the X-configuration antibody molecule.

[0098] The X-configuration antibody heavy and light chain sequences disclosed in step 2 of the above method can be constructed using any method known in the art. For example, the coding sequences of each domain can be obtained first, and the corresponding X-configuration antibody heavy and light chain coding sequences can be constructed using molecular biology methods, or the X-configuration antibody heavy and light chain coding sequences can be directly synthesized.

[0099] Optionally, in step 2 of the above method, the method further includes transfecting suitable host cells with coding sequences encoding the X-configuration antibody heavy chain and light chain, and expressing the corresponding antibody heavy chain and light chain molecules under conditions suitable for simultaneous expression of the antibody heavy chain and light chain.

[0100] In any of the foregoing embodiments, the variable regions (VH and VL) of the antibody are derived from any antibody molecule that recognizes an antigen. In one embodiment, the antibody variable regions can be derived from different species, such as mammals commonly used to produce antibodies, such as rodents, dogs, camels, horses, cattle, sheep, etc. In one embodiment, the antibody variable regions are derived from animal antibodies, humanized antibodies, chimeric antibodies, or human antibodies. In a specific embodiment, the VH and VL are derived from the same antibody molecule.

[0101] In any of the foregoing embodiments, the linker sequence includes a connector. In another embodiment, the linker sequence includes a hinge region. In yet another embodiment, the linker sequence includes both a connector and a hinge region. In one embodiment, the hinge region and the Fc polypeptide sequence to which it is linked are homologous or heterologous.

[0102] In any of the foregoing embodiments, the IgG Fc sequence may be derived from an antibody that provides the heavy chain variable region, or may be selected from the Fc sequence of any IgG antibody known in the prior art, such as a common or germline IgG Fc sequence.

[0103] In any of the foregoing embodiments, the IgA Fc sequence may be derived from an antibody that provides the heavy chain variable region, or may be selected from the Fc sequence of any IgA antibody known in the prior art, such as a common or germline IgA Fc sequence.

[0104] In any of the foregoing embodiments, the CH1 and / or CL are homologous to the corresponding antibody variable regions (VH and VL). In another embodiment, the CH1 and / or CL in the X-configuration antibody are heterologous to the corresponding VH and / or VL. In one embodiment, the CH1 and / or CL in the X-configuration antibody may be derived from commonly used CH1 and CL known in the art, such as universal CH1 and CL.

[0105] Thirdly, the present invention provides a method for modifying monoclonal antibodies into X-configuration antibody molecules, wherein the obtained X-configuration antibody molecule comprises two identical antibody heavy chains and two identical antibody light chains, and the antibody heavy chains and light chains of the X-configuration molecule sequentially comprise the following structures:

[0106] (1) The antibody heavy chain, from N-terminus to C-terminus, sequentially contains the antibody heavy chain variable region (VH)-CH1-linker sequence-IgG Fc sequence; the antibody light chain, from N-terminus to C-terminus, sequentially contains the antibody light chain variable region (VL)-light chain constant region (CL)-linker sequence-IgA Fc sequence; or

[0107] (2) The antibody heavy chain contains, from the N-terminus to the C-terminus, the antibody heavy chain variable region (VH)-CH1-linker sequence-IgA Fc sequence, and the antibody light chain contains, from the N-terminus to the C-terminus, the antibody light chain variable region (VL)-light chain constant region (CL)-linker sequence-IgG Fc sequence.

[0108] Among them, VH and VL recognize the same target antigen, and VH-CH1 and VL-CL assemble to form the corresponding Fab. Thus, the X-configuration antibody molecule forms a total of 2 Fabs with the same antigen binding site.

[0109] Two identical IgG Fc sequences dimerize to form an IgG Fc region; two identical IgA Fc sequences dimerize to form an IgA Fc region.

[0110] In one specific implementation, the monoclonal antibody may be an antibody molecule that recognizes any antigen.

[0111] In one embodiment, the linker sequence includes a connector. In another embodiment, the linker sequence includes a hinge region. In yet another embodiment, the linker sequence includes both a connector and a hinge region. In one embodiment, the hinge region and the Fc polypeptide sequence to which it is linked are homologous or heterologous.

[0112] In one specific embodiment, the IgG Fc sequence is derived from the monoclonal antibody, or may be selected from the Fc sequence of any IgG antibody known in the prior art, such as a common or germline IgG Fc sequence.

[0113] In one specific implementation, the IgA Fc sequence may be derived from the monoclonal antibody or selected from the Fc sequence of any IgA antibody known in the prior art, such as a common or germline IgA Fc sequence.

[0114] In one embodiment, the monoclonal antibody provides CH1 and / or CL from the X-configuration antibody. In another embodiment, CH1 and / or CL from the X-configuration antibody may be homologous to or heterologous to the corresponding VH and / or VL. In one embodiment, CH1 and / or CL from the X-configuration antibody may be derived from commonly used CH1 and CL known in the art, such as universal CH1 and CL.

[0115] In one embodiment, the present invention provides a method for modifying a monoclonal antibody into an X-configuration antibody molecule having the structure described in any of the above embodiments, wherein...

[0116] A. When the monoclonal antibody is an IgG molecule, the method includes...

[0117] (1) Modify the light chain sequence of the monoclonal antibody into an X-configuration antibody light chain sequence with the structure VL-CL-linker sequence-IgA Fc sequence, wherein VL and CL are derived from the monoclonal antibody, or VL is derived from the monoclonal antibody and CL is derived from a commonly used sequence in the prior art;

[0118] (2) Express the heavy chain of the monoclonal antibody and the light chain of the X-configuration antibody obtained in step (1), and assemble them into an X-configuration antibody under suitable conditions, wherein the two heavy chain polypeptides and the two chimeric light chain polypeptides are assembled into an X-configuration antibody in the form of a tetramer, wherein VH-CH1 and VL-CL are assembled to form the corresponding Fab, the same IgG Fc sequence contained in the two heavy chains is dimerized to form an IgG Fc region; the same IgA Fc sequence contained in the two X-configuration antibody light chains is dimerized to form an IgA Fc region, thereby obtaining the corresponding X-configuration antibody molecule;

[0119] (3) Optionally, the X-configuration antibody molecule is purified.

[0120] Alternatively, the method may include:

[0121] (1) The heavy chain sequence of the monoclonal antibody is modified into an X-configuration antibody heavy chain sequence with the structure VH-CH1-linker sequence-IgAFc sequence by replacing the Fc sequence of the antibody heavy chain with the IgA Fc sequence, and the light chain sequence of the monoclonal antibody is modified into an X-configuration antibody light chain sequence with the structure VL-CL-linker sequence-IgG Fc sequence.

[0122] (2) Express the X-configuration antibody heavy chain and X-configuration antibody light chain obtained in step (1), and assemble them into X-configuration antibody under appropriate conditions. The two heavy chain polypeptides and the two light chain polypeptides are assembled into X-configuration antibody in the form of tetramers. VH-CH1 and VL-CL are assembled to form the corresponding Fab. The same IgG Fc sequence contained in the two X-configuration antibody light chains dimerizes to form the IgG Fc region. The same IgA Fc sequence contained in the two X-configuration antibody heavy chains dimerizes to form the IgA Fc region, thereby obtaining the corresponding X-configuration antibody molecule.

[0123] (3) Optionally, the X-configuration antibody molecule is purified.

[0124] B. When the monoclonal antibody is an IgA molecule, the method includes:

[0125] (1) Modify the light chain sequence of the monoclonal antibody into an X-configuration antibody light chain sequence with the structure VL-CL-linking sequence-IgG Fc sequence;

[0126] (2) Express the heavy chain of the monoclonal antibody and the light chain of the X-configuration antibody obtained in step (1), and assemble them into an X-configuration antibody under suitable conditions, wherein the two heavy chain polypeptides and the two chimeric light chain polypeptides are assembled into an X-configuration antibody in the form of a tetramer, wherein VH-CH1 and VL-CL are assembled to form the corresponding Fab, the same IgA Fc sequence contained in the two heavy chains is dimerized to form an IgA Fc region; the same IgG Fc sequence contained in the two X-configuration antibody light chains is dimerized to form an IgG Fc region, thereby obtaining the corresponding X-configuration antibody molecule;

[0127] (3) Optionally, the X-configuration antibody molecule is purified.

[0128] Alternatively, the method may include:

[0129] (1) The heavy chain sequence of the monoclonal antibody is modified into an X-configuration antibody heavy chain sequence with the structure VH-CH1-linker sequence-IgG Fc sequence by replacing the Fc sequence of the antibody heavy chain with the IgG Fc sequence, and the light chain sequence of the monoclonal antibody is modified into an X-configuration antibody light chain sequence with the structure VL-CL-linker sequence-IgA Fc sequence.

[0130] (2) Express the X-configuration antibody heavy chain and X-configuration antibody light chain obtained in step (1), and assemble them into X-configuration antibody under appropriate conditions. The two heavy chain polypeptides and the two light chain polypeptides are assembled into X-configuration antibody in the form of tetramers. VH-CH1 and VL-CL are assembled to form the corresponding Fab. The same IgA Fc sequence contained in the two X-configuration antibody light chains dimerizes to form IgA Fc region. The same IgG Fc sequence contained in the two X-configuration antibody heavy chains dimerizes to form IgG Fc region, thereby obtaining the corresponding X-configuration antibody molecule.

[0131] (3) Optionally, the X-configuration antibody molecule is purified.

[0132] C. When the monoclonal antibody is another type of immunoglobulin molecule, the method includes:

[0133] (1) Modify the light chain sequence of the monoclonal antibody into an X-configuration antibody light chain sequence with the structure VL-CL-linker sequence-IgA Fc sequence, and modify the heavy chain sequence of the monoclonal antibody into an X-configuration antibody heavy chain sequence with the structure VH-CH1-linker sequence-IgG Fc sequence, or

[0134] The light chain sequence of the monoclonal antibody was modified into an X-configuration antibody light chain sequence with the structure VL-CL-linker sequence-IgG Fc sequence, and the heavy chain sequence of the monoclonal antibody was modified into an X-configuration antibody heavy chain sequence with the structure VH-CH1-linker sequence-IgA Fc sequence.

[0135] (2) Express the X-configuration antibody heavy chain and X-configuration antibody light chain obtained in step (1), and assemble them into an X-configuration antibody under suitable conditions. The two heavy chain polypeptides and the two chimeric light chain polypeptides are assembled into an X-configuration antibody in the form of a tetramer. VH-CH1 and VL-CL are assembled to form the corresponding Fab. The same Fc sequence contained in the two heavy chains is dimerized. The same Fc sequence contained in the two X-configuration antibody light chains is dimerized, thereby obtaining the corresponding X-configuration antibody molecule.

[0136] (3) Optionally, the X-configuration antibody molecule is purified.

[0137] In one specific implementation, the method further includes first obtaining the heavy chain coding sequence and the light chain coding sequence of the monoclonal antibody to be modified.

[0138] The X-configuration antibody heavy / light chain sequence can be obtained by any method known in the art. For example, the coding sequences of each domain can be obtained first, and the corresponding X-configuration antibody heavy / light chain coding sequences can be constructed using molecular biology methods, or the X-configuration antibody heavy / light chain coding sequences can be directly synthesized.

[0139] In step (2) of the above method, optionally, the sequences encoding the X-configuration antibody heavy chain and the sequences encoding the X-configuration antibody light chain are transfected into suitable host cells, and the corresponding antibody heavy chain and light chain molecules are expressed under conditions suitable for simultaneous expression of antibody heavy chain and light chain.

[0140] In one specific implementation, the method further includes first obtaining the heavy chain coding sequence and the light chain coding sequence of the monoclonal antibody to be modified.

[0141] The X-configuration antibody light chain sequence of the X-configuration antibody heavy chain can be obtained by any method known in the art. For example, the coding sequences of each domain can be obtained first, and the corresponding coding sequences of the X-configuration antibody heavy chain and X-configuration antibody light chain can be constructed by molecular biology methods, or the coding sequences of the X-configuration antibody heavy chain and X-configuration antibody light chain can be directly synthesized.

[0142] In step 2 of the above method, optionally, the sequences encoding the X-configuration antibody heavy chain and the sequences encoding the X-configuration antibody light chain are transfected into suitable host cells, and the corresponding antibody heavy chain and light chain molecules are expressed under conditions suitable for simultaneous expression of the antibody heavy chain and light chain.

[0143] In one implementation, the variable regions (VH and VL) of the antibody are derived from any antibody molecule that recognizes an antigen.

[0144] In any of the foregoing embodiments, the linker sequence comprises a connector, such as any connector known in the art. In another embodiment, the linker sequence comprises a hinge region. In yet another embodiment, the linker sequence comprises both a connector and a hinge region. In one embodiment, the hinge region and the Fc polypeptide sequence to which it is linked are homologous or heterologous.

[0145] In one embodiment, the IgG Fc sequence of the X-configuration antibody may be derived from the monoclonal antibody, or may be selected from the Fc sequence of any IgG antibody known in the art, such as a common or germline IgG Fc sequence.

[0146] In one embodiment, the IgA Fc sequence of the X-configuration antibody may be derived from the monoclonal antibody or selected from the Fc sequence of any IgA antibody known in the art, such as a common or germline IgA Fc sequence.

[0147] In one embodiment, the monoclonal antibody provides CH1 and / or CL from the X-configuration antibody. In another embodiment, CH1 and / or CL from the X-configuration antibody may be homologous to or heterologous to the corresponding VH and / or VL. In one embodiment, CH1 and / or CL from the X-configuration antibody may be derived from commonly used CH1 and CL known in the art, such as universal CH1 and CL.

[0148] Fourthly, the present invention provides a derived X-configuration antibody molecule comprising two identical antibody heavy chains and two identical antibody light chains, wherein the antibody heavy chains and light chains of the X-configuration molecule respectively comprise the following structures:

[0149] The antibody heavy chain, from N-terminus to C-terminus, sequentially contains the antibody heavy chain variable region (VH)-CH1-linker sequence-IgG Fc sequence; and

[0150] The antibody light chain, from N-terminus to C-terminus, sequentially includes the SIRPα extracellular region, the antibody light chain variable region (VL), the light chain constant region (CL), the linker sequence, and the IgA Fc sequence.

[0151] In this process, the VH and CH1 of a heavy chain interact with the VL and CL of a light chain to assemble into corresponding Fabs, thereby forming two Fabs with the same antigen-binding sites in the X-configuration antibody molecules derived therefrom.

[0152] Two identical IgG Fc sequences dimerize to form an IgG Fc region; two identical IgA Fc sequences dimerize to form an IgA Fc region.

[0153] In one embodiment, the variable regions (VH and VL) of the antibody are derived from any antibody molecule that recognizes an antigen. In another embodiment, the antibody variable regions are derived from antibodies from different species that recognize any antigen, said species being mammals, such as mammals commonly used to produce antibodies, such as rodents, dogs, camels, horses, cattle, sheep, etc. In yet another embodiment, the antibody variable regions are derived from animal antibodies, humanized antibodies, chimeric antibodies, or human antibodies.

[0154] In one embodiment, the linker sequence includes a connector. In another embodiment, the linker sequence includes a hinge region. In yet another embodiment, the linker sequence includes both a connector and a hinge region. In one embodiment, the hinge region and the Fc polypeptide sequence linked thereto are homologous or heterologous.

[0155] In one embodiment, a linker is included between the SIRPα extracellular region and the antibody's light chain variable region (VL). In a specific embodiment, the linker can be any of the linker sequences mentioned in this application.

[0156] In one embodiment, the IgG Fc sequence is selected from the Fc sequence of IgG1, IgG2, IgG3 or IgG4, and the IgA Fc is selected from the IgA1 Fc sequence or the IgA2 Fc sequence. Preferably, the IgG Fc is the Fc sequence of IgG1 and the IgA Fc is the IgA1 Fc sequence.

[0157] In one embodiment, the IgG Fc sequence comprises the Fc sequence shown in SEQ ID NO:21, or comprises an Fc sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the Fc sequence shown in SEQ ID NO:21.

[0158] In one embodiment, the IgA Fc sequence comprises the Fc sequence shown in SEQ ID NO:22, or comprises an Fc sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the Fc sequence shown in SEQ ID NO:22.

[0159] In one specific embodiment, the linking sequence of the molecular light chain includes a linker sequence selected from G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)6, (G4S)8, or 218 linkers.

[0160] In one specific implementation, the hinge region is selected from SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.

[0161] Fifthly, the present invention provides a method for preparing a derived X-configuration antibody molecule, comprising:

[0162] (1) Obtain the coding sequences or amino acid sequences of the heavy chain variable region and light chain variable region of the derived X-configuration antibody molecule.

[0163] (2) Construct the corresponding X-configuration antibody heavy chain sequence and light chain sequence of the derived X-configuration antibody molecule as described in any of the fourth aspects.

[0164] (3) Under suitable conditions, express the antibody heavy chain and light chain sequences obtained in step 2, wherein two heavy chain polypeptides and two light chain polypeptides are assembled into X-configuration antibody in the form of tetramers, wherein VH-CH1 and VL-CL are assembled to form the corresponding Fab, two identical IgG Fc sequences are dimerized to form IgG Fc region; two identical IgA Fc sequences are dimerized to form IgAFc region, thereby obtaining the corresponding derived X-configuration antibody molecule;

[0165] (4) Optionally, the derived X-configuration antibody molecule is purified.

[0166] In a sixth aspect, the present invention provides a method for modifying an antibody into any of the derived X-configuration antibody molecules as described in the fourth aspect, comprising:

[0167] (1) Modify the light chain sequence of the monoclonal antibody to a structure of SIRPα extracellular region - antibody light chain variable region (VL) - light chain constant region (CL) - linker sequence - IgA Fc sequence;

[0168] (2) Under suitable conditions, express the antibody heavy chain and the X-configuration antibody light chain obtained in step (1), wherein the two heavy chain polypeptides and the two chimeric light chain polypeptides are assembled into an X-configuration antibody in the form of a tetramer, wherein VH-CH1 and VL-CL are assembled to form the corresponding Fab, and the same Fc sequence contained in the two heavy chains is dimerized; the same Fc sequence contained in the two derived X-configuration antibody light chains is dimerized, thereby obtaining the corresponding X-configuration antibody molecule;

[0169] (3) Optionally, the derived X-configuration antibody molecule is purified.

[0170] In a seventh aspect, the present invention provides a nucleic acid encoding the X-configuration antibody molecule of this application, a derived X-configuration antibody molecule, a vector containing the nucleic acid, and a host cell containing the nucleic acid or the vector.

[0171] Eighthly, the present invention provides a pharmaceutical composition comprising an X-configuration antibody molecule of the present application and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising an X-configuration antibody molecule derived from the present application and a pharmaceutically acceptable carrier.

[0172] In one embodiment, the pharmaceutical composition further comprises other therapeutic agents.

[0173] In a ninth aspect, the present invention provides the use of the X-configuration antibody molecule of the present application in the preparation of a medicament for treating a disease, the use of the X-configuration antibody molecule derived from the present application in the preparation of a medicament for treating a disease, and the use of the pharmaceutical composition of the present application in the preparation of a medicament for treating a disease.

[0174] In one embodiment, the disease can be any disease that can be treated with antibodies. In another embodiment, the disease is cancer.

[0175] In a tenth aspect, the present invention provides a method for treating a disease, comprising administering a therapeutically effective amount of an X-configuration antibody molecule of the present invention, a therapeutically effective amount of an X-configuration antibody molecule derived from the present invention, or a therapeutically effective amount of a pharmaceutical composition of the present invention to a subject in need.

[0176] In one embodiment, the disease can be any disease that can be treated with antibodies. In another embodiment, the disease is cancer.

[0177] In some embodiments, the methods and uses described herein further include administering an effective amount of at least one additional therapeutic agent, such as a chemotherapeutic agent, a radiotherapy agent, or a biological macromolecule to the individual. In one embodiment, the biological macromolecule is, for example, a variety of monoclonal antibody drugs.

[0178] The aforementioned combination therapy includes combined administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administration, wherein the administration of the antibody molecule of the present invention may occur before, simultaneously with and / or after the administration of other therapeutic agents and / or adjuvants.

[0179] The X-configuration antibody constructed in this invention has the following advantages:

[0180] - Binds to antigen molecules with high affinity;

[0181] - It binds to the receptor FcαRI (CD89) and effectively recruits neutrophils as effector cells to kill tumor cells;

[0182] - It binds to the receptor FcγR, effectively recruiting NK cells and macrophages as effector cells to kill tumor cells;

[0183] - It binds to the receptor FcRn, achieving a longer antibody half-life. Brief description of the attached diagram

[0184] Figure 1 Schematic diagram of the structure of an X-configuration antibody. (A) Schematic diagram of the heavy and light chain structure of an X-configuration antibody. (B) The quaternary assembly of an X-configuration antibody. (C) The function of an X-configuration antibody in eliminating cancer cells.

[0185] Figure 2 Expression and purification of the X-configuration Rituximab antibody. (A) Schematic diagram of the heavy and light chain structures of the X-configuration Rituximab antibody. (B) Non-reducing SDS-PAGE electrophoresis results of the X-configuration Rituximab antibody sample. "Affinity chromatography" represents the sample purified by Protein A affinity chromatography, and "molecular sieve chromatography" represents the sample purified by molecular sieve chromatography. (C) Reducing SDS-PAGE electrophoresis results of the X-configuration Rituximab antibody sample. The sample used is the same as the sample used in the "molecular sieve chromatography" lane in Figure B. (D) UV (UV280) absorption peak shape during the molecular sieve chromatography purification process. The main peak is located at approximately 11.5 ml, corresponding to a molecular weight of approximately 200 kDa.

[0186] Figure 3 Two-dimensional reconstruction results of X-configuration Rituximab antibody under negative staining electron microscopy. Using a total of 9201 images of chimeric antibody particles, two-dimensional reconstruction was performed, and the chimeric antibody particles were divided into 15 classes, representing X-configuration Rituximab antibodies at different angles and in different conformations.

[0187] Figure 4 The effect of linker length and sequence on the assembleability of X-configuration antibodies. The peak shapes of X-configuration Rituximab antibody purified by molecular sieve chromatography are shown for chimeric light chains containing 3×G4S sequence (A), 218 linker (B), 4×G4S sequence (C), 6×G4S sequence (D), or 8×G4S sequence (E). Figure D is related to... Figure 2 The same data for D series.

[0188] Figure 5 Tumor cell killing effect of X-configuration Rituximab antibody. (A) Using neutrophils as effector cells, the X-configuration antibody, IgA type and IgG type Rituximab antibody at different concentrations showed their effects on Ramos cells (CD20). + (A) The ADCC killing effect of the above three antibody conformations on Ramos cells, using NK cells as effector cells. (C) The ADCP killing effect of the above three antibody conformations on Ramos cells, using macrophages as effector cells.

[0189] Figure 6 Tumor suppressive activity of the .X configuration Rituximab antibody in humanized FcαRI transgenic mice. (A) Bioluminescence imaging (BLI) of Eg7-hCD20-Luc tumor cells before treatment (day 2) and 24 hours after treatment (day 3). (B) Quantification of bioluminescence signal in panel A. (C) Inhibition of cancer cells by treatment. Tumor size change (%) = (Total count after treatment - Total count before treatment) / Total count before treatment × 100%.

[0190] Figure 7 Expression, purification, and in vivo activity of the X-configuration anti-mouse CD19 antibody. (A) Non-reducing SDS-PAGE electrophoresis results of the X-configuration anti-mouse CD19 antibody. "Affinity chromatography" represents the sample purified by Protein A affinity chromatography, and "molecular sieve chromatography" represents the sample purified by molecular sieve chromatography. (B) OD280 peak shape during molecular sieve chromatography purification. (C) The ability of IgA and X-configuration anti-mouse CD19 antibodies to clear mouse B cells in humanized FcαRI transgenic mice.

[0191] Figure 8 Expression and purification of Trastuzumab antibodies in the X and trans-X configurations. (A) Schematic diagram of the heavy and light chain structures of Trastuzumab antibody in the X configuration (Trastuzumab X-body). (B) Non-reducing SDS-PAGE electrophoresis results and OD280 peak shape of molecular sieve chromatography for Trastuzumab antibody in the X configuration. (C) Schematic diagram of the heavy and light chain structures of Trastuzumab antibody in the trans-X configuration (Trastuzumab RX-body). (D) Non-reducing SDS-PAGE electrophoresis results and OD280 peak shape of molecular sieve chromatography for Trastuzumab antibody in the trans-X configuration. All electrophoresis samples were obtained after purification by Protein A affinity chromatography and molecular sieve chromatography. (E) Schematic diagram of the heavy chain and chimeric light chain structures of Trastuzumab antibody without linkers, and its non-reducing SDS-PAGE electrophoresis results (F).

[0192] Figure 9 Tumor cell killing activity of X-configuration Trastuzumab antibody. The figure shows the tumor cell killing effect of X-configuration, IgA, and IgG Trastuzumab antibodies at different concentrations on SKBR3 (Her2) cells when neutrophils are used as effector cells. + ADCC damage effect.

[0193] Figure 10Tumor suppressive activity of .X configuration Trastuzumab antibody in humanized FcαRI transgenic mice. (A) Tumor volume change curve of MC38-hHer2 cells. (B) Survival time curve of MC38-hHer2 tumor-bearing mice. (C) Tumor volume change curve of MB49-hHer2 cells. (D) Survival time curve of MB49-hHer2 cell tumor-bearing mice. Invention Details

[0194] I. Definition

[0195] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will become apparent from this specification and the accompanying drawings, and from the appended claims.

[0196] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value.

[0197] As used herein, the terms “comprising” or “including” mean that the stated elements, integers or steps are included, but do not exclude any other elements, integers or steps.

[0198] The term "antibody" is used in the broadest sense herein to refer to a protein containing an antigen-binding site, encompassing natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, intact antibodies, and antibody fragments. The term "antibody heavy chain" is used in the broadest sense herein to encompass the heavy chains of various natural antibodies, including artificially modified or genetically engineered heavy chains, such as chimeric heavy chains, heavy chain conjugates, and heavy chain derivatives. The term "antibody light chain" is used in the broadest sense herein to encompass the light chains of various natural antibodies, including artificially modified or genetically engineered light chains, such as chimeric light chains, light chain conjugates, and light chain derivatives.

[0199] The terms "whole antibody," "intact antibody," and "immunoglobulin" are used interchangeably in this document. An immunoglobulin typically comprises at least two full-length heavy chains (H) and two full-length light chains (L), but in some cases may include fewer chains; for example, naturally occurring antibodies in camels may consist only of heavy chains. An immunoglobulin heavy chain contains a heavy chain variable region (VH, also called the heavy chain variable domain) and a heavy chain constant region (CH, also called the heavy chain constant domain), while the light chain contains a light chain variable region (VL, also called the light chain variable domain) and a light chain constant region (CL, also called the light chain constant domain).

[0200] Immunoglobulins can be classified into five classes based on the amino acid sequence of the constant region of the immunoglobulin heavy chain: IgA, IgD, IgE, IgG, and IgM. Some of these classes can be further subdivided into subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4 subclasses, and IgA into IgA1 and IgA2 subclasses. Based on the amino acid sequence of the constant domain of the immunoglobulin light chain, immunoglobulins can be classified into one of two types: κ and λ.

[0201] IgG immunoglobulins are essentially heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. The constant region of the heavy chain of IgG contains three domains: CH1, CH2, and CH3, with CH1 and CH2 linked by a hinge region. Typically, the CH2 and CH3 domains form the Fc region of IgG immunoglobulins, mediating various effector functions through their interaction with their receptor (FcγR).

[0202] IgA immunoglobulins can be produced as monomers, dimers, or secreted forms, and comprise two subclasses: IgA1 and IgA2. IgA1 and IgA2 bind to their receptor CD89 with similar affinities. Human IgA1 and IgA2 share 90% sequence identity; the main difference lies in their hinge regions: IgA1 has a longer hinge region with multiple glycosylation sites. The heavy chain constant region of IgA contains three domains: CH1, CH2, and CH3, with CH1 and CH2 linked by the hinge region. IgA mediates various effector functions through its binding to the CD89 receptor, such as ADCC mediated by neutrophils and ADCP mediated by macrophages.

[0203] In some embodiments, the IgA antibody may be monomeric. In some embodiments, the IgA heavy chain constant region comprises the three domains CH1, CH2, and CH3 of IgA1; in other embodiments, the IgA heavy chain constant region comprises the three domains CH1, CH2, and CH3 of IgA2. In other embodiments, the IgA heavy chain constant region comprises chimeric sequences, such as a hinge region derived from IgA2, and CH1, CH2, and CH3 derived from IgA1.

[0204] The term "hinge region" refers to the continuous amino acid sequence connecting the C-terminus of the CH1 domain and the N-terminus of the CH2 domain of an immunoglobulin. This region is rich in proline and does not form an α-helix, thus allowing it to easily extend and bend, altering the distance between the two arms of the "Y" shape. This facilitates complementary binding between the antibody's antigen-binding site and the antigenic epitope, and allows for the simultaneous binding of two antigenic epitopes to both arms. It also facilitates the exposure of the effector binding sites of the immunoglobulin molecule.

[0205] The terms "variable region" or "variable domain" (heavy chain variable region VH, light chain variable region VL) refer to the structural domains of the heavy or light chain of an antibody involved in antibody-antigen binding. The variable domains of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three complementarity-determining regions. In some cases, a single VH or VL domain may be sufficient to provide antigen-binding specificity.

[0206] The hinge regions of the five classes of immunoglobulins and their subclasses are not entirely the same. Among them, the heavy chains of IgG, IgA, and IgD have hinge regions between CH1 and CH2, while the heavy chains of IgM and IgE do not have hinge regions.

[0207] The terms “Fc polypeptide sequence,” “Fc sequence,” and “Fc fragment” are used interchangeably herein to refer to a C-terminal polypeptide fragment of an immunoglobulin heavy chain. The “Fc polypeptide sequence” of a native immunoglobulin contains two or three constant domains: a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in native antibodies, the Fc polypeptide sequences of IgG, IgA, and IgD immunoglobulins contain a CH2 and CH3 domain of a heavy chain; the Fc polypeptide sequences of IgM and IgE immunoglobulins contain a CH2, CH3, and CH4 domain of a heavy chain. Fc sequences can include wild-type Fc sequences and variant Fc sequences, and can originate from various antibody classes (e.g., IgG, IgA, IgE, IgD, IgM) and subclasses (e.g., IgG1, IgG2, IgG3, or IgG4).

[0208] The C-terminal lysine (residue 447 in the EU numbering system) of the Fc polypeptide sequence can be removed, for example, during antibody production or purification, or by recombinantly modifying the nucleic acid encoding the antibody heavy chain. Therefore, compositions of complete antibodies can include antibody populations with all K447 residues removed, antibody populations without K447 residues removed, and antibody populations comprising a mixture of antibodies with and without K447 residues. Unless otherwise stated herein, amino acid residues in the Fc polypeptide sequence or the constant region of the heavy chain are numbered according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0209] The terms “Fc domain” and “Fc region” are used interchangeably in this paper. They refer to a structure composed of two Fc polypeptide sequences, which are usually formed by the interaction between the two Fc polypeptide sequences through disulfide bonds, non-peptide bonds and / or forces (e.g. van der Waals forces, hydrophobic forces, hydrophilic forces).

[0210] The Fc domain of an antibody mediates several important effector functions. The term "effector function" refers to those biological activities attributable to the Fc region of an immunoglobulin that vary with immunoglobulin isotype. Examples of immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and cytokine secretion. Unless otherwise stated, the Fc domain used in the X-type chimeric antibodies described herein retains at least one or all of the functional properties of the Fc domain in its original donor antibody.

[0211] In some embodiments, one or more other amino acid modifications may be introduced into the disclosed Fc domain and / or Fc polypeptide sequence, thereby creating corresponding variants to alter antibody effector function. For example, the Fc domain and / or Fc polypeptide sequence may contain one or more amino acid substitutions that enhance ADCC, such as substitutions at positions 298, 333, and / or 334 of the Fc polypeptide sequence (EU number).

[0212] The term "effective cell" refers to a cell that expresses one or more Fc receptors and performs effector functions. For example, ADCC can be mediated by activating different effector cells expressing different Fc receptors. Cells that mediate ADCC function include, for example, NK cells, macrophages, neutrophils, peripheral blood mononuclear cells, monocytes, and cytotoxic T cells. Effector cells can originate from the natural environment, such as blood.

[0213] "Fc receptor" or "FcR" refers to a molecule that binds to the Fc domain of an antibody. In some embodiments, the FcR is a natural human FcR. In some embodiments, the FcR is a recombinant FcR. In one specific embodiment, the FcR of the X-type antibody molecule disclosed in this application comprises a receptor (FcγR) for binding the Fc region of IgG and a receptor (FcαRI(CD89)) for binding the Fc region of IgA. FcγR, including FcγRI(CD64), FcγRII(CD32), and FcγRIII(CD16), has different cellular expression profiles. For example, FcγRIIIA(CD16A) is expressed on macrophages, monocytes, natural killer cells (NK cells), and other cells. FcαRI is constitutively expressed in various myeloid cells, such as neutrophils, macrophages, Kuppfer cells, monocytes, and granulocytes.

[0214] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated immune response in which certain cytotoxic cells, with Fc receptors on their surface, recognize antibodies bound to target cells. This allows the cytotoxic cells to specifically bind to target cells carrying antigens and activate effector cells of the immune system, thereby lysing the target cells. ADCC can be mediated by, for example, natural killer (NK) cells, macrophages, neutrophils, and eosinophils (eosinophils).

[0215] The term "antibody-dependent phagocytosis" or "ADCP" refers to a cellular response in which antibodies binding to target cells bind to corresponding receptors on the surface of macrophages, inducing macrophage activation, thereby internalizing the target cells and degrading the phagosomes through acidification. For example, ADCP can be mediated by the binding of the IgG Fc region to its FcγR, or by the binding of the IgAFc region to its FcαR.

[0216] The term "complement-dependent cytotoxicity" or "CDC" refers to complement-mediated cytotoxicity, in which the Fc effector domain of an antibody that binds to the target activates a series of complement cascade reactions, creating pores in the target cell membrane, thereby leading to target cell death.

[0217] The terms "linker" or "peptide linker," etc., are used interchangeably in this application and refer to a peptide comprising one or more consecutive amino acids, said amino acids being, for example, small amino acid residues or hydrophilic amino acid residues (e.g., glycine, serine, threonine, proline, aspartic acid, asparagine, etc.). Linkers typically comprise 5-50 amino acids in length, for example, 10, 15, 20, 25, or 30 amino acid lengths. Those skilled in the art will understand that many commonly used peptide linkers can be used in embodiments of the present invention.

[0218] An example of a linker is the amino acid sequence (G4S)n, where n is an integer equal to or greater than 1, for example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, or 9. In this application, (G4S)n is sometimes also described as n×G4S, for example (G4S)6, also called 6×G4S, representing the sequence GGGGSGGGGSGGGGSGGGGSGGGSGGGS (SEQ ID NO: 15). Other examples of linkers may include amino acid variations in the sequence, such as GGGSG, GGSGG, GSGGG, or SGGGG, and may also include peptides containing amino acid residues other than G or S, such as GGTGS, GTSPGG, GNGGGS, etc., including mixtures of different peptide sequences, such as G4S-GGSGG-G4S-SGGGG, etc. The linker can also be, for example, the following amino acid sequences: GGG, DGGGS, TGEKP, GGRR, EGKSSGSGSESKVD, KESGSVSSEQLAQFRSLD, GGRRGGGS, LRQRDGERP, LRQKDGGGSERP, and GSTGSGSGKPGSGEGSTKG.

[0219] Another connector example in this application is GSTSGGSGKPGSGEGSTKG (SEQ ID NO:16), also referred to in this application as "218 connector".

[0220] The terms “flexible linker peptide,” “linker sequence,” or “linker peptide” are used interchangeably in this application and refer to one or more amino acid sequences containing a linker and hinge region.

[0221] In one implementation, the connection sequence includes a connector, such as the connector sequence described above.

[0222] In another embodiment, the linker sequence includes an antibody hinge region, such as an IgG hinge region sequence containing "CPPC" (e.g., the amino acid sequence "EPKSCDKTHTCPPCP(SEQ ID NO:19)" or "EPKSSDKTHTCPPCP(SEQ ID NO:20)"), or an IgA hinge region sequence VPSTPPTPSPSTPPTPSPS, VPPPPP.

[0223] In yet another embodiment, the linker sequence includes a connector and an antibody hinge region.

[0224] Alternatively, computer programs can be used to simulate the three-dimensional structure of proteins and peptides, or phage display methods can be used to rationally design suitable linking sequences that can be used to link the various structural domains of the antibody molecules of this invention.

[0225] The terms “linked,” “fused to,” “fused,” or “fused,” or other similar expressions, are used interchangeably. These terms refer to the connection of two or more elements or components together by any means, including chemical conjugation or recombination, either directly via peptide bonds or via linkers, typically linking two or more elements or components within a frame.

[0226] As used herein, the terms “binding” or “specific binding” mean that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antigen-binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art.

[0227] "Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y can usually be represented by the dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art.

[0228] The "percentage of identity (%)" for an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to those in the specific amino acid sequence shown in this specification, after comparing the candidate sequence with the specific amino acid sequence shown herein and, if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of sequence identity. In some embodiments, the invention contemplates variants of the antibody molecules of the invention that have a considerable degree of identity with respect to the antibody molecules and their sequences specifically disclosed herein, for example, an identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or higher. These variants may contain conserved modifications.

[0229] For polypeptide sequences, “conservative modifications” include substitutions, deletions, or additions to the polypeptide sequence that result in the replacement of a certain amino acid with a chemically similar amino acid. Tables providing conserved substitutions of functionally similar amino acids are well known in the art. Variations of such conserved modifications are supplementary to, and not excluded from, the polymorphic variants, interspecies homologs, and alleles of this invention. The following eight groups contain amino acids that are conserved substitutions for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, for example, Creighton, Proteins (1984)). In some implementations, the term "conservative sequence modification" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing an amino acid sequence.

[0230] The term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including progeny cells of this type. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny. Host cells can be any type of cell system that can be used to produce the antibody molecules of this invention, including eukaryotic cells, such as mammalian cells, insect cells, and yeast cells; and prokaryotic cells, such as *E. coli* cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0231] The term "expression vector" refers to a vector containing recombinant polynucleotides and an expression control sequence that effectively links the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including clomids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0232] The terms “individual” or “subject” are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, an individual is a human.

[0233] The term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in an individual undergoing treatment. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. In some embodiments, the antibody molecules of this invention are used to delay disease development or to slow disease progression.

[0234] The term "antitumor effect" or tumor-suppressing effect refers to biological effects that can be demonstrated through a variety of means, including but not limited to, for example, reduction in tumor volume, reduction in the number of tumor cells, reduction in tumor cell proliferation, or reduction in tumor cell survival. The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid tumors and liquid tumors.

[0235] II. Immunocytoconjugates and immunoderivatives

[0236] This invention also relates to molecules fused with the X-configuration antibody of this invention and other substances (“immunodermal derivatives”, also referred to as “derived X-configuration antibody molecules”). In some embodiments, the other substances are, for example, receptor molecules, receptor ligands, cell membrane surface molecules, or other antibody fragments. In one embodiment, the other substance is a SIRPα molecule, such as the SIRPα extracellular domain. In one embodiment, the substance may be fused to the N-terminus or C-terminus of the antibody heavy or light chain. In a specific embodiment, the substance is fused to the N-terminus of the antibody light chain. In yet another embodiment, the substance is linked to the antibody terminus via a linker sequence. In a specific embodiment, the linker sequence comprises a adapter.

[0237] In some embodiments, the immune derivative is used to prevent or treat tumors. In some embodiments, the tumor is cancer. In some embodiments, the immune derivative is used to prevent or treat infections, such as chronic infections, including bacterial infections, viral infections, fungal infections, protozoan infections, etc.

[0238] This invention also relates to molecules (“immunoconjugates”) formed by conjugating the X-configuration antibody of this invention with other substances. In some embodiments, the other substances are, for example, therapeutic agents (such as cytotoxic agents). Cytotoxic agents include any agent that is harmful to cells. Examples of cytotoxic agents (e.g., chemotherapeutic agents) suitable for forming immunoconjugates are known in the art. For example, cytotoxic agents include, but are not limited to: radioactive isotopes; growth inhibitors; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof; and various known antitumor or anticancer agents.

[0239] III. Pharmaceutical Compositions and Kits

[0240] In one aspect, the present invention provides compositions, such as pharmaceutical compositions, comprising an X-configuration antibody molecule, a derived X-configuration antibody molecule, an immunoderive, or an immunoconjugate formulated with a pharmaceutically acceptable carrier as described herein. As used herein, a "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. The pharmaceutical compositions of the present invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion). In some embodiments, the X-configuration antibody molecule of the present invention is the sole active ingredient in the pharmaceutical composition. In other embodiments, the pharmaceutical composition may comprise an antibody molecule as described herein with one or more therapeutic agents.

[0241] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), dispersions or suspensions, liposomes, and suppositories. Preferred forms depend on the intended administration method and therapeutic use. Various dosing regimens are considered herein, including, but not limited to, single or multiple administrations at different time points, bolus administration, and pulsatile infusion.

[0242] The pharmaceutical compositions of the present invention may comprise a “therapeutic effective amount” or a “preventative effective amount” of the X-configuration antibody molecule described herein. A “therapeutic effective amount” refers to the amount that effectively achieves the desired therapeutic outcome at the required dose and for the required duration. The therapeutic effective amount can vary depending on various factors such as disease state, individual age, sex, and weight. A therapeutic effective amount is any amount in which any toxic or harmful effects are less than the beneficial therapeutic effect. Relative to untreated subjects, the “therapeutic effective amount” preferably inhibits a measurable parameter (e.g., tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more preferably at least about 80%. The ability of the X-configuration antibody molecule of the present invention to inhibit measurable parameters (e.g., tumor volume) can be evaluated in animal model systems that predict efficacy in human tumors.

[0243] "Prophylactic effective dose" refers to the amount of medication administered at the required dose for the required duration to effectively achieve the desired preventive outcome. Typically, because prophylactic doses are administered in subjects before or at an early stage of the disease, the prophylactic effective dose is less than the therapeutic effective dose.

[0244] Kits containing the X-configuration antibody molecules described herein are also within the scope of this invention. The kit may include one or more other elements, such as: instructions for use; other reagents, such as markers or reagents for conjugation; a pharmaceutically acceptable carrier; and a device or other material for administration to a subject. In one embodiment, the present invention provides therapeutic kits, preventative kits, and diagnostic kits comprising the antibody molecules described herein and instructions for use.

[0245] IV. Uses and methods of the molecules of this invention

[0246] Since the X-configuration antibody provided in this application may include antigen-binding sites (VH / VL) targeting any target, the X-configuration antibody of this application has a wide range of therapeutic uses. Depending on the specific target it targets, those skilled in the art will understand the specific disease to which the specific X-configuration antibody is applicable. For example, the X-configuration antibody of this application can be used to treat inflammatory diseases, infections caused by pathogens such as bacteria, fungi, and viruses, tumors, and autoimmune diseases.

[0247] In one aspect, this application provides a method of treating a disease, comprising administering an X-configuration antibody disclosed herein to a subject, preferably a mammal, more preferably a human. In one embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the subject. In treatment, the X-configuration antibody disclosed herein may be used alone or in combination with other agents. For example, the X-configuration antibody reported herein may be co-administered with at least one additional therapeutic agent.

[0248] In one embodiment, the present invention provides a method of treating cancer, comprising administering an X-configuration antibody disclosed herein to a subject. In one embodiment, the cancer can be any cancer currently supported by immunotargeted therapy, such as bone cancer, lung cancer, liver cancer, gastric cancer, colon cancer, breast cancer, lymphoma, such as B-cell lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma [NHL], precursor B-cell lymphoblastic leukemia / lymphoma, mature B-cell tumor, B-cell chronic lymphocytic leukemia / small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma, follicular lymphoma, cutaneous centrofollicular lymphoma, marginal zone B-cell lymphoma). Pilocytic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasmacytoma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia and anaplastic large cell lymphoma, multiple myeloma, melanoma, renal cell carcinoma, pancreatic cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, ovarian cancer, uterine cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, squamous cell carcinoma, medulloblastoma, pituitary adenoma, astrocytoma and other cancers.

[0249] On the other hand, this invention provides the combination of X-configuration antibodies with other drugs. The combination of the X-configuration antibodies of this invention with other drugs can produce additive or synergistic effects in treating diseases. Other drugs can be first-line or second-line drugs commonly used in the prior art for treating specific diseases. Examples include chemotherapeutic agents, cytotoxic agents, antibodies, and small molecule drugs. In one specific embodiment, the drug combination can be the combination of the X-configuration antibody of this invention with antibody molecules targeting other targets. The combined molecules can be administered to the subject simultaneously, before, or after using the X-configuration antibody of this application.

[0250] In one specific embodiment, the present invention discloses the combined use of an X-configuration antibody and an anti-CD47 antibody.

[0251] In another aspect, the present invention provides the use of an X-configuration antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is used to treat cancer.

[0252] In one aspect, the present invention provides a diagnostic method for detecting the presence of a relevant antigen in biological samples, such as serum, semen, or urine, or tissue biopsy samples (e.g., from hyperplastic or cancerous lesions), either in vitro or in vivo. The diagnostic method comprises: (i) contacting the sample (and optionally, a control sample) with an antibody molecule as described herein, under conditions that allow for interaction, or administering the antibody molecule to a subject; and (ii) detecting the formation of a complex between the antibody molecule and the sample (and optionally, the control sample). The formation of the complex indicates the presence of the relevant antigen and may demonstrate the suitability or need for the treatment and / or prevention described herein.

[0253] V. Sequence of the exemplary X antibody molecule of the present invention

[0254] Table A shows the amino acid sequence of the CDR of the antibody in Example X of this invention (based on Kabat numbering rules).

[0255]

[0256] Example

[0257] The following are examples of the methods and compositions of the present invention to aid in understanding the invention. It should be understood that the examples should not be construed as limiting the scope of the invention in any way. Based on the disclosure in this specification, various modifications can be made by those skilled in the art without departing from the spirit of the invention.

[0258] Unless otherwise expressly indicated, the present invention will be practiced using conventional chemical, biochemical, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology and cell biology methods within the art.

[0259] Example 1. Construction, expression, and purification of X-configuration antibody.

[0260] As an example of the X-configuration antibody in this application, this embodiment is constructed based on the known anti-CD20 antibody Rituximab, and the IgA Fc sequence is constructed based on the IgA1 Fc sequence. The linker sequence (including the adapter and hinge region) between the Fab and Fc of the chimeric light chain of the X-configuration Rituximab antibody described in this embodiment is an optimized sequence, and the relevant data on the design and selection of the linker sequence are described in subsequent embodiments.

[0261] 1. Construct genes encoding the heavy chain and chimeric light chain of the X-configuration Rituximab antibody.

[0262] Based on the disclosed information in the prior art, the gene sequence information encoding the heavy and light chains of Rituximab, as well as the gene information encoding the CH2+CH3 domain (Fc polypeptide sequence) of the human IgA1 constant region, were obtained. Based on the gene sequence encoding the Rituximab light chain and the gene information of the CH2+CH3 domain of the human IgA1 constant region, the gene sequence encoding the chimeric light chain of the X-configuration Rituximab antibody of this application was designed.

[0263] When constructing the chimeric light chain, a linker sequence needs to be added between the light chain constant region and the IgA1 Fc polypeptide sequence. As verified in subsequent examples, the inventors found that the linker is crucial for the assembly of the X-configuration antibody, but within a certain length range, its specific sequence composition has little impact on the expression and function of the chimeric light chain, and can be selected according to the actual situation. Exemplary linker sequences include 3×G4S-8×G4S sequences, 218 linkers, etc. This example selected a 6×G4S linker, i.e. (GGGGSGGGGSGGGGSGGGGSGGGGS, SEQ ID NO: 15) for illustration. On the other hand, since the flexible hinge region of the antibody often has important functions such as providing disulfide bonds in addition to helping to avoid the influence of steric hindrance, it is often indispensable. As verified in subsequent examples, although the hinge region of natural IgA1 (VPSTPPTPSPSTPPTPSPS, SEQ ID NO: 17) does not affect the assembly of the X-configuration antibody molecule, it has a significant impact on the binding of the antibody molecule to FcγR. Therefore, in this embodiment, the inventors replaced the hinge region of IgA1 with the hinge region of IgA2 (VPPPPP, SEQ ID NO:18). Thus, in this embodiment, the inventors constructed a mating light chain with the structure of Rituximab light chain (VL+CL) - 6×G4S joint - IgA2 hinge region - IgA1 Fc. Figure 2 A).

[0264] Based on the obtained coding gene information, the X-configuration Rituximab antibody heavy chain gene and chimeric light chain gene were synthesized separately (commissioned to Suzhou Genewiz Biotechnology Co., Ltd.). Then, Clon was used... II (NovoPro, catalog number: C112-01) recombinant reaction system: According to the manufacturer's instructions, the obtained gene coding sequences were ligated into the mammalian cell expression vector pTT5 (NovoPro, catalog number: V001466) to obtain plasmids containing genes encoding the Rituximab heavy chain and corresponding plasmids containing genes encoding the Rituximab chimeric light chain.

[0265] The coding gene in the obtained plasmid was sequenced for verification, thus confirming that the correct coding gene was obtained. The heavy chain amino acid sequence of the X-configuration Rituximab antibody encoded by the gene is shown below:

[0266] QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSSLTSEDSAVYYCARSTYYGGDWYFNVWGA GTTVTVSAASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:1)

[0267] The IgG1 Fc polypeptide sequence used in this embodiment is:

[0268] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO:21).

[0269] The encoded chimeric light chain amino acid sequence is shown below:

[0270] QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKS GNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMMVGHEALPLAFTQKTIDRLAGK(SEQ ID NO:2).

[0271] The IgA1 Fc polypeptide sequence used in this embodiment is:

[0272] CCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEV HLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMMVGHEALPLAFTQKTIDRLAGK(SEQ IDNO: 22).

[0273] 2. Expression of Rituximab antibody with X configuration

[0274] HEK293F cells (Thermo Fisher Scientific, catalog number R79007) were passaged in FreeStyle. TM Culture medium (Gibco) TM (Item No.: 12338-018). Use fresh FreeStyle the day before transfection. TMThe culture medium was used to adjust the cell density to 5-7.5 × 10⁻⁶. 5 Cells / ml. On the day of transfection, cells grew to 1-1.5 × 10⁻⁶ cells / ml. 6 Cells / ml

[0275] Take 1 / 20 of the final volume of FreeStyle transfected HEK293F cells TM The culture medium was used as the transfection buffer. 20 μg of the recombinant plasmids containing the coding gene sequences of the heavy chain and the chimeric light chain, prepared above, were added to each ml of transfection buffer at a mass ratio of 1:1. The mixture was then mixed. 60 μg of polyethyleneimine (PEI; Polysciences, catalog number: 23966-1) was added to each ml of transfection buffer, and the mixture was mixed. The mixture was incubated at room temperature for 20 minutes. Then, the PEI / DNA mixture was gently poured into the HEK293F cell suspension and mixed. The mixture was then placed in a shaker and cultured under the following conditions: 8% CO2, 36.5℃, and 120 rpm.

[0276] The antibody heavy chain gene and chimeric light chain gene of this application are expressed in HEK293F cells to produce corresponding heavy chain polypeptides and chimeric light chain polypeptides. It is known in the art that the natural IgG Fc sequence does not interact with the IgA Fc sequence. Since the C-terminus of each heavy chain polypeptide contains the same IgG Fc sequence, the interaction of the two IgG Fc sequences promotes the homodimerization of the IgG Fc sequences of the two heavy chain polypeptide chains to form an IgG Fc region. Similarly, since the C-terminus of each chimeric light chain polypeptide contains the same IgA Fc sequence, the interaction of the two IgA Fc sequences promotes the homodimerization of the IgA Fc sequences of the two chimeric light chain polypeptide chains to form an IgA Fc region. Simultaneously, VH and CH1 located at the N-terminus of one heavy chain also interact with the corresponding light chains (VL and CL) located at the N-terminus of one chimeric light chain targeting the same target, assembling to form the corresponding Fab, thereby forming... Figure 1 Figure B shows a complete cloverleaf X-configuration antibody composed of four polypeptide chains. A complete cloverleaf X-configuration antibody simultaneously possesses two antibody Fab regions, an IgA-type antibody Fc region, and an IgG-type antibody Fc region.

[0277] 3. Purification of the X-configuration Rituximab antibody

[0278] HEK293F cells expressing the X-configuration Rituximab antibody were cultured for 5 days. The culture was then collected and centrifuged at 4000 rpm for 30 minutes. The supernatant was collected and filtered through a 0.45 μM filter. The filtered supernatant was mixed with PBS buffer at a 1:1 volume ratio and subjected to Protein A (GE Healthcare, catalog number: 17528004) affinity chromatography. After the antibody bound to the affinity chromatography column, the column was washed with approximately 10 column volumes of PBS buffer, followed by elution with 0.1 M glycine-HCl pH 3.0 buffer. The eluent was collected and immediately adjusted to pH 7.4 with 1 M Tris-HCl pH 8.0 buffer.

[0279] Using the AKTA protein purification system (GE Healthcare, model: The antibody eluent purified by Protein A affinity chromatography at pH 7.4 was dialyzed into PBS buffer and subjected to size exclusion chromatography (Superdex 200 increase 10 / 300GL molecular sieve column; GE Healthcare, catalog number: 28990944) according to the manufacturer's instructions to further purify the antibody. Gel electrophoresis was used to detect and verify the antibodies obtained each time.

[0280] Purification results are as follows Figure 2 As shown, the main bands of the X-configuration Rituximab antibody purified by Protein A affinity chromatography, and the X-configuration Rituximab antibody further purified by molecular sieve, were located at approximately 200 kDa in non-reducing SDS-PAGE assays. Figure 2 (Indicated by arrow B), and after molecular sieve chromatography, the antibody purity is greatly improved. Theoretically, the molecular weights of the heavy chain and chimeric light chain polypeptides of the X-configuration Rituximab antibody are approximately 49 kDa. Considering the glycosylation of the polypeptide, the theoretical molecular weights of both are approximately 50 kDa. Therefore, the theoretical molecular weight of the corresponding tetramer molecule should be approximately 200 kDa. It is evident that... Figure 2 The nonreducing PAGE results of B preliminarily confirm that the X-configuration Rituximab antibody expressed in this embodiment formed Figure 1 The four-leaf clover X configuration antibody shown in B. Figure 2 The results of C indicate that in reduced SDS-PAGE (using a reducing agent such as mercaptoethanol or DTT and heating the sample to reduce it), each polypeptide chain of the depolymerized cloverleaf X configuration Rituximab antibody is located at approximately 50 kDa. Figure 2 (Indicated by arrow C) This is consistent with the theoretical value.

[0281] Figure 2 Figure D shows the UV (UV280) absorption peak shape during purification using Superdex 200 increase 10 / 300GL molecular sieve chromatography. The main peak of the X-configuration Rituximab antibody is located at approximately 11.5 ml, corresponding to a molecular weight of approximately 200 kDa, further confirming that the X-configuration Rituximab antibody expressed in this embodiment forms the desired cloverleaf X configuration. In non-reduced and reduced SDS-PAGE, the main peak bands are located at approximately 200 kDa and approximately 50 kDa, respectively, both consistent with theoretical values.

[0282] Example 2. Structural imaging identification of X-configuration antibodies

[0283] First, use the glow discharge cleaning system (PELCO Products Inc., model: easiGlow) TM The carbon-supported membrane was treated with a 300-mesh copper mesh (EMS, catalog number: FCF300-Cu-50). Then, 3.5 μl of purified X-configuration Rituximab antibody solution was added to the carbon-supported membrane, and after one minute, it was blotted dry with filter paper. The carbon-supported membrane was then cleaned sequentially with three small drops of 1% (w / v) uranium acetate and air-dried. Micrographs were taken using a transmission electron microscope (Thermo Fisher Scientific, model: Talos F200C) equipped with a Ceta CCD camera. The image resolution was [resolution missing]. The photomicrographs were recorded using EPU software (ThermoFisher Scientific) with an underfocus range of -1.5 to -2.5 μm, for a total of 200 images.

[0284] The collected image data were evaluated using CTF using CTFFIND4 software, and then reference-free Gaussian automatic picking was performed using the Lanplacian function in Relion-3.0 software. Initially, a total of 33,580 particles were picked. After three rounds of two-dimensional reconstruction, classification, and discarding, 9,102 chimeric antibody particles were finally classified into 15 classes, representing X-configuration Rituximab antibodies under different angles and conformations. Figure 3 The images of the chimeric antibody particles visually demonstrate that the X-configuration antibody described in this patent has formed... Figure 1 The four-leaf clover X configuration shown in B.

[0285] Example 3. Effects of linkers of different lengths and sequences on X-configuration antibodies

[0286] This embodiment discusses the influence of linkers of different lengths and sequences in the chimeric light chain of the X-configuration antibody on the formation of the X-configuration antibody, in order to analyze linkers suitable for the chimeric light chain of the X-configuration antibody. It should be noted that, as verified in subsequent embodiments, linkers of a certain length are essential elements for the assembly of the X-configuration antibody.

[0287] Following the method described in Example 1, chimeric light chain sequences containing different linkers were constructed, thereby obtaining Rituximab antibodies with different X-configurations. In addition to the 6×G4S linker disclosed in Example 1, the linkers also included 3×G4S, 4×G4S, 8×G4S, and 218 linkers. The obtained antibodies were purified sequentially by Protein A affinity chromatography and molecular sieve chromatography, and the polymerization state of the X-configuration antibodies was identified.

[0288] The results are as follows Figure 4 As shown, the X-configuration antibodies containing the different linkers described above all exhibit a main peak position of approximately 11.5 ml in molecular sieve chromatography, corresponding to a molecular weight of approximately 200 kDa, consistent with the X-configuration Rituximab antibody containing the 6×G4S linker described in Example 1. Combined with the structural imaging evidence provided in Example 2, the above molecular sieve chromatography results indicate that the tested linker sequences enable the corresponding antibodies to assemble primarily in a cloverleaf X configuration.

[0289] The linkers used in the X-configuration antibody chimeric light chains tested here range in length from 15 to 40 amino acid residues; their sequences include both G4S sequences containing only glycine and serine, and 218 linkers containing multiple amino acid residues. This demonstrates that peptide sequences of appropriate length and with a certain degree of flexibility, without specific higher-order structures, commonly used in the prior art, can be applied to the X-configuration antibody of this application. When the flexible linker is constructed between the X-configuration antibody light chain and the corresponding Fc sequence, it facilitates the assembly of the antibody light chain with the corresponding VH into Fab, promotes the formation of homodimers of the corresponding Fc sequence, and does not adversely affect the biological function of the corresponding domains.

[0290] Example 4. Recognition and binding of X-configuration antibodies to antigens and corresponding receptors

[0291] This embodiment utilizes surface plasmon resonance (SPR) technology to detect the binding of the X-configuration antibody to the antigen (CD20) and to the corresponding receptors.

[0292] First, the anti-His tag antibody was immobilized on a Series S CM5 chip (Cytiva, catalog number: 29-1049-88) using an amino-coupled reagent (Cytiva, catalog number: BR-1000-50). The chip was then mounted on a surface plasmon resonance (SPR) instrument (GE Healthcare, model: Biacore T200). His-tag-containing antigens (CD20) or different Fc receptors were diluted to a concentration of 400 nM with PBS buffer and flowed through the chip at a flow rate of 10 μl / min for 60 s for sample capture. Channel F1 was used as a reference channel and did not capture any antigen or receptor. Channels F2, F3, and F4 were used to capture antigens or different Fc receptors, respectively. The X-configuration antibody, or the IgG or IgA antibody used as a control, was dissolved in PBS buffer (usually pH 7.4, except when determining the affinity constant of the antibody with FcRn, which uses pH 6.0 PBS buffer). A 2-fold serial dilution gradient was prepared, with end concentrations ranging from 500 nM to 15.625 nM (500 nM, 250 nM, 125 nM, ..., 15.625 nM). The different types and concentrations of antibodies were injected sequentially into channels F1, F2, F3, and F4 as the mobile phase, binding for 120 s and dissociation for 240 s. The antibody was then eluted with 10 mM Glycine-HCl (pH 1.5) regeneration buffer at a flow rate of 30 μl / min for 30 s to regenerate the chip. Antibody injection and chip regeneration cycles were alternated. Using the instrument's accompanying software, Biacore T200 Evaluation Software, a steady-state binding model or kinetic analysis was established to fit all the binding response curves, thus obtaining the corresponding binding constant K. a and dissociation constant K d The affinity constant K was obtained through calculation. D (K D =K d / K a ).

[0293] The IgG control used in this test is the Rituximab antibody molecule. The IgA control was constructed by the inventors based on information about Rituximab, by fusing the Rituximab antibody F(ab)2 sequence with the Fc region of IgA. Its antibody heavy chain sequence is as follows:

[0294] QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSSLTSEDSAVYYCARSTYYGGDWYFNVWGAG TTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVVPSTPPTPS PSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNT FRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGK(SEQ ID NO:9).

[0295] The light chain sequence is as follows:

[0296] QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:10).

[0297] The SPR test results are shown in Table 1 below. The X-configuration antibody retained its binding ability to antigen CD20, receptor FcαRI (CD89), FcRn, and various FcγRs. The affinity of the X-configuration antibody for each test molecule was comparable to that of the corresponding control IgA or IgG antibody, with no order of magnitude difference, and even superior to both natural antibodies in some indicators.

[0298] Table 1. Determined affinity of X-configuration Rituximab antibody

[0299]

[0300] ND: Undetectable

[0301] These results indicate that each structural part (Fab, IgG Fc domain, and IgAFc domain) of the X-configuration antibody constructed in this application maintains the function of the corresponding structure of the wild-type natural antibody, and has the potential to exert further physiological functions. For example, the X-configuration antibody constructed in this application retains the affinity for binding to the antigen, while retaining the binding ability of the IgG Fc region to the corresponding receptors (FcγRIII, FcγRII, FcγRI) and to FcRn, and retaining the binding ability of the IgA Fc region to the corresponding receptor (CD89).

[0302] Example 5. Tumor cell killing effect of X-configuration Rituximab antibody

[0303] Rituximab is a recombinant monoclonal antibody that specifically targets the CD20 antigen and exerts its effects through multiple effector functions. Ramos cells (ATCC catalog number: CRL-1596), as human B-lymphoma cells, naturally possess CD20 molecules on their cell surface, and therefore can serve as target cells for Rituximab antibodies to detect the effector functions of Rituximab.

[0304] 1. Tumor cell killing effect using neutrophils as effector cells

[0305] IgA antibodies can recruit neutrophils and exert tumor-killing effects by binding their Fc domain to the receptor FcαRI (CD89).

[0306] In a solution containing 10% fetal bovine serum (FBS; Gibco) TM RPMI-1640 medium (Gibco, catalog number: 16000-044) TM Ramos tumor cells were cultured in RPMI-1640 medium (catalog number: 11875-085). Neutrophils were isolated from human blood using the MACSxpress Whole Blood Neutrophil Isolation Kit (Miltenyi Biotec, catalog number: 130-104-434) according to the manufacturer's instructions, and then counted. The isolated cells were cultured in RPMI-1640 medium containing 10% FBS, resulting in a cell count of 2.5 × 10⁶ cells / mL. 5 The number of neutrophils and cells was 10. 4Ramos tumor cells were mixed in 96-well culture dishes. Rituximab X-configuration antibody, Rituximab antibody, or IgA-type Rituximab antibody, prepared in RPMI-1640 medium containing 10% FBS, were added to the cells to final concentrations of 100, 10, 1, 0.1, and 0.01 μg / ml, respectively. The volume of the mixture was adjusted to 200 μl with RPMI-1640 medium containing 10% FBS. Each mixture was divided into triplicate and incubated at 37°C and 5% CO2 for 4 hours. The supernatant was then collected and... The non-radioactive cytotoxicity assay kit (Promega, catalog number: G1780) was used to quantitatively detect the release of lactate dehydrogenase (LDH) according to the manufacturer's instructions. LDH is a stable cytoplasmic enzyme that is released during cell lysis, and its amount is proportional to the number of dead cells; therefore, it was used to analyze the degree of lysis of Ramos cells. The LDH released from lysing a corresponding number of Ramos cells using the lysis buffer provided in the kit served as a 100% control.

[0307] Test results are as follows Figure 5 As shown in Figure A, the ADCC killing effect of neutrophils as effector cells on Ramos cells increases with increasing antibody concentration, demonstrating that this killing effect is dose-dependent. The killing effect of Rituximab antibody X-configuration on Ramos cells mediated at all concentrations is significantly higher than that of IgG Rituximab antibody, approximately two to three times higher; it is also higher than that of IgA Rituximab antibody. These results demonstrate that the X-configuration antibody can effectively recruit neutrophils as effector cells to kill tumor cells.

[0308] 2. Tumor cell killing effect using NK cells as effector cells

[0309] IgG antibodies can recruit NK cells and exert ADCC effects by binding their Fc domain to the receptor FcγR.

[0310] Human peripheral blood mononuclear cells (PBMCs) were isolated from human blood using Ficoll-Paque Plus reagent (GE Healthcare, catalog number: 17-1440-02) according to the manufacturer's instructions, and then treated with EasySep. TM Human NK cell isolation kit (STEMCELL) TM Technologies (product number: 19855) isolated NK cells from PBMCs and counted them according to the manufacturer's instructions.

[0311] The isolated cells were cultured in RPMI-1640 medium containing 10% FBS, with a cell count of 5 × 10⁶. 4 The number of NK cells was 10. 4 Ramos tumor cells were mixed in 96-well culture dishes. The ADCC killing effect of Rituximab antibodies of configuration X, IgG, or IgA on Ramos cells was detected using the method described in Section 1 of this embodiment.

[0312] Test results are as follows Figure 5 As shown in Figure B, the ADCC killing effect of NK cells as effector cells against Ramos cells generally increases with increasing antibody concentration, demonstrating that this killing effect is antibody-mediated and dose-dependent. The ADCC killing effect of the X-configuration Rituximab antibody against Ramos cells was higher than that of the IgG and IgA Rituximab antibodies at all concentrations. These results demonstrate that the X-configuration antibody can also effectively recruit NK cells as effector cells to kill tumor cells.

[0313] 3. Tumor cell killing effect using macrophages as effector cells

[0314] PBMCs were separated from human blood using the method described in Section 2 of this embodiment, and then CD14 was separated from the PBMCs using CD14 MicroBeads (Miltenyi Biotec, catalog number: 130-050-201) according to the manufacturer's instructions. + Mononuclear cells. The isolated CD14... + Mononuclear cells were cultured in RPMI-1640 medium containing 20% ​​FBS, and macrophage colony-stimulating factor (M-CSF) was added to a final concentration of 50 ng / ml. Macrophages were formed after induction at 37°C and 5% CO2 for 2 weeks.

[0315] The obtained macrophages were labeled with the green fluorescent dye CSFE (Biolegend, catalog number: 423801) according to the manufacturer's instructions, while Ramos cells were labeled with the red fluorescent dye PKH26 (Sigma Aldrich, catalog number: mini26) according to the manufacturer's instructions. The number of labeled cells was 5 × 10⁶. 4 The number of macrophages and cells was 10. 4Ramos cells were mixed and Rituximab antibodies of configuration X, IgG, or IgA were added at the concentrations described in Section 5.1 of this Example. After culturing in RPMI-1640 medium containing 10% FBS at 37°C and 5% CO2 for 4 hours, the proportion of PKH26 / CSFE red-green fluorescent double-positive cells was detected by flow cytometry (BD Biosciences, model: LSR Fortessa) to characterize the phagocytic killing effect of macrophages on the ADCP of Ramos cells.

[0316] Test results are as follows Figure 5 As shown in Figure C, the ADCP phagocytic and killing effect of macrophages as effector cells on Ramos cells generally increases with increasing antibody concentration, demonstrating that this killing effect is dose-dependent. Since macrophages possess both FcαRI and FcγR, all types of Rituximab antibodies—X-configuration, IgG, and IgA—have a strong recruitment effect on macrophages. Comparatively, the ADCP effect mediated by IgG-type Rituximab antibodies is weaker than that of X-configuration or IgA-type Rituximab antibodies, while the ADCP effect mediated by X-configuration antibodies is slightly stronger than that of IgA-type antibodies in most cases. These results demonstrate that X-configuration antibodies can effectively recruit more macrophages as effector cells based on the interaction of both IgG and IgA Fc domains with their corresponding receptors, thereby triggering a stronger ADCP effect to kill tumor cells.

[0317] Example 6. Tumor suppressive activity of the X-configuration Rituximab antibody in animals.

[0318] This embodiment demonstrates in animal studies that the X-configuration antibody exhibits superior activity compared to the IgG and IgA antibodies in inhibiting hematologic malignancies.

[0319] In humanized FcαRI transgenic mice (Nanmo Biotechnology, catalog number: NM-KI-200063), monocytes (including macrophages) and granulocytes (including neutrophils) express humanized FcαRI receptors on their surfaces. These receptors can bind to antibodies carrying human IgAFc and act as effector cells to kill target cells. The Eg7 cell line (ATCC, catalog number: CRL-2113) is a mouse T-lymphoma cell line. Based on the Eg7 cell line, human CD20 genes and luciferase reporter genes were introduced separately using lentiviruses to obtain Eg7-hCD20-Luc cells. These cells highly express human CD20 antigen on their surface and express luciferase intracellularly, thus oxidizing luciferin to emit fluorescence, making the cells visible.

[0320] Eg7-hCD20-Luc cells in the logarithmic growth phase were digested and counted, and then injected into the peritoneum of humanized FcαRI transgenic mice at a concentration of approximately 3 × 10⁻⁶. 6 The mice were inoculated with cells at a rate of [number] cells per mouse. Forty-eight hours later (day two), mice were randomly divided into four groups of three: a negative control group receiving PBS, an experimental group receiving the X-configuration Rituximab antibody (as described in this application), and a positive control group receiving either IgG or IgA Rituximab antibody. Tumors were visualized by intraperitoneal injection of 100 μL of 0.025 g / mL fluorescein (Promega, catalog number: P1041), and tumor images were recorded using a small animal in vivo optical 3D imaging system (PerkinElmer, model: IVIS Spectrum). Subsequently, mice were injected intraperitoneally with PBS, or with the X-configuration Rituximab antibody, IgG, or IgA Rituximab antibody at a dose of 10 mg / kg. Twenty-four hours later (day three), tumor images were again recorded using the small animal in vivo optical 3D imaging system, and the mice were subsequently euthanized in accordance with animal welfare and ethical requirements.

[0321] Tumor images showed that, compared to before treatment (day 2), tumor cells in the control mice injected with PBS further increased significantly after treatment (day 3). Figure 6 Injection of IgG or IgA type Rituximab antibodies significantly delayed tumor growth, and tumors in mice showed a slight reduction in size. Figure 6 Injection of the X-configuration Rituximab antibody significantly reduced tumor size in mice, with a reduction of over 70%. Figure 6 C). These results demonstrate that the X-configuration antibody can inhibit hematologic malignancies in mice, and its effect is superior to that of IgG and IgA antibodies.

[0322] Example 7. Expression, purification, and in vivo activity of X-configuration anti-mouse CD19 antibody.

[0323] This embodiment uses a known anti-mouse CD19 (Anti-mCD19) antibody as an example to verify the universality of the X-configuration antibody construction method and the activity of the X-configuration antibody in animals.

[0324] 1. Construct genes encoding the heavy chain and chimeric light chain of the X-configuration anti-mouse CD19 antibody.

[0325] Based on the publicly available light and heavy chain sequences of the Fab region of murine anti-mouse CD19 antibody, and following the method described in Example 1, a chimeric light chain gene sequence for an X-configuration anti-mouse CD19 antibody was designed, sequentially comprising the light chain sequence of murine anti-mouse CD19 antibody - 6×G4S linker - IgA2 hinge region - IgA1 Fc sequence, i.e., replacing the VL-CL sequence of Rituximab in sequence 2 (SEQ ID NO:2) with the VL-CL sequence of murine anti-mouse CD19. In addition, a gene sequence was designed sequentially comprising the variable region sequence of the heavy chain of murine anti-mouse CD19 antibody - CH1 - hinge region - human IgG1 Fc sequence, i.e., replacing the VH sequence of Rituximab in sequence 1 (SEQ ID NO:1) with the VH sequence of murine anti-mouse CD19, as the chimeric heavy chain gene sequence for the X-configuration anti-mouse CD19 antibody.

[0326] The chimeric heavy chain gene and chimeric light chain gene of the X-configuration anti-mouse CD19 antibody described above were synthesized separately (contracted to Suzhou Genewise Biotechnology Co., Ltd.). Following the method described in Example 1, they were constructed into mammalian cell expression plasmids. The coding genes in the obtained plasmids were sequenced for verification, thus confirming the correct coding gene sequence. The chimeric heavy chain amino acid sequence of the X-configuration anti-mouse CD19 antibody encoded by the gene is shown below:

[0327] EVQLQQSGAELVRPGTSVKLSCKVSGDTITFYYMHFVKQRPGQGLEWIGRIDPEDESTKYSEKFKNKATLTADTSSNTAYLKLSSLTSEDTATYFCIYGGYYFDYWGQGVMV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:3)

[0328] The encoded chimeric light chain amino acid sequence is shown below:

[0329] DIQMTQSPASLSSTSLGETVTIQCQASEDIYSGLAWYQQKPGKSPQLLIYGASDLQDGVPSRFSGSGSGTQYSLKITSMQTEDEGVYFCQQGLTYPRTFGGGTKLELKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKS GNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMMVGHEALPLAFTQKTIDRLAGK(SEQ ID NO:4).

[0330] 2. Expression and purification of X-configuration anti-mouse CD19 antibody

[0331] Following the method described in Example 1, the X-configuration anti-mouse CD19 antibody was expressed and purified. The X-configuration anti-mouse CD19 antibody purified by Protein A affinity chromatography, and the X-configuration anti-mouse CD19 antibody further purified by molecular sieve, showed a main band at approximately 200 kDa in non-reducing SDS-PAGE assays. Figure 7 (Indicated by arrow A) and after molecular sieve chromatography, the antibody purity was greatly improved. During the purification process using Superdex 200 increase 10 / 300GL molecular sieve chromatography, the main absorption peak of the X-configuration anti-mouse CD19 antibody under ultraviolet light (UV280) was located at approximately 11.5 ml, corresponding to a molecular weight of approximately 200 kDa. The above data proves that the X-configuration anti-mouse CD19 antibody expressed in this embodiment also exists in tetrameric form, that is, forming the desired cloverleaf X configuration.

[0332] 3. Target cell clearance activity of the X-configuration anti-mouse CD19 antibody in animals.

[0333] In humanized FcαRI transgenic mice (Nanmo Biotechnology, catalog number: NM-KI-200063), monocytes (including macrophages) and granulocytes (including neutrophils) express humanized FcαRI receptors on their surfaces, which can bind to antibodies carrying human IgA Fc and act as effector cells to kill target cells. On the other hand, CD19 is a surface CD molecule expressed by B cells; all B cell lines except plasma cells, including malignant B cells, express this molecule. Therefore, this embodiment tested the biological activity of the X-configuration anti-mouse CD19 antibody carrying human IgA Fc, specifically its activity in mediating the clearance of B cells in humanized FcαRI transgenic mice.

[0334] Humanized FcαRI transgenic mice were randomly divided into groups of five for the experiment. Mice were intraperitoneally injected with either X-configuration anti-mouse CD19 antibody or positive control IgA-type anti-mouse CD19 antibody at a dose of 8 mg / kg. PBS solution served as the negative control. Blood samples were collected from the tail vein at 43, 91, 115, 165, 213, and 285 hours post-injection. Lymphocytes in the blood samples were labeled with FITC-tagged mouse CD45 antibody (Biolegend, catalog number: 157607) and B cells were labeled with APC-tagged mouse CD19 antibody (Biolegend, catalog number: 115511) according to the manufacturer's instructions. The percentage of B cells among all lymphocytes was analyzed using flow cytometry (BD Biosciences, model: LSR Fortessa).

[0335] The amino acid sequence of the positive control IgA type anti-mouse CD19 antibody is as follows:

[0336] Anti-mouse CD19 IgA antibody heavy chain sequence:

[0337] EVQLQQSGAELVRPGTSVKLSCKVSGDTITFYYMHFVKQRPGQGLEWIGRIDPEDESTKYSEKFKNKATLTADTSSNTAYLKLSSLTSEDTATYFCIYGGYYFDYWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGK(SEQ ID NO: 11)

[0338] Light chain sequence of anti-mouse CD19 IgA antibody:

[0339] DIQMTQSPASLSTSLGETVTIQCQASEDIYSGLAWYQQKPGKSPQLLIYGASDLQDGVPSRFSGSGSGTQYSLKITSMQTEDEGVYFCQQGLTYPRTFGGGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 12).

[0340] The results are shown in Figure 7As shown in Figure C, the number of B cells in the peripheral blood of FcαRI transgenic mice in the negative control group remained stable at approximately 60% of all lymphocytes, without changing over time. In transgenic mice injected with either the X-configuration or IgA-type anti-mouse CD19 antibody, the number of B cells in the peripheral blood decreased to only about 5% of all lymphocytes at 43 hours, demonstrating that both antibodies effectively cleared their targeted cells in vivo. By 91 hours, the percentage of B cells in the peripheral blood of both antibody-injected groups slightly recovered to approximately 10%. Subsequently, the percentage of B cells in the peripheral blood of transgenic mice injected with IgA-type anti-mouse CD19 antibody rapidly recovered, reaching nearly 60% by 165 hours, essentially reaching the level of the control group, and remained relatively constant thereafter. However, the percentage of B cells in the peripheral blood of transgenic mice injected with the X-configuration anti-mouse CD19 antibody recovered slowly, falling below 30% by 165 hours, and remaining significantly lower than the negative control group and the IgA-type antibody group until 285 hours. These results demonstrate that the X-configuration antibody exerts its effect for a longer period compared to IgA antibodies, reflecting its longer half-life. Because of the cross-reactivity between the Fc terminus of human IgG antibodies and the mouse FcRn receptor, this type of antibody maintains a longer half-life in mice. The longer half-life of the X-configuration antibody in mice proves that its IgG Fc can function effectively in animals.

[0341] In summary, the B-cell clearance experiment in humanized FcαRI transgenic mice demonstrated that all parts of the X-configuration antibody constructed in this application (including the IgA Fc region, IgG Fc region, and Fab terminus) function normally in animals. It is evident that even against variable regions from different species, the X-configuration antibody constructed in this embodiment can still retain its target recognition function. Furthermore, given the combined effect of the simultaneously functioning IgG Fc and IgA Fc domains, it can recruit more effector cells, thereby more effectively killing tumor cells expressing the target antigen.

[0342] Example 8. Expression, purification and functional characterization of X-configuration and trans-X-configuration Trastuzumab antibodies

[0343] This embodiment uses the known anti-Her2 antibody trastuzumab as an example to further verify the universality of the X-configuration antibody construction method. It also verifies the influence of Fc domains of different immunoglobulin classes attached to the antibody light and heavy chains on the assembly and biological function of X-configuration antibodies. This embodiment further demonstrates that if the chimeric light chain is not linked to the Fc domain via a hinge region without a linker, it will be difficult to assemble it with the heavy chain into an X-configuration antibody.

[0344] 1. Expression, purification and functional characterization of X-configuration Trastuzumab antibody

[0345] This section, based on the known anti-Her2 antibody trastuzumab, uses the same construction scheme as in the previous examples to further verify the universality of the X-configuration antibody construction method.

[0346] 1.1. Constructing genes encoding the heavy chain and chimeric light chain of the X-configuration Trastuzumab antibody.

[0347] Based on the publicly available light and heavy chain sequences of IgG type Trastuzumab antibody, and following the method described in Example 1, a chimeric light chain of Trastuzumab antibody with an X-configuration, sequentially comprising the light chain sequence of Trastuzumab antibody - 6×G4S linker - IgA2 hinge region - IgA1 Fc sequence, was designed. Figure 8 A) The VL-CL sequence of Rituximab in sequence 2 (SEQ ID NO:2) is replaced with the VL-CL sequence of Trastuzumab. A gene sequence is then designed containing the heavy chain variable region sequence of Trastuzumab antibody, CH1, hinge region, and human IgG1 Fc sequence, i.e., the VH sequence of Rituximab in sequence 1 (SEQ ID NO:1) is replaced with the VH sequence of Trastuzumab, serving as the chimeric heavy chain gene sequence for the X-configuration Trastuzumab antibody.

[0348] The heavy chain gene and chimeric light chain gene of the X-configuration Trastuzumab antibody were synthesized separately (contracted to Suzhou Genewise Biotechnology Co., Ltd.). Following the method described in Example 1, they were constructed into mammalian cell expression plasmids. Sequencing verification was performed on the encoding genes in the obtained plasmids, confirming the correct sequence of the encoding genes. The heavy chain amino acid sequence of the X-configuration Trastuzumab antibody encoded by the gene is shown below:

[0349] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 5).

[0350] The encoded chimeric light chain amino acid sequence is shown below:

[0351] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGS GGGGSGGGGSVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKS GNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMMVGHEALPLAFTQKTIDRLAGK(SEQ ID NO:6).

[0352] In addition, the laboratory constructed an IgA antibody containing the antigen recognition site of Trastuzumab as a control, wherein the heavy chain sequence of the anti-human Her2 IgA antibody is shown in SEQ ID NO:7, and the light chain sequence of the anti-human Her2 IgA antibody is shown below:

[0353] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:14).

[0354] 1.2. Expression and purification of X-configuration Trastuzumab antibody

[0355] Following the method described in Example 1, the X-configuration Trastuzumab antibody was expressed and purified. The X-configuration Trastuzumab antibody, purified by Protein A affinity chromatography and further purified by molecular sieve, showed a band at approximately 200 kDa in non-reducing SDS-PAGE assay. Figure 8 (Indicated by arrow B), and with high purity. During purification using Superdex 200 increase 10 / 300GL molecular sieve chromatography, the main absorption peak of the X-configuration Trastuzumab antibody under ultraviolet light (UV280) was located at approximately 11.5 ml, corresponding to a molecular weight of approximately 200 kDa. The data demonstrates that the X-configuration Trastuzumab antibody expressed in this example also exists in tetrameric form, i.e., forming the desired cloverleaf X configuration.

[0356] 1.3. Recognition and binding of X-configuration Trastuzumab antibodies to antigens and corresponding receptors

[0357] Following the method described in Example 4, the binding of the X-configuration Trastuzumab antibody to the antigen (Her2), receptor FcαRI (CD89), and the important receptor FcγRIIIa (F158 type) of IgG Fc was detected using SPR technology.

[0358] The results are shown in Table 2 below. The affinity of the X-configuration Trastuzumab antibody for antigen Her2 was at the same level as that of the control IgA or IgG antibodies. The affinity of the X-configuration Trastuzumab antibody for CD89 was comparable to that of the IgA antibody, and its affinity for FcγRIIIa was comparable to that of the IgG antibody. These results indicate that each structural part (Fab, IgG Fc domain, and IgA Fc domain) of the X-configuration Trastuzumab antibody constructed and purified in this embodiment maintained the function of the corresponding structure of the wild-type natural antibody. Therefore, the X-configuration antibody constructed in this embodiment targeting a different target not only retains its target recognition function, but also, due to the combined effect of the simultaneously functioning IgG Fc and IgA Fc domains, can recruit more effector cells, thereby more effectively killing tumor cells expressing the target antigen.

[0359] Table 2. Determined affinity of X-configuration Trastuzumab antibody

[0360]

[0361] ND: Undetectable

[0362] 2. Expression, purification and functional characterization of trans-X conformation Trastuzumab antibody

[0363] To verify whether the Fc domains of different immunoglobulin classes attached to the antibody light and heavy chains affect the assembly and biological function of X-configuration antibodies, this section constructs an X-configuration antibody molecule with an IgA Fc peptide intercalated on the antibody heavy chain and an IgG Fc peptide intercalated on the antibody light chain. For the purpose of distinguishing it from the X-configuration antibody molecule constructed in the previous examples with an IgG Fc peptide intercalated on the antibody heavy chain and an IgA Fc peptide intercalated on the antibody light chain, the antibody constructed in this example is called a trans-X-configuration antibody (RX-body), but it still possesses the same functional characteristics as... Figure 1 The X configuration described in B. Therefore, the qualifier "trans" in the description "trans X configuration" cannot be interpreted as a denial of the overall configuration structure of the antibody.

[0364] In this embodiment, an X-configuration antibody with an IgA Fc polypeptide in the heavy chain and an IgG Fc polypeptide in the light chain was constructed based on Trastuzumab antibody, and its assembly capability and function were verified.

[0365] 2.1. Constructing genes encoding the heavy chain and chimeric light chain of the trans-X configuration Trastuzumab antibody.

[0366] Based on the publicly available light and heavy chain sequences of IgG-type Trastuzumab antibodies, a heavy chain gene sequence for IgA1-type Trastuzumab antibodies was designed. This gene sequence sequentially contains the VH-CH1-IgA1 hinge region-IgA1Fc sequence of the Trastuzumab antibody. Figure 8 C). On the other hand, a gene sequence for a chimeric light chain of a trans-X conformation Trastuzumab antibody was designed, sequentially comprising the light chain sequence of the Trastuzumab antibody, a 6×G4S linker, an IgG1 hinge region, and an IgG1 Fc sequence. Figure 8 C).

[0367] The heavy chain gene and chimeric light chain gene of the trans-X conformation Trastuzumab antibody were synthesized separately (contracted to Suzhou Genewiz Biotechnology Co., Ltd.). Following the method described in Example 1, they were constructed into mammalian cell expression plasmids. The coding genes in the obtained plasmids were sequenced for verification, thus confirming the correct coding gene sequence. The heavy chain amino acid sequence of the trans-X conformation Trastuzumab antibody encoded by the gene is shown below:

[0368] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGK(SEQ ID NO: 7)

[0369] The encoded chimeric light chain amino acid sequence is shown below:

[0370] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 8).

[0371] 2.2. Expression and purification of trans-X conformation Trastuzumab antibody

[0372] The expression and purification methods for the trans-X configuration of Trastuzumab antibody were as described in Example 1. The trans-X configuration of Trastuzumab antibody, purified by Protein A affinity chromatography and further purified by molecular sieve, showed a major band at approximately 200 kDa in non-reducing SDS-PAGE assays. Figure 8 (Indicated by arrow D). During purification using Superdex 200 increase 10 / 300GL molecular sieve chromatography, the main UV (UV280) absorption peak of the trans-X configuration Trastuzumab antibody was located at approximately 11.5 ml, corresponding to a molecular weight of approximately 200 kDa. This data demonstrates that the trans-X configuration Trastuzumab antibody expressed in this embodiment can also assemble in tetrameric form, i.e., form the desired cloverleaf X configuration.

[0373] Compared with the X-configuration Trastuzumab antibody described in this embodiment ( Figure 8 B), the molecular sieve chromatography peak shape diagram shows that the trans-X configuration Trastuzumab antibody purified by Protein A affinity chromatography contains a large number of polymeric components (B). Figure 8D). This may be because the trans-X configuration antibody in this embodiment uses the IgA1 hinge region containing multiple natural glycosylation sites, which affects antibody assembly due to steric hindrance, thus resulting in lower assembly efficiency compared to assembly using the IgA2 hinge region.

[0374] 2.3. Recognition and binding of trans-X configuration Trastuzumab antibody to antigens and corresponding receptors

[0375] Following the method described in Example 4, the binding of the trans-X configuration Trastuzumab antibody to the antigen (Her2), receptor FcαRI (CD89), and the important receptor FcγRIIIa (F158 type) of IgG Fc was detected using SPR technology.

[0376] The results are shown in Table 3 below. The affinity of the trans-X configuration Trastuzumab antibody for antigen Her2 was at the same level as that of the control IgA or IgG antibodies described in this example. Compared with the IgA antibody, the trans-X configuration Trastuzumab antibody showed a higher affinity for CD89. However, compared with the IgG antibody, although the trans-X configuration Trastuzumab antibody showed affinity for FcγRIIIa, the affinity was lower, only slightly higher than one-tenth of that of the IgG antibody.

[0377] Table 3. Determined affinity of trans-X configuration Trastuzumab antibody

[0378]

[0379] ND: Undetectable

[0380] The hinge region of natural IgA1 (VPSTPPTPSPSTPPTPSPS, SEQ ID NO:17) contains multiple glycosylation sites, while the hinge region of natural IgA2 (VPPPPP, SEQ ID NO:18) lacks glycosylation sites. The lower receptor affinity exhibited by the trans-X configuration Trastuzumab antibody may be due to the steric hindrance of site glycosylation in the IgA1 hinge region affecting the antibody-receptor interaction; however, the Fab region, which binds the antigen, is unaffected because it is far from the IgA1 hinge region.

[0381] In summary, the construction method of embedding IgA Fc peptides on the antibody heavy chain and IgG Fc peptides on the antibody light chain can also form antibody molecules that exist mainly in the X configuration. The IgA1 hinge region has no effect on the function of the Fab and IgA Fc domains, but it significantly affects the binding of the IgG Fc domain to FcγR, demonstrating that the choice of the antibody hinge region is closely related to the effective functioning of each structural part of the antibody molecule. Therefore, when FcγR is required to participate in the corresponding function, the IgA2 hinge region is preferred, even when using IgA1 Fc; when applied to specific scenarios where FcγR participation is undesirable, the IgA1 hinge region is preferred.

[0382] 3. Lacking a linker, it is difficult to form X-configuration antibodies.

[0383] The foregoing embodiments demonstrate that linkers within a certain length range in the chimeric light chain have a relatively small impact on the formation of the X-configuration antibody. This section demonstrates that this specific length of linker is an essential element for the assembly of the X-configuration antibody.

[0384] 3.1. Construct a gene encoding a chimeric light chain of a Trastuzumab antibody without a linker.

[0385] Design a gene sequence containing a chimeric light chain sequentially comprising the light chain sequence of Trastuzumab antibody, the IgG1 hinge region, and the IgG1 Fc sequence. Figure 8 E), that is, removing the linker from the above-mentioned trans-X configuration Trastuzumab antibody chimeric light chain.

[0386] The gene for the chimeric light chain of the Trastuzumab antibody without the linker was synthesized (commissioned by Suzhou Genewiz Biotechnology Co., Ltd.). Following the method described in Example 1, it was constructed into a mammalian cell expression plasmid. The coding gene in the obtained plasmid was sequenced for verification, thus confirming the correct sequence of the coding gene. The amino acid sequence of the chimeric light chain encoded by the gene is shown below:

[0387] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 23).

[0388] 3.2. Expression and purification of trastuzumab antibody without linker

[0389] Following the method described in Example 1, the adapter-free Trastuzumab antibody composed of SEQ ID NO: 7 and SEQ ID NO: 23 was expressed and purified. After purification by Protein A affinity chromatography, the adapter-free Trastuzumab antibody showed no obvious main band in non-reducing SDS-PAGE assay, especially at approximately 200 kDa. Figure 8 (indicated by arrow F) No obvious bands ( Figure 8 (F). This result demonstrates that trastuzumab antibodies without a linker cannot assemble in tetrameric form, i.e., form the desired cloverleaf X configuration. Compared to the trans-X configuration trastuzumab antibody, the lack of a linker in the chimeric light chain alone prevents the formation of the X configuration, indicating that the linker is an indispensable element for the correct assembly of X-configuration antibodies.

[0390] As can be seen from all the data in this embodiment, the design of the chimeric light chain cannot simply link the antibody Fab light chain to the Fc via the antibody hinge region. The presence or absence of a linker between the antibody light chain and the Fc plays a decisive role in whether the X-configuration antibody can be formed. The choice of the antibody hinge region is crucial to whether each structural part of the X-configuration antibody can function properly.

[0391] Example 9. Tumor cell killing effect of X-configuration Trastuzumab antibody using neutrophils as effector cells

[0392] Trastuzumab is a recombinant monoclonal antibody that specifically targets the Her2 antigen and exerts its effects through multiple effector functions. SKBR3 cells (ATCC catalog number: HTB-30), as human breast cancer cells, naturally possess Her2 molecules on their cell surface, and therefore can serve as target cells for Trastuzumab antibody to detect its effector functions.

[0393] The X-configuration Trastuzumab antibody used in this embodiment is the X-configuration Trastuzumab antibody described in Part 1 of Example 8. The IgG and IgA Trastuzumab antibodies used as controls are also the IgG and IgA Trastuzumab antibodies used as controls in Example 8. The experimental method is the same as the method described in Example 5 for detecting the tumor cell killing effect of the X-configuration Rituximab antibody using neutrophils as effector cells. The only difference is that the culture medium used to culture SKBR3 cells is McCoy's 5a medium (Gibco). TM (Item No.: 16600-082).

[0394] Test results are as follows Figure 9 As shown, the ADCC killing effect of neutrophils as effector cells on SKBR3 cells increases with increasing antibody concentration, demonstrating that this killing effect is antibody dose-dependent. The killing effect of X-configuration Trastuzumab antibody on SKBR3 cells is higher than that of IgG-type Trastuzumab antibody at all concentrations, and also higher than that of IgA-type Trastuzumab antibody at most concentrations. These results demonstrate that the X-configuration antibody can effectively recruit neutrophils as effector cells to kill tumor cells.

[0395] Example 10. Tumor suppressive activity of X-configuration Trastuzumab antibody in animals.

[0396] This embodiment demonstrates in animal studies that the X-configuration antibody exhibits superior activity compared to IgG and IgA antibodies in inhibiting solid tumors.

[0397] 1. Inhibitory effect of X-configuration Trastuzumab antibody on MC38-hHer2 tumors

[0398] In humanized FcαRI transgenic mice (Nanmo Biotechnology, catalog number: NM-KI-200063), humanized FcαRI receptors are expressed on the surface of monocytes (including macrophages) and granulocytes (including neutrophils), which can bind to antibodies carrying human IgA Fc and act as effector cells to kill target cells. On the other hand, the MC38-hHer2 cell line is a stable transfected cell line with high expression of human Her2 antigen on its cell surface. This cell line was developed by the inventors using conventional methods in the mouse MC38 cell line (Cellonco...). TM Based on the cell line obtained by introducing the human Her2 gene using lentivirus (catalog number: IOC-ZP093), this example uses the same antibody as in Example 9 for experiments.

[0399] MC38-hHer2 cells in the logarithmic growth phase were digested and counted. Approximately 5 × 10⁶ MC38-hHer2 cells were subcutaneously inoculated into the backs of approximately 11-week-old humanized FcαRI transgenic mice after shaving their backs. 5 One tumor per animal. Tumor size is monitored one week after inoculation, and is maintained when the tumor reaches approximately 50 mm in size. 3 At approximately 10 days, mice were randomly divided into 4 groups of 5 mice each. Each group received an intraperitoneal injection of either X-configuration Trastuzumab antibody, IgG or IgA type Trastuzumab antibody, or PBS as a negative control. The antibody concentration was 10 mg / kg, administered every three days for a total of 3 injections. Tumor size was measured every three days, and the tumor volume was calculated using the formula: length × width. 2 ×0.5. When the tumor size in mice exceeds 2000 mm. 3 In accordance with animal welfare and ethical requirements, the mice were euthanized.

[0400] Tumor size measurements showed that, compared to PBS injection, injection of X-configuration Trastuzumab antibody significantly (statistically significant) delayed tumor growth. Figure 10 A). Meanwhile, the tumor suppressor activity of the X-configuration Trastuzumab antibody was significantly (statistically significant) higher than that of the IgG and IgA-configuration Trastuzumab antibodies. Figure 10 A). The corresponding mouse survival curves also showed that, compared with tumor-bearing mice injected with IgG or IgA type Trastuzumab antibodies, tumor-bearing mice injected with X configuration Trastuzumab antibodies had a significantly (statistically significant) longer survival time. Figure 10 B).

[0401] 2. Inhibitory effect of X-configuration Trastuzumab antibody on MB49-hHer2 malignant tumors

[0402] The MB49-hHer2 cell line is a stable transfected cell line that highly expresses the human Her2 antigen on its cell surface. This cell line was obtained by the inventors using conventional methods, by introducing the human Her2 gene into the murine malignant tumor MB49 cell line (Merck, catalog number: SCC148) using lentivirus.

[0403] Following the method described above for detecting the inhibitory effect of X-configuration Trastuzumab antibody on MC38-hHer2 tumors, the inventors investigated the inhibitory effect of X-configuration Trastuzumab antibody on MB49-hHer2 malignant tumors in humanized FcαRI transgenic mice (Nanmo Biotechnology, catalog number: NM-KI-200063). The results showed that compared to PBS injection, injection of X-configuration Trastuzumab antibody significantly (statistically significant) delayed tumor growth. Figure 10 C). Meanwhile, the X-configuration Trastuzumab antibody was able to delay tumor growth better than IgG and IgA Trastuzumab antibodies, especially the X-configuration Trastuzumab antibody showed a significantly (statistically significant) tumor-suppressive effect compared to the IgA Trastuzumab antibody. Mouse survival curves also showed that tumor-bearing mice injected with the X-configuration Trastuzumab antibody had a longer median survival time compared to those injected with PBS or IgG and IgA Trastuzumab antibodies. Figure 10 D).

[0404] In summary, these results demonstrate that X-configuration antibodies possess superior solid tumor inhibitory activity compared to other types of antibodies.

Claims

1. An X-configuration antibody molecule, comprising two identical antibody heavy chains and two identical antibody light chains, wherein the antibody heavy chains and antibody light chains respectively comprise the following structures: (1) The antibody heavy chain, from N-terminus to C-terminus, sequentially contains the antibody heavy chain variable region (VH) - CH1 - IgG hinge region - IgG Fc sequence, and the antibody light chain, from N-terminus to C-terminus, sequentially contains the antibody light chain variable region (VL) - light chain constant region (CL) - linker - IgA hinge region - IgA Fc sequence; or (2) The antibody heavy chain contains the antibody heavy chain variable region (VH)-CH1-IgA hinge region-IgA Fc sequence from N-terminus to C-terminus, and the antibody light chain contains the antibody light chain variable region (VL)-light chain constant region (CL)-adaptor-IgG hinge region-IgG Fc sequence from N-terminus to C-terminus. in, The VH and CH1 of a heavy chain interact with the VL and CL of a light chain to assemble into corresponding Fabs, thus forming two Fabs with the same antigen binding site in the X-configuration antibody molecule. Two identical IgG Fc sequences dimerize to form an IgG Fc region; two identical IgA Fc sequences dimerize to form an IgA Fc region. The linker is a polypeptide sequence of 15-40 amino acid residues in length and with a certain degree of flexibility, without a specific higher-order structure.

2. The X-configuration antibody molecule of claim 1, wherein the IgG Fc sequence is selected from any Fc sequence of the IgG1, IgG2, IgG3 and IgG4 subclasses; wherein the IgA Fc sequence is selected from any sequence of the IgA subclass.

3. The X-configuration antibody molecule of claim 2, wherein the IgG Fc sequence comprises a modification; and the IgA Fc sequence comprises a modification.

4. The X-configuration antibody molecule of claim 2 or 3, wherein CH2 and CH3 of the IgG Fc sequence are derived from the same Fc sequence but different Fc sequences; and CH2 and CH3 of the IgA Fc sequence are derived from the same Fc sequence but different Fc sequences.

5. The X-configuration antibody molecule of any one of claims 1-2, wherein the IgG Fc sequence is the Fc sequence of IgG1.

6. The X-configuration antibody molecule of claim 5, wherein the IgG Fc comprises the Fc sequence shown in SEQ ID NO:

21.

7. The X-configuration antibody molecule of claim 5, wherein the IgG Fc comprises an Fc sequence having at least 90% identity with the Fc sequence shown in SEQ ID NO:

21.

8. The X-configuration antibody molecule of any one of claims 1-2, wherein the IgA Fc is selected from the IgA1 Fc sequence or the IgA2 Fc sequence.

9. The X-configuration antibody molecule of claim 8, wherein the IgA Fc comprises the Fc sequence shown in SEQ ID NO:

22.

10. The X-configuration antibody molecule of claim 8, wherein the IgA Fc comprises an Fc sequence having at least 90% identity with the Fc sequence shown in SEQ ID NO:

22.

11. The X-configuration antibody molecule of claim 1, wherein the hinge region and the Fc polypeptide sequence linked thereto are homologous or heterologous.

12. The X-configuration antibody molecule of claim 1, wherein the linker is selected from the sequence (G4S)n or shown in SEQ ID NO:16, wherein n is an integer equal to or greater than 1.

13. The X-configuration antibody molecule of claim 1 or 11, wherein the hinge region is selected from SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:

20.

14. The X-configuration antibody molecule of claim 1 or 2, wherein the CH1 and / or CL and the VH and / or VL linked thereto are homologous or heterologous, or the CH1 and / or CL and the Fc sequence linked thereto are homologous or heterologous, or the CH1 and / or CL are known common CH1 and CL.

15. The X-configuration antibody molecule of claim 1 or 2, wherein... (1) Antibody VH contains the three heavy chain CDRs shown in SEQ ID NO:44, and antibody VL contains the three light chain CDRs shown in SEQ ID NO:45, wherein antibodies VH and VL specifically recognize the CD20 antigen; (2) Antibody VH contains the three heavy chain CDRs shown in SEQ ID NO:46, and antibody VL contains the three light chain CDRs shown in SEQ ID NO:47, wherein antibodies VH and VL specifically recognize the CD19 antigen; or (3) Antibody VH contains the three heavy chain CDRs shown in SEQ ID NO:48, and antibody VL contains the three light chain CDRs shown in SEQ ID NO:49, wherein antibodies VH and VL specifically recognize Her2 antigen.

16. The X-configuration antibody molecule of claim 1 or 2, wherein (1) Antibody VH comprises VH CDR1 shown in SEQ ID NO:26, VH CDR2 shown in SEQ ID NO:27 and VH CDR3 shown in SEQ ID NO:28, and antibody VL comprises VL CDR1 shown in SEQ ID NO:29, VLCDR2 shown in SEQ ID NO:30 and CDR3 shown in SEQ ID NO:31, wherein the antibodies VH and VL specifically recognize the CD20 antigen; (2) Antibody VH comprises VH CDR1 shown in SEQ ID NO: 32, VH CDR2 shown in SEQ ID NO: 33, and VH CDR3 shown in SEQ ID NO: 34, and antibody VL comprises VL CDR1 shown in SEQ ID NO: 35, VL CDR2 shown in SEQ ID NO: 36, and VL CDR3 shown in SEQ ID NO: 37, wherein antibodies VH and VL specifically recognize the CD19 antigen; or (3) The antibody VH comprises VH CDR1 shown in SEQ ID NO: 38, VH CDR2 shown in SEQ ID NO: 39 and VH CDR3 shown in SEQ ID NO: 40, and the antibody VL comprises VL CDR1 shown in SEQ ID NO: 41, VL CDR2 shown in SEQ ID NO: 42 and VL CDR3 shown in SEQ ID NO: 43, wherein the antibodies VH and VL specifically recognize the Her2 antigen.

17. The X-configuration antibody molecule of claim 15 or 16, wherein... (1) Antibody VH contains the sequence shown in SEQ ID NO:44, and antibody VL contains the sequence shown in SEQ ID NO:45; (2) Antibody VH contains the sequence shown in SEQ ID NO: 46, and antibody VL contains the sequence shown in SEQ ID NO: 47; or (3) Antibody VH contains the sequence shown in SEQ ID NO:48, and antibody VL contains the sequence shown in SEQ ID NO:

49.

18. The X-configuration antibody molecule of claim 15 or 16, wherein (1) Antibody VH contains a sequence that is at least 90% identical to SEQ ID NO:44, and antibody VL contains a sequence that is at least 90% identical to SEQ ID NO:45; (2) Antibody VH contains a sequence having at least 90% identity with SEQ ID NO:46, and antibody VL contains a sequence having at least 90% identity with SEQ ID NO:47; or (3) Antibody VH contains a sequence that is at least 90% identical to SEQ ID NO:48, and antibody VL contains a sequence that is at least 90% identical to SEQ ID NO:

49.

19. The X-configuration antibody molecule of claim 15 or 16, wherein (1) The antibody heavy chain contains the sequence shown in SEQ ID NO:1 and the antibody light chain contains the sequence shown in SEQ ID NO:2; (2) The antibody heavy chain contains the sequence shown in SEQ ID NO:3 and the antibody light chain contains the sequence shown in SEQ ID NO:4; (3) The antibody heavy chain comprises the sequence shown in SEQ ID NO:5, and the antibody light chain comprises the sequence shown in SEQ ID NO:6; or (4) The antibody heavy chain contains the sequence shown in SEQ ID NO:7 and the antibody light chain contains the sequence shown in SEQ ID NO:

8.

20. The X-configuration antibody molecule of claim 15 or 16, wherein... (1) The antibody heavy chain contains a sequence that has at least 90% identity with SEQ ID NO:1, and the antibody light chain contains a sequence that has at least 90% identity with SEQ ID NO:2; (2) The antibody heavy chain contains a sequence having at least 90% identity with SEQ ID NO:3, and the antibody light chain contains a sequence having at least 90% identity with SEQ ID NO:4; (3) The antibody heavy chain contains a sequence having at least 90% identity with SEQ ID NO:5, and the antibody light chain contains a sequence having at least 90% identity with SEQ ID NO:6; or (4) The antibody heavy chain contains a sequence that is at least 90% identical to SEQ ID NO:7, and the antibody light chain contains a sequence that is at least 90% identical to SEQ ID NO:

8.

21. A method for preparing an X-configuration antibody molecule as described in any one of claims 1-20, comprising: (1) Obtain the coding sequences or amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody molecule. (2) Construct the corresponding X-configuration antibody heavy chain sequence and light chain sequence of the X-configuration antibody molecule as described in any one of claims 1-20. (3) Under suitable conditions, express the antibody heavy chain and light chain sequences obtained in step 2, wherein two heavy chain polypeptides and two light chain polypeptides are assembled into X-configuration antibody in the form of tetramers, wherein VH-CH1 and VL-CL are assembled to form the corresponding Fab, two identical IgG Fc sequences are dimerized to form IgG Fc region; two identical IgA Fc sequences are dimerized to form IgA Fc region, thereby obtaining the corresponding X-configuration antibody molecule; (4) Optionally, the X-configuration antibody molecule is purified.

22. A method for modifying a monoclonal antibody into an X-configuration antibody molecule as described in any one of claims 1-20, comprising: (1) The light chain sequence of the monoclonal antibody is modified into an X-configuration antibody light chain sequence with the structure VL-CL-linker-IgA hinge region-IgA Fc sequence, and the heavy chain sequence of the antibody is modified into an X-configuration antibody heavy chain sequence with the structure VH-CH1-IgG hinge region-IgG Fc sequence, or The light chain sequence of the monoclonal antibody was modified into an X-configuration antibody light chain sequence with the structure VL-CL-linker-IgG hinge region-IgG Fc sequence, and the heavy chain sequence of the monoclonal antibody was modified into an X-configuration antibody heavy chain sequence with the structure VH-CH1-IgA hinge region-IgA Fc sequence. (2) Under suitable conditions, express the X-configuration antibody light chain and heavy chain obtained in step (1), wherein the two heavy chain polypeptides and the two chimeric light chain polypeptides are assembled into X-configuration antibody in the form of tetramers, wherein VH-CH1 and VL-CL are assembled to form the corresponding Fab, and the same Fc sequence contained in the two heavy chains is dimerized; the same Fc sequence contained in the two X-configuration antibody light chains is dimerized, thereby obtaining the corresponding X-configuration antibody molecule; (3) Optionally, the X-configuration antibody molecule is purified.

23. A pharmaceutical composition comprising an X-configuration antibody molecule of any one of claims 1-20 and a pharmaceutically acceptable carrier.

24. Use of the X-configuration antibody molecule of any one of claims 1-20 in the preparation of a medicament for treating cancer in a subject.

Images