Molecule binding to amino acid linker and use thereof

Abstract

The present application relates to a monoclonal antibody specifically binding to the amino acid linker G4S, and the use of the antibody in the detection of the amino acid linker G4S or in the construction of a bispecific antibody and a universal CAR-T composition.

Description

Amino acid linker binding molecules and uses thereof

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese patent application number CN202311692347.2, filed on December 8, 2023, the entire contents of which are incorporated herein by reference. Field of the Invention

[0003] The present application relates to a molecule that specifically binds to an amino acid linker, and the use of the molecule in detecting amino acid linkers, or constructing bispecific antibodies and universal CAR-T compositions. Background Art

[0004] In recombinant biomacromolecules, linkers are often used to connect two or more functional modules to form a single, integrated structure. For example, linkers can be used to connect proteins, nucleic acids, and / or polypeptides to achieve specific functional combinations or enhancements. In some cases, linkers also serve to separate two or more functional modules, allowing each module to perform its own function without interfering with each other.

[0005] The choice of linker for recombinant biomacromolecules depends primarily on the type of molecule to be connected, the stability requirements of the connection, and the specific needs of the subsequent application. Amino acid linkers, such as the glycine-serine linker (i.e., GS linker), are among the most commonly used linkers. They can be designed in different sequence combinations and lengths in the form of (GnS)m, where n and m can be integers as low as 1. (G2S)2, (G2S)4, (G3S)3, (G4S)1, (G4S)2, and (G4S)3 are some of the more commonly used GS linkers, often used to connect different functional domains of proteins or peptides, and they are characterized by flexibility and scalability. These linker sequences are widely used in many research fields, such as protein engineering and drug delivery systems.

[0006] GS linkers are commonly used to connect the heavy chain variable region (VH) and light chain variable region (VL) in single-chain variable fragments (scFvs). Compared to classic IgG antibody molecules, scFvs offer advantages such as smaller molecular weight, stronger penetrating power, lower immunogenicity, and higher specificity. Consequently, they are attracting increasing attention in applications such as targeted therapy, diagnostic imaging, and cell therapy. In targeted therapy, drugs or toxins can be linked to scFvs, leveraging their specificity and low immunogenicity to deliver the drugs to target tissues, thereby achieving specific killing of target cells. In diagnostic imaging, due to their strong penetrating power and short half-life, scFvs exhibit a higher distribution index in tumor tissue during radiological imaging compared to full-length antibodies, resulting in less harm to the body. In cell therapy, scFvs, as the extracellular domain of chimeric antigen receptor T cells (CAR-T), determine the targeting and efficacy of CAR-T cells. In biomonitoring, due to their high affinity and ease of preparation, scFvs are often used to monitor hazardous substances in water and food. In addition, due to its simple structure, scFv is often used in the construction of bispecific antibodies.

[0007] GS linkers are also commonly used to connect two or more nanobodies to improve half-life and single-target recognition specificity, or to construct bispecific or multispecific antibodies targeting two or more antigens to better target certain cells in a specific environment, such as tumor cells in the tumor microenvironment. CARs also contain two or more nanobodies connected by GS linkers in the extracellular domain to reduce the risk of off-target effects.

[0008] Many recombinant proteins containing GS linkers, such as the aforementioned scFv, or CARs or multispecific antibodies containing two or more nanobodies, do not contain an Fc region, making it impossible to use the most commonly used universal Fc-targeted antibodies to detect the presence or quantify these recombinant antibodies. Currently, protein L and anti-Fab antibodies are commonly used to detect proteins containing scFv, but they all have certain limitations. For example, protein L mainly targets the light chain of the κ subtype and cannot detect other subtypes, and has problems such as high non-specificity and low detection rate. Anti-Fab antibodies are mostly polyclonal antibodies with large batch-to-batch variability, and have problems such as low versatility and poor specificity. Therefore, other highly specific and versatile methods are needed to detect scFv antibodies and the like. Summary of the Invention

[0009] The inventors of the present application have prepared and screened antibodies that specifically bind to GS connectors. These antibodies can locate, quantify, or amplify signals of proteins containing GS connectors by specifically binding to proteins such as scFv and chimeric antigen receptors (CARs), thereby detecting the expression, metabolism, and distribution of proteins containing GS connectors. Compared to traditional methods using protein L, anti-Fab antibodies, target antigens, antibodies targeting idiotypic antibodies, etc., the antibodies of the present application i) have certain versatility, that is, they are applicable to most proteins containing GS connectors, without considering the target of the protein, the subtype of the Fc region contained in the protein, the subtype of the antibody light chain, or the form of the antibody (single domain antibody or scFv, etc.), and are applicable to most GS connectors except (G4S) 1, ii) have higher binding specificity for proteins containing GS connectors, and the detection accuracy is higher than that of the protein L detection method. The clustering ability of CAR-T cells based on scFv containing GS connectors is stronger than that of anti-Fab antibodies, and iii) have higher binding sensitivity to proteins containing GS connectors, and can detect proteins containing 2pmol of GS connectors.

[0010] In addition, the GS antibody of the present application can also be used for the construction of universal CAR-T cells. Specifically, the GS antibody of the present application or its antigen-binding portion thereof such as scFv form (referred to as anti-GS-CAR) can be included in the extracellular domain of CAR, and an antibody or its antigen-binding portion thereof with a GS linker and targeting disease-associated antigens (referred to as disease-targeted antibody) can be constructed. In this way, T cells expressing anti-GS-CAR can recognize cells expressing disease-associated antigens through disease-targeted antibodies and trigger an attack on cells expressing disease-associated antigens. Alternatively, a GS linker (referred to as GS linker-CAR) can be included in the extracellular domain of CAR, and a bispecific molecule (referred to as GS linker+disease dual-targeted antibody) comprising the GS antibody of the present application or its antigen-binding portion thereof and an antibody or its antigen-binding portion thereof targeting disease-associated antigens can be constructed. In this way, T cells expressing GS linker-CAR can recognize cells expressing disease-associated antigens through GS linker+disease dual-targeted antibody and trigger an attack on cells expressing disease-associated antigens. Constructing the above-mentioned universal CAR-T cells and collocating corresponding disease-targeted antibodies or GS linkers+disease dual-targeted antibodies can reduce the cost of cell therapy to a certain extent.

[0011] In addition, the GS antibody of the present application can also be used for the purification of proteins containing GS linkers. Specifically, the antibody of the present invention can be coupled to a solid phase carrier, and the solid phase carrier includes magnetic beads, resins, agarose beads, etc. The prokaryotic or eukaryotic lysate or cell culture supernatant of the protein containing the GS linker is contacted with the solid phase carrier containing the GS antibody. At this time, the protein containing the GS linker will be specifically captured by the GS antibody, and then the foreign proteins that do not bind to the GS antibody are washed away. Finally, the protein containing the GS linker is separated from the GS antibody by changing the pH value, salt ion concentration, etc., and a protein containing the GS linker with higher purity can be obtained. This method provides a new purification strategy for proteins containing GS linkers, which simplifies the purification process of such proteins.

[0012] Thus, in a first aspect, the present application provides an isolated monoclonal antibody or an antigen-binding portion thereof that can specifically bind to the GS linker. The antibody or antigen-binding portion thereof of the present application can be rabbit-derived, chimeric, or humanized.

[0013] The antibodies or antigen-binding portions thereof of the present application may comprise i) a heavy chain variable region, which may comprise VH CDR1, VH CDR2 and VH CDR3, wherein VH CDR1, VH CDR2 and VH CDR3 may respectively comprise (1) NYAIM (SEQ ID NO: 1), VIYANGDPYCASWAKG (SEQ ID NO: 5), and GGF; (2) NYDMY (SEQ ID NO: 2), VSYKNGRAHYASWAKG (SEQ ID NO: 8), and GPL; (3) NYDMY (SEQ ID NO: 2), VSYASGRTYSVRWAKG (SEQ ID NO: 10), and GPL; (4) NYDMY (SEQ ID NO: 2), VIYKNGNAHSASWARG (SEQ ID NO: 12), and GPF; (5) NHAIM (SEQ ID NO: 3), VIYSNGNPYCARWVKG (SEQ ID NO: 7), and GGF; (6) NYDMY (SEQ ID NO: 2), VSYKNGRAHYASWAKG (SEQ ID NO: 8), and GPL; NO: 2), VIYVNGNTHYASWAKG (SEQ ID NO: 6), and GPF; (7) NYDMY (SEQ ID NO: 2), CIYTGSGGKRYANWAKG (SEQ ID NO: 9), and GPF; or (8) TNAMT (SEQ ID NO: 4), TITISGNKYYASWAKG (SEQ ID NO: 11), and GVVQSLVL (SEQ ID NO: 13); or ii) a light chain variable region, which light chain variable region may contain VL CDR1, VL CDR2 and VL CDR3, wherein VL CDR1, VL CDR2 and VL CDR3 may respectively contain (1) QSSQSTYNKNDLV (SEQ ID NO: 14), GISTLDS (SEQ ID NO: 20), and LGGFSCSSGDCGA (SEQ ID NO: 25); (2) QASQSVWNNNDLV (SEQ ID NO: 17), DASTLSS (SEQ ID NO: 18), and LGGFSCSSGDCGA (SEQ ID NO: 29); NO: 21), and LGGFSCSTGDCGA (SEQ ID NO: 28); (3) QASQTVWKNNDLV (SEQ ID NO: 18), DASTLSS (SEQ ID NO: 21), and LGGFSCSSGDCGA (SEQ ID NO: 25); (4) QASQSVWNNNDLV (SEQ ID NO: 17), DASTLSS (SEQ ID NO: 25); NO: 21), and LGGFSCSRGDCGS (SEQ ID NO: 31);(5) KTSQSTYNNNDLV (SEQ ID NO: 16), GVSTLDS (SEQ ID NO: 22), and LGGFRCSSGDCGA (SEQ ID NO: 27); (6) QASQSVWKNKDLV (SEQ ID NO: 15), DASTLSS (SEQ ID NO: 21), and LGGFSCSRGDCGA (SEQ ID NO: 26); (7) QASQSVWNNNDLV (SEQ ID NO: 17), DASILSS (SEQ ID NO: 23), and LGGFSCRSGDCGA (SEQ ID NO: 29); or (8) RSSQNVYNNNGLG (SEQ ID NO: 19), DAADLAS (SEQ ID NO: 24), and AGGYSSGSIDNT (SEQ ID NO: 30). Variants of the above antibodies or antigen-binding portions are also provided, comprising up to about three amino acid residue substitutions, e.g., one, two, or three amino acid residue substitutions, in each CDR compared to the above antibodies or antigen-binding portions thereof. ;

[0014] The isolated monoclonal antibody or antigen-binding portion thereof of the present application can comprise a heavy chain variable region and a light chain variable region, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 can respectively comprise (1) NYAIM (SEQ ID NO: 1), VIYANGDPYCASWAKG (SEQ ID NO: 5), GGF, QSSQSIYNKNDLV (SEQ ID NO: 14), GISTLDS (SEQ ID NO: 20), and LGGFSCSSGDCGA (SEQ ID NO: 25); (2) NYDMY (SEQ ID NO: 2), VSYKNGRAHYASWAKG (SEQ ID NO: 8), GPL, QASQSVWNNNDLV (SEQ ID NO: 17), DASTLSS (SEQ ID NO: 21), and LGGFSCSTGDCGA (SEQ ID NO: 28); (3) NYDMY (SEQ ID NO: 2), VSYASGRTYSVRWAKG (SEQ ID NO: 30), GGF, QSSQSIYNKNDLV (SEQ ID NO: 31), GISTLDS (SEQ ID NO: 32), and LGGFSCSSGDCGA (SEQ ID NO: 33); NO: 10), GPL, QASQTVWKNNDLV (SEQ ID NO: 18), DASTLSS (SEQ ID NO: 21), and LGGSCSSSGDCGA (SEQ ID NO: 25); (4) NYDMY (SEQ ID NO: 2), VIYKNGNAHSASWARG (SEQ ID NO: 12), GPF, QASQSVWNNNDLV (SEQ ID NO:17), DASTLSS (SEQ ID NO:21), and LGGFSCSRGDCGS (SEQ ID NO:31); (5) NHAIM (SEQ ID NO:3), VIYSNGNPYCARWVKG (SEQ ID NO:7), GGF, KTSQSIYNNNDLV (SEQ ID NO:16), GVSTLDS (SEQ ID NO:22), and LGGFRCSSGDCGA (SEQ ID NO:22) ID NO: 27); (6) NYDMY (SEQ ID NO: 2), VIYVNGNTHYASWAKG (SEQ ID NO: 6), GPF, QASQSVWKNKDLV (SEQ ID NO: 15), DASTLSS (SEQ ID NO: 21), and LGGFSCSRGDCGA (SEQ ID NO: 26);(7) NYDMY (SEQ ID NO: 2), CIYTGSGGKRYANWAKG (SEQ ID NO: 9), GPF, QASQSVWNNNDLV (SEQ ID NO: 17), DASILSS (SEQ ID NO: 23), and LGGFSCRSGDCGA (SEQ ID NO: 29); or (8) TNAMT (SEQ ID NO: 4), TITISGNKYYASWAKG (SEQ ID NO: 11), GVVQSLVL (SEQ ID NO: 13), RSSQNVYNNNGLG (SEQ ID NO: 19), DAADLAS (SEQ ID NO: 24), and AGGYSSGSIDNT (SEQ ID NO: 30). Also provided are variants of the above antibodies or antigen-binding portions thereof, comprising up to about three amino acid residue substitutions, e.g., one, two, or three amino acid residue substitutions, in each CDR compared to the above antibodies or antigen-binding portions thereof.

[0015] The heavy chain variable region of the antibody or antigen-binding portion thereof of the present application can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 32, 35, 37, 39, 34, 33, 36, or 38. The amino acid sequences of SEQ ID NO: 32, 35, 37, 39, 34, 33, 36, and 38 can be encoded by the nucleotide sequences set forth in SEQ ID NO: 48, 51, 53, 55, 50, 49, 53, and 54, respectively.

[0016] The light chain variable region of the antibody or antigen-binding portion thereof of the present application can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 40, 43, 45, 47, 42, 41, 44, or 46. The amino acid sequences of SEQ ID NO: 40, 43, 45, 47, 42, 41, 44, and 46 can be encoded by the nucleotide sequence set forth in SEQ ID NO: 56, 59, 61, 63, 58, 57, 60 or 62, respectively.

[0017] The antibodies or antigen-binding portions thereof of the present application may comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region may respectively comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to (1) SEQ ID NOs: 32 and 40; (2) SEQ ID NOs: 35 and 43; (3) SEQ ID NOs: 37 and 45; (4) SEQ ID NOs: 39 and 47; (5) SEQ ID NOs: 34 and 42; (6) SEQ ID NOs: 33 and 41; (7) SEQ ID NOs: 36 and 44; or (8) SEQ ID NOs: 38 and 46.

[0018] In some embodiments, the isolated monoclonal antibody or antigen-binding portion thereof of the present application can comprise a heavy chain variable region and a light chain variable region, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 can comprise the amino acid sequences set forth in NYAIM (SEQ ID NO: 1), VIYANGDPYCASWAKG (SEQ ID NO: 5), GGF, QSSQSIYNKNDLV (SEQ ID NO: 14), GISTLDS (SEQ ID NO: 20), and LGGFSCSSGDCGA (SEQ ID NO: 25), respectively. The heavy chain variable region can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 32. The light chain variable region can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:40.

[0019] In some embodiments, the isolated monoclonal antibody or antigen-binding portion thereof of the present application can comprise a heavy chain variable region and a light chain variable region, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 can comprise the amino acid sequences set forth in NYDMY (SEQ ID NO: 2), VSYKNGRAHYASWAKG (SEQ ID NO: 8), GPL, QASQSVWNNNDLV (SEQ ID NO: 17), DASTLSS (SEQ ID NO: 21), and LGGFSCSTGDCGA (SEQ ID NO: 28), respectively. The heavy chain variable region can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 35. The light chain variable region can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:43.

[0020] In some embodiments, the isolated monoclonal antibody or antigen-binding portion thereof of the present application can comprise a heavy chain variable region and a light chain variable region, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 can comprise the amino acid sequences set forth in NYDMY (SEQ ID NO: 2), VSYASGRTYSVRWAKG (SEQ ID NO: 10), GPL, QASQTVWKNNDLV (SEQ ID NO: 18), DASTLSS (SEQ ID NO: 21), and LGGFSCSSGDCGA (SEQ ID NO: 25), respectively. The heavy chain variable region can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 37. The light chain variable region can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:45.

[0021] In some embodiments, the isolated monoclonal antibody or antigen-binding portion thereof of the present application can comprise a heavy chain variable region and a light chain variable region, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 can comprise the amino acid sequences set forth in NYDMY (SEQ ID NO: 2), VIYKNGNAHSASWARG (SEQ ID NO: 12), GPF, QASQSVWNNNDLV (SEQ ID NO: 17), DASTLSS (SEQ ID NO: 21), and LGGFSCSRGDCGS (SEQ ID NO: 31), respectively. The heavy chain variable region can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 39. The light chain variable region can comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:47.

[0022] The isolated monoclonal antibody or antigen-binding portion thereof of the present application may comprise a heavy chain constant region and / or a light chain constant region, wherein the N-terminus of the heavy chain constant region is linked to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is linked to the C-terminus of the light chain variable region. The heavy chain constant region may be an IgG, IgD, IgA, IgM, or IgE heavy chain constant region, or a functional fragment thereof, such as a fragment comprising the hinge region, CH2, and CH3 of the heavy chain constant region. In one embodiment, the heavy chain constant region may comprise the amino acid sequence set forth in SEQ ID NO: 64. The light chain constant region may be a kappa or lambda light chain constant region. In some embodiments, the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO: 66. The amino acid sequences set forth in SEQ ID NOs: 64 and 66 may be encoded by the nucleotide sequences set forth in SEQ ID NOs: 65 and 67, respectively.

[0023] In some embodiments, the antibodies or antigen-binding portions thereof of the present application comprise two heavy chains and two light chains, or are composed of two heavy chains and two light chains, wherein each heavy chain comprises the aforementioned heavy chain constant region sequence, heavy chain variable region sequence, and / or CDR sequence, and each light chain comprises the aforementioned light chain constant region sequence, light chain variable region sequence, and / or CDR sequence. In some embodiments, the antibodies or antigen-binding portions thereof of the present application may be Fab, F(ab')2 fragment, Fv, scFv, or (scFv)2, etc.

[0024] In some embodiments, the antibody or antigen-binding portion thereof of the present application may be a full-length antibody. In some embodiments, the antibody or antigen-binding portion thereof of the present application may be a single-chain antibody (scFv).

[0025] The antibody or its antigen-binding portion thereof of the present application may include a detection substance. For example, the antibody or its antigen-binding portion thereof of the present application may be fused with a detection substance (e.g., via recombinant expression) or coupled with a detection substance. The antibody or its antigen-binding portion thereof of the present application may include a detection substance at any position that does not affect the antigen binding of the antibody or its antigen-binding portion thereof, such as the heavy chain constant region, the light chain constant region, the framework (FR) region of the heavy chain variable region, or the framework region of the light chain variable region. In particular, the antibody or its antigen-binding portion thereof may include a detection substance in the framework region of the heavy chain constant region or the light chain constant region. The detection substance may be a fluorescent marker or a chemiluminescent marker. The fluorescent marker may be fluorescein isothiocyanate (FITC), or phycoerythrin (PE), allophycocyanin (APC), or other dyes that can emit fluorescence under the irradiation of excitation light, such as iFluor, Alexa fluor, Bright violet dye, etc. The chemiluminescent marker may be a peroxidase (e.g., horseradish peroxidase (HRP)) or alkaline phosphatase (AP), etc.

[0026] The present application also provides a bispecific molecule comprising an antibody or antigen-binding portion thereof that specifically binds to a GS linker as described herein. For example, a bispecific molecule as described herein may comprise i) an antibody or antigen-binding portion thereof that specifically binds to a GS linker as described herein, and ii) an antibody or antigen-binding portion thereof that targets a disease-associated antigen. i) and ii) may be linked together, for example, by fusion through recombinant expression, or bound together via, for example, a disulfide bond or an amino group. The disease-associated antigen may be, for example, a tumor-associated antigen. In particular, in the bispecific molecule as described herein, the antibody or antigen-binding portion thereof that specifically binds to a GS linker as described herein may be humanized. In particular, in the bispecific molecule as described herein, the antibody or antigen-binding portion thereof that targets a disease-associated antigen may be humanized or humanized.

[0027] The present application also provides a chimeric antigen receptor (CAR), which may comprise a) an extracellular domain, which may comprise an antibody or antigen-binding portion thereof that specifically binds to a GS linker of the present application, particularly an antibody of the present application in the form of an scFv, b) a transmembrane region, and c) an intracellular signaling domain. The present application also provides immune cells, such as T cells, comprising the above-mentioned chimeric antigen receptor. In particular, in the chimeric antigen receptor of the present application, the antibody or antigen-binding portion thereof that specifically binds to a GS linker of the present application may be humanized.

[0028] The present application also includes nucleic acid molecules encoding the antibodies or antigen-binding portions thereof, bispecific molecules, or chimeric antigen receptors of the present application. The nucleic acid molecules of the present application may comprise the nucleotide sequence set forth in any one of SEQ ID NOs: 48-63. The present application may also provide an expression vector and a host cell. The expression vector may comprise the nucleic acid molecule of the present application. The host cell may comprise the expression vector of the present application or have the nucleic acid molecule of the present application integrated into its genome.

[0029] The application also provides a method for preparing the antibody or its antigen-binding portion thereof, bispecific molecule or chimeric antigen receptor using the host cell of the application, comprising: (i) expressing the antibody or its antigen-binding portion thereof, bispecific molecule or chimeric antigen receptor in the host cell, and (ii) isolating the antibody or its antigen-binding portion thereof, bispecific molecule or chimeric antigen receptor from the host cell or its culture. In some embodiments, the method can also include coupling the detection substance to the antibody or its antigen-binding portion thereof of the application. In particular, the method can also include contacting the detection substance with the antibody or its antigen-binding portion thereof of the application, so that the detection substance is coupled to the antibody or its antigen-binding portion thereof of the application.

[0030] The present application also provides a composition comprising the antibody or antigen-binding portion thereof, bispecific molecule, chimeric antigen receptor, nucleic acid molecule, expression vector, or host cell of the present application.

[0031] In some embodiments, the antibody or antigen-binding portion thereof of the present application may include a detection substance, such as a fluorescent marker or a chemiluminescent marker. For example, the antibody or antigen-binding portion thereof of the present application may be fused with a detection substance (e.g., via recombinant expression), or coupled with a detection substance. The antibody or antigen-binding portion thereof of the present application may include a detection substance at any position that does not affect the antigen binding of the antibody or its antigen-binding portion, such as the heavy chain constant region, the light chain constant region, the framework region of the heavy chain variable region, or the framework region of the light chain variable region. In particular, the antibody or antigen-binding portion thereof may include a detection substance in the framework region of the heavy chain constant region or the light chain constant region.

[0032] In some embodiments, the antibody or antigen-binding portion thereof of the present application may comprise a chemiluminescent label, and the composition may further comprise a reagent that causes the chemiluminescent label to emit light. In some embodiments, the chemiluminescent label may be horseradish peroxidase, and the reagent that causes the chemiluminescent label to emit light may be a peroxide (e.g., H2O2) and 3,3'-diaminobenzidine (DAB), or a peroxide (e.g., H2O2) and 3,3',5,5'-tetramethylbenzidine (TMB). In some embodiments, the chemiluminescent label may be alkaline phosphatase, and the reagent that causes the chemiluminescent label to emit light may be 4-nitrophenylphosphate disodium salt hexahydrate (pNPP).

[0033] In some embodiments, the antibody or its antigen-binding portion thereof of the present application comprises a heavy chain constant region (such as an Fc region), and the composition further comprises an antibody that specifically binds to the heavy chain constant region (such as an Fc region) and contains a detection substance. In some embodiments, the antibody or its antigen-binding portion thereof of the present application comprises a heavy chain constant region (such as an Fc region), and the composition further comprises an antibody that specifically binds to the heavy chain constant region (such as an Fc region) and contains a fluorescent marker. The fluorescent marker can be fluorescein isothiocyanate. In some embodiments, the antibody or its antigen-binding portion thereof of the present application comprises a heavy chain constant region (such as an Fc region), and the composition further comprises an antibody that specifically binds to the heavy chain constant region (such as an Fc region) and contains a chemiluminescent marker, and the composition further comprises a reagent that makes the chemiluminescent marker emit light.

[0034] In some embodiments, the composition can be a kit, further comprising instructions for using the kit.

[0035] The composition of the present application may comprise i) a bispecific molecule of the present application, and ii) a T cell expressing a chimeric antigen receptor, wherein the chimeric antigen receptor comprises a) an extracellular domain comprising a GS linker, b) a transmembrane region, and c) an intracellular signaling domain. The chimeric antigen receptor may comprise a GS linker at any suitable position in the extracellular domain. In some embodiments, the extracellular domain of the chimeric antigen receptor may comprise a non-antigen binding single-chain antibody comprising a GS linker. In some embodiments, the extracellular domain of the chimeric antigen receptor may comprise a non-antigen binding single-chain antibody comprising a GS linker between its heavy chain variable region and light chain variable region.

[0036] Alternatively, the composition of the present application may comprise i) a T cell expressing the chimeric antigen receptor of the present application, and ii) an antibody or antigen-binding portion thereof that is linked to a GS linker and targets a disease-associated antigen. The GS linker may be linked to the antibody or antigen-binding portion thereof that targets a disease-associated antigen via a peptide, such as a peptide of 10-30 amino acids. The disease-associated antigen may be, for example, a tumor-associated antigen. The antibody or antigen-binding portion thereof that targets a disease-associated antigen may be human or humanized. The GS linker may be any GS linker, particularly a GS linker of 6 amino acids or more in length, particularly a GS linker of 6-20 amino acids in length, particularly a GS linker of 6-15 amino acids in length, including, but not limited to, (G2S)2, (G2S)4, (G3S)3, (G4S)2, and (G4S)3. In particular, the GS linker may be (G4S)3 or (G4S)2. The antibody or antigen-binding portion thereof that targets a disease-associated antigen may not comprise a GS linker.

[0037] In another aspect, the present application provides a method for detecting a molecule containing a GS linker in a sample, comprising:

[0038] i) contacting the sample with the antibody or antigen-binding portion thereof of the present application,

[0039] ii) detecting the presence of the antibody or antigen-binding portion thereof in the sample,

[0040] The presence of the antibody or antigen-binding portion thereof in the sample indicates the presence of a molecule containing a GS linker in the sample.

[0041] Alternatively, the present application provides a method for quantifying molecules containing a GS linker in a sample, comprising:

[0042] i) contacting the sample with the antibody or antigen-binding portion thereof of the present application,

[0043] ii) detecting the presence of the antibody or antigen-binding portion thereof in the sample,

[0044] The amount of GS linker-containing molecules in the sample is quantified based on the amount of the antibody or antigen-binding portion thereof in the sample.

[0045] Alternatively, the present application provides a method for purifying a molecule containing a GS linker in a sample, comprising:

[0046] i) contacting the sample with a solid phase carrier coupled with the antibody or antigen-binding portion thereof of the present application,

[0047] ii) separating the sample molecules bound to the solid phase carrier in i) from the sample,

[0048] iii) eluting the sample molecules bound to the solid support from the solid support,

[0049] The solid phase carrier includes magnetic beads, resin, and agarose beads; the sample molecules bound to the solid phase carrier are molecules containing GS linkers.

[0050] The molecule containing a GS linker can be any molecule containing a GS linker, such as a recombinant protein or a nucleic acid-protein complex. In some embodiments, the molecule containing a GS linker can be an antibody containing a GS linker or a chimeric antigen receptor containing a GS linker. The antibody containing a GS linker can be a monospecific antibody, a bispecific antibody, or a multispecific antibody, and can include scFv, Fab, nanobody, etc.

[0051] The GS linker may be any GS linker, in particular a GS linker of 6 amino acids or more in length, in particular a GS linker of 6-20 amino acids in length, in particular a GS linker of 6-15 amino acids in length, including, but not limited to, (G2S)2, (G2S)4, (G3S)3, (G4S)2, and (G4S)3. In particular, the GS linker may be (G4S)2 or (G4S)3.

[0052] The methods of the present application can use one or more antibodies of the present application, or antigen-binding portions thereof.

[0053] The method of the present application may include, before step i), a step of selecting an antibody or an antigen-binding portion thereof based on the GS linker.

[0054] When the GS linker is (G2S)2, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 in the present application can be selected to include (1) NYDMY (SEQ ID NO: 2), VIYVNGNTHYASWAKG (SEQ ID NO: 6), GPF, QASQSVWKNKDLV (SEQ ID NO: 15), DASTLSS (SEQ ID NO: 21), and LGGFSCSRGDCGA (SEQ ID NO: 26), respectively; (2) NHAIM (SEQ ID NO: 3), VIYSNGNPYCARWVKG (SEQ ID NO: 7), GGF, KTSQSIYNNNDLV (SEQ ID NO: 16), GVSTLDS (SEQ ID NO: 22), and LGGFRCSSGDCGA (SEQ ID NO: 27); (3) NYDMY (SEQ ID NO: 2), VSYKNGRAHYASWAKG (SEQ ID NO: 6), GPF, QASQSVWKNKDLV (SEQ ID NO: 15), DASTLSS (SEQ ID NO: 21), and LGGFSCSRGDCGA (SEQ ID NO: 26), respectively. NO: 8), GPL, QASQSVWNNNDLV (SEQ ID NO: 17), DASTLSS (SEQ ID NO: 21), and LGGFSCSTGDCGA (SEQ ID NO: 28); or (4) NYDMY (SEQ ID NO: 2), VSYASGRTYSVRWAKG (SEQ ID NO: 10), GPL, QASQTVWKNNDLV (SEQ ID NO: 18), DASTLSS (SEQ ID NO: 21), and LGGFSCSSGDCGA (SEQ ID NO: 25), or an antigen-binding portion thereof.

[0055] When the GS linker is (G2S)4, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 in the present application can be selected to include (1) NYDMY (SEQ ID NO: 2), VIYVNGNTHYASWAKG (SEQ ID NO: 6), GPF, QASQSVWKNKDLV (SEQ ID NO: 15), DASTLSS (SEQ ID NO: 21), and LGGFSCSRGDCGA (SEQ ID NO: 26), respectively; (2) NHAIM (SEQ ID NO: 3), VIYSNGNPYCARWVKG (SEQ ID NO: 7), GGF, KTSQSIYNNNDLV (SEQ ID NO: 16), GVSTLDS (SEQ ID NO: 22), and LGGFRCSSGDCGA (SEQ ID NO: 27); (3) NYDMY (SEQ ID NO: 2), VSYKNGRAHYASWAKG (SEQ ID NO: 6), GPF, QASQSVWKNKDLV (SEQ ID NO: 15), DASTLSS (SEQ ID NO: 21), and LGGFSCSRGDCGA (SEQ ID NO: 26), respectively. NO: 8), GPL, QASQSVWNNNDLV (SEQ ID NO: 17), DASTLSS (SEQ ID NO: 21), and LGGFSCSTGDCGA (SEQ ID NO: 28); or (4) NYDMY (SEQ ID NO: 2), VSYASGRTYSVRWAKG (SEQ ID NO: 10), GPL, QASQTVWKNNDLV (SEQ ID NO: 18), DASTLSS (SEQ ID NO: 21), and LGGFSCSSGDCGA (SEQ ID NO: 25), or an antigen-binding portion thereof.

[0056] When the GS linker is (G3S)3, the antibodies comprising the amino acid sequences shown in VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 in the present application, respectively, (1) NYAIM (SEQ ID NO: 1), VIYANGDPYCASWAKG (SEQ ID NO: 5), GGF, QSSQSIYNKNDLV (SEQ ID NO: 14), GISTLDS (SEQ ID NO: 20), and LGGFSCSSGDCGA (SEQ ID NO: 25); or (2) NHAIM (SEQ ID NO: 3), VIYSNGNPYCARWVKG (SEQ ID NO: 7), GGF, KTSQSIYNNNDLV (SEQ ID NO: 16), GVSTLDS (SEQ ID NO: 22), and LGGFRCSSGDCGA (SEQ ID NO: 27), or their antigen-binding portions can be selected.

[0057] When the GS linker is (G4S)2, any antibody or antigen-binding portion thereof described in the present application can be selected.

[0058] When the GS linker is (G4S)3, any antibody or antigen-binding portion thereof described herein can be selected, particularly wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise (1) NHAIM (SEQ ID NO: 3), VIYSNGNPYCARWVKG (SEQ ID NO: 7), GGF, KTSQSIYNNNDLV (SEQ ID NO: 16), GVSTLDS (SEQ ID NO: 22), and LGGFRCSSGDCGA (SEQ ID NO: 27), respectively; (2) NYDMY (SEQ ID NO: 2), VSYKNGRAHYASWAKG (SEQ ID NO: 8), GPL, QASQSVWNNNDLV (SEQ ID NO: 17), DASTLSS (SEQ ID NO: 21), and LGGFSCSTGDCGA (SEQ ID NO: 28); or (3) NYAIM (SEQ ID NO: 1), VIYANGDPYCASWAKG (SEQ ID NO: 9), GGF, KTSQSIYNNNDLV (SEQ ID NO: 19), GVSTLDS (SEQ ID NO: 22), and LGGFRCSSGDCGA (SEQ ID NO: 27), respectively. NO: 5), GGF, QSSQSIYNKNDLV (SEQ ID NO: 14), GISTLDS (SEQ ID NO: 20), and LGGFSCSSGDCGA (SEQ ID NO: 25), or an antigen-binding portion thereof.

[0059] The sample can be any sample containing a molecule with a GS linker, such as a sample containing an antibody, protein sample, or small molecule sample with a GS linker, or a sample containing a cell with a GS linker (such as a cell containing a CAR with a GS linker). In some embodiments, the sample can include cells treated with an antibody containing a GS linker. In some embodiments, the sample can include cells introduced with a vector, wherein the vector contains a sequence for expressing a chimeric antigen receptor containing a GS linker.

[0060] The antibody or its antigen-binding portion thereof of the present application may include a detection substance. For example, the antibody or its antigen-binding portion thereof of the present application may be fused with a detection substance (e.g., through recombinant expression), or coupled with a detection substance. The antibody or its antigen-binding portion thereof of the present application may not affect any position of the antigen binding of the antibody or its antigen-binding portion thereof, for example, in the framework region of the heavy chain constant region, the light chain constant region, the heavy chain variable region, or the light chain variable region. The detection substance is included in the framework region of the heavy chain constant region or the light chain constant region. The detection substance may be a fluorescent marker or a chemiluminescent marker. The fluorescent marker may be fluorescein isothiocyanate. The chemiluminescent marker may be a peroxidase (e.g., horseradish peroxidase (HRP)) or alkaline phosphatase (AP).

[0061] The method of the present application can use the antibody or antigen-binding portion thereof of the present application containing a fluorescent marker, and determine whether the antibody or antigen-binding portion thereof is present in the sample, or the amount of the antibody or antigen-binding portion thereof present, by the fluorescent signal.

[0062] The method of the present application can use the antibody of the present application or its antigen-binding portion containing a chemiluminescent marker, and contact the antibody of the present application or its antigen-binding portion with a reagent that causes the chemiluminescent marker to emit light, and judge whether the antibody or its antigen-binding portion is present in the sample, or how much antibody or its antigen-binding portion is present, by the chemiluminescent signal. In some embodiments, the method of the present application can use the antibody of the present application or its antigen-binding portion containing horseradish peroxidase, and contact the antibody of the present application or its antigen-binding portion with peroxide (such as H2O2) and 3,3'-diaminobenzidine (DAB), or peroxide (such as H2O2) and 3,3',5,5'-tetramethylbenzidine (TMB). In some embodiments, the method of the present application can use the antibody of the present application or its antigen-binding portion containing alkaline phosphatase, and contact the antibody of the present application or its antigen-binding portion with 4-nitrophenylphosphate disodium salt hexahydrate (pNPP).

[0063] The methods of the present application can be used to detect the presence or amount of a protein containing a GS linker in a sample by, for example, Western blotting, to detect the presence or amount of cells bound by a protein containing a GS linker in a sample by, for example, flow cytometry, to detect the presence or amount of cells expressing a protein containing a GS linker in a sample by, for example, flow cytometry, or to detect the presence or amount of cells expressing a protein containing a GS linker via viral transduction in a sample by, for example, flow cytometry.

[0064] The present application also relates to a method for treating a disease in a subject in need thereof, comprising administering to the subject a composition of the present application, wherein the composition comprises i) a bispecific molecule of the present application, and ii) a T cell expressing a chimeric antigen receptor, wherein the chimeric antigen receptor comprises a) an extracellular domain comprising a GS linker, b) a transmembrane region, and c) an intracellular signaling domain, or i) a T cell expressing the chimeric antigen receptor of the present application, and ii) an antibody or antigen-binding portion thereof that is linked to a GS linker and targets a disease-associated antigen. The disease may be associated with a disease-associated antigen targeted by the bispecific molecule or the antibody or antigen-binding portion thereof that contains a GS linker and targets a disease-associated antigen in the composition. The disease-associated antigen may be a tumor-associated antigen.

[0065] The present application also protects the use of the antibodies or antigen-binding portions thereof, or the compositions of the present application, in detecting or quantifying proteins containing a GS linker, particularly in detecting the presence or amount of a protein containing a GS linker in a sample by, for example, Western blotting, detecting the presence or amount of cells bound by a protein containing a GS linker in a sample by, for example, flow cytometry, detecting the presence or amount of cells expressing a protein containing a GS linker in a sample by, for example, flow cytometry, or detecting the presence or amount of cells expressing a protein containing a GS linker by viral transduction in a sample by, for example, flow cytometry.

[0066] The present application also protects the use of the bispecific molecule, chimeric antigen receptor (CAR), CAR-T, or composition of the present application in treating a disease, or in preparing a medicament for treating a disease.

[0067] It should be noted that in this application, especially in the claims, terms such as "comprising" and "including" may have the meanings assigned to them by the Chinese Patent Law; and terms such as "essentially consisting of..." have the meanings assigned to them by the Chinese Patent Law, such as allowing the existence of elements not explicitly stated, but excluding elements existing in the prior art or elements that affect the basic or new characteristics of the invention.

[0068] Based on the following specific description and examples, other features and advantages of the current disclosure will become clearer, and specific description and examples should not be interpreted as restrictive. The contents of all documents, Genbank records, patents and published patent applications cited in this application are expressly included in this article by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] The following detailed description is given by way of example but is not intended to limit the present invention to the specific embodiments described, and can be better understood in conjunction with the accompanying drawings.

[0070] Figure 1 shows the binding ability of the antibodies R177.B8 (A), R177.B11 (B), R177.D13 (C), R177.E4 (D), R177.G3 (E), R177.G5 (F), R177.G12 (G), and R177.I8 (H) of the present application to different GS linkers in ELISA detection.

[0071] Figure 2 shows the binding of the present application antibodies R177.B8 (A), R177.B11 (B), R177.G3 (C), R177.G5 (D), R177.D13 (E), R177.E4 (F), R177.G12 (G), and R177.I8 (H) to proteins containing a GS linker in Western blot detection.

[0072] FIG3 shows the percentage of cells bound to the antibody containing a GS linker detected by the antibody of the present application in the total cells.

[0073] FIG. 4 shows the detection of lentivirus-transduced cells by the antibodies of the present application.

[0074] FIG5 shows cells expressing chimeric antigen receptors with a GS linker detected by the antibodies of the present application.

[0075] FIG6 shows the SDS-PAGE electrophoresis results of the antibody-purified scFv containing a GS linker of the present application. DETAILED DESCRIPTION

[0076] Unless otherwise specified, the terms used herein have the ordinary meanings in dictionaries, textbooks, technical reference books, or as generally understood by those skilled in the art. The following descriptions of certain terms are intended only to facilitate understanding of this application and are not intended to be limiting of these terms unless otherwise specified.

[0077] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0078] The term "or" refers to a single element of the listed alternative elements unless the context clearly dictates otherwise.

[0079] The term "comprise" or "include" means that the elements, integers or steps are included, but does not exclude the addition of any other elements, integers or steps. In this article, when the term "comprise" or "include" is used, unless otherwise indicated, combinations of the elements, integers or steps mentioned are also covered.

[0080] "GS linker" refers to a peptide composed of glycine and serine in the form of (GnS)m, where n and m can be integers with a minimum length of 1. In particular, the GS linker in the present application refers to a GS linker with a length of 6 amino acids or more, in particular a GS linker with a length of 6-20 amino acids, and in particular a GS linker with a length of 6-15 amino acids, including but not limited to (G2S)2, (G2S)4, (G3S)3, (G4S)2, and (G4S)3.

[0081] The "detection substance" in this application refers to a substance or molecule containing a group that can be detected by the naked eye or an instrument. The group that can be detected by the naked eye or an instrument can be, for example, a fluorescent marker, a luminescent marker, an immunodetectable marker, a radiolabel, a chemical marker, a nucleic acid marker, or a polypeptide marker. Thus, the detection substance can be a fluorescent marker containing a fluorescent group, or a chemiluminescent marker containing a group that can directly develop color or enzymatically develop color. By including (e.g., coupling) a detection substance on the antibody or antigen-binding portion thereof of the present application, the antibody or antigen-binding portion thereof of the present application can be located or quantified.

[0082] As used herein, "coupled" may refer to the state in which two organic chemical groups are linked by a chemical reaction, or the state in which two substances are attached to each other by any reaction or chemical bond. For example, the test substance may be linked to the antibody or antigen-binding portion thereof by reacting with a side chain group on an amino acid of the antibody or antigen-binding portion thereof, or the test substance may be attached to the antibody or antigen-binding portion thereof by, for example, van der Waals bonds.

[0083] The term "non-antigen-binding single-chain antibody" as used herein refers to a scFv-like peptide that has a single-chain antibody structure, i.e., a heavy chain variable region-linker-light chain variable region structure, and does not specifically bind to any disease-associated antigen. Such a "non-antigen-binding single-chain antibody" may comprise a heavy chain-like variable region (i.e., a peptide with an amino acid length similar to that of an antibody heavy chain variable region), a GS linker, and a light chain-like variable region (i.e., a peptide with an amino acid length similar to that of an antibody light chain variable region).

[0084] The term "antibody" as used herein is intended to include IgG, IgA, IgD, IgE and IgM full-length antibodies and any antigen-binding fragments (i.e., antigen-binding portions) thereof. A full-length antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains connected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (V H The heavy chain constant region is composed of three domains, namely C H1 、C H2 and C H3 Each light chain consists of a light chain variable region (V L or VL) and the light chain constant region. The light chain constant region consists of a domain C L Composition. H and V L The V domains can be further divided into regions of hypervariability, termed complementarity determining regions (CDRs), separated by more conserved framework regions (FRs). H and V L It is composed of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including binding to a variety of immune system cells (e.g., effector cells) and the first component (C1q) of the traditional complement system. A "functional fragment" of an antibody constant region refers to a fragment in the constant region that retains certain desired functions, such as the Fc fragment in the heavy chain constant region.

[0085] As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a GS linker). It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included in the "antigen-binding portion" of an antibody include (i) a Fab fragment, which is comprised of a V L 、V H 、C L and C H1 (ii) F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) V H and C H1 (iv) an Fd fragment consisting of an antibody single-arm V L and V H scFv fragment composed of V HH composed of dAb fragments (Ward et al., (1989) Nature 341: 544-546); (vi) isolated complementarity determining regions (CDRs); and (vii) dAb-V L , a fragment comprising a single variable domain and a heavy chain constant domain. In addition, although the two domains V L and V H Encoded by different genes, they can be recombinantly linked via a synthetic linker that makes the two into a single protein chain, where V L and V H The regions are paired to form monovalent molecules. These single-chain antibodies are also intended to be included in the meaning of the term. These antibody fragments can be obtained by common techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

[0086] "Single-chain antibody" or "scFv" refers to an antibody format composed of the heavy chain variable region of an antibody connected directly or through a linker to the light chain variable region.

[0087] "Nanobodies" or "V H H" refers to a single antigen-binding polypeptide comprising a single monomeric variable domain comprising three complementarity determining regions (CDRs), which is capable of binding to an antigen without the need for pairing with other corresponding CDR-containing polypeptides.

[0088] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities. For example, an isolated antibody that specifically binds to a GS linker protein is substantially free of antibodies that specifically bind to antigens other than the GS linker. An isolated antibody is substantially free of other cellular material and / or chemicals.

[0089] The term "monoclonal antibody" or "mAb" or "monoclonal antibody composition" refers to an antibody molecule preparation of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope.

[0090] The term "rabbit-derived antibody" refers to an antibody whose variable region framework and CDR region are derived from rabbit germline immunoglobulin sequences. In addition, if the antibody contains a constant region, it is also derived from a rabbit germline immunoglobulin sequence. The rabbit-derived antibody of the present application may contain amino acid residues that are not encoded by rabbit germline immunoglobulin sequences, such as mutations introduced by random mutations or point mutations in vitro or by somatic mutations in vivo. However, the term "rabbit-derived antibody" does not include antibodies in which CDR sequences derived from other mammalian species are inserted into the rabbit framework sequence.

[0091] The term "chimeric antibody" refers to an antibody that combines genetic material from one species with genetic material from another species. In particular, a chimeric antibody in this application refers to an antibody that is derived from a combination of non-human genetic material and human genetic material.

[0092] The term "humanized antibody" refers to an antibody derived from a non-human (eg, rabbit) species whose protein sequence has been altered to increase similarity to naturally occurring antibodies in humans.

[0093] The terms "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen."

[0094] As used herein, the term "specifically recognizes" or "specifically binds to" a target, such as a GS linker, means that an antibody or antigen-binding fragment can distinguish between a GS linker and a reference molecule, such as another amino acid peptide, and has a binding affinity or activity to the GS linker that is, for example, 1-fold, 5-fold, 10-fold, etc., higher than the other reference molecule. Specificity determination methods include, but are not limited to, SPR, Western blotting, ELISA, RIA, ECL, IRMA testing, and peptide scanning.

[0095] "Sequence identity" herein refers to the percentage of nucleotides / amino acids in a sequence that are identical to the nucleotides / amino acid residues in a reference sequence after sequence alignment, with spaces introduced, if necessary, to achieve the maximum percentage of sequence identity between the two sequences. Those skilled in the art can perform pairwise sequence alignment or multiple sequence alignment to determine the percentage of sequence identity between two or more nucleic acid or amino acid sequences by various methods, such as using computer software such as Clustal Omega, T-coffee, Kalign, and MAFFT.

[0096] "Chimeric antigen receptor" or "CAR" refers to a recombinant protein that contains both antigen recognition function and immune cell activation function, which is generally recombinantly expressed in immune cells so that the immune cells produce cytotoxicity to cells where the target antigen is located, such as tumor cells, when they recognize and bind to the target antigen. CAR generally comprises (a) an extracellular antigen binding domain, (b) a transmembrane domain, and (c) an intracellular signal transduction domain, wherein the extracellular antigen binding domain comprises an scFv or nanobody that specifically binds to the target antigen. In some embodiments of the present application, the extracellular antigen binding domain may comprise a GS linker, which may be, for example, located in an scFv, or between two or more nanobodies.

[0097] The term "EC 50 ", also known as half-maximal effect concentration, refers to the drug concentration that causes 50% of the maximum effect.

[0098] The term "IC50 ” refers to the half-inhibitory concentration, which is the concentration of the drug or inhibitor required to inhibit a specified biological process by half.

[0099] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cows and horses, are preferred.

[0100] The term "therapeutically effective amount" refers to an amount of the present invention sufficient to prevent or alleviate the symptoms associated with a disease or disorder (e.g., cancer). The therapeutically effective amount is related to the disease being treated, and those skilled in the art can readily determine the actual effective amount.

[0101] The antibodies or antigen-binding portions thereof that specifically bind to the GS linker of the present application can bind to a variety of GS linkers with high binding affinity, including, but not limited to, (G2S)2, (G2S)4, (G3S)3, (G4S)2, and (G4S)3. In particular, the antibodies or antigen-binding portions thereof of the present application can specifically bind to (G4S)2 or (G4S)3.

[0102] Compared to traditional methods using protein L, target antigens, antibodies targeting idiotypic antibodies, etc., the antibodies or antigen-binding portions thereof of the present application i) have a certain degree of versatility, that is, they are applicable to most proteins containing GS linkers, regardless of the target of the protein, the subtype of the Fc region contained in the protein, the subtype of the antibody light chain, or the form of the antibody (single domain antibody or scFv, etc.), and are applicable to most GS linkers except G4S, ii) they have high binding specificity for proteins containing GS linkers, the detection accuracy is higher than that of the protein L detection method, and the discrimination between samples containing GS linker proteins and samples not containing GS linker proteins, such as negative and positive CAR-T cells with or without GS linker scFv, is stronger than that of anti-Fab antibodies, iii) they have high binding sensitivity for proteins containing GS linkers, and can detect about 2 pmol of proteins with GS linkers. In particular, the antibodies of the present application are monoclonal antibodies. In addition, the antibodies or antigen-binding portions thereof can be, for example, rabbit-derived, chimeric, or humanized.

[0103] The heavy chain variable region and light chain variable region sequences or sequence numbers of the antibodies or antigen-binding portions thereof of the present application are listed in Table 1. The heavy chain variable region CDRs and light chain variable region CDRs are identified using the Kabat numbering system, and the CDR sequences or sequence numbers identified thereby are listed in Table 1. The heavy chain variable region CDRs and light chain variable region CDRs of the antibodies or antigen-binding portions thereof of the present application can also be identified using the IMGT, Chothia, AbM, or Contact numbering systems based on the full-length variable region sequences.

[0104] V of other antibodies bound to the GS linker H and / or V L The sequence (or CDR sequence) can be the same as the V H and / or V L Sequences (or CDR sequences) are "mixed and paired". Preferably, when V H and V L When (or CDRs therein) are mixed and paired, a specific V H / V L V in pairing H The sequence can be approximated by the structure of V H Similarly, specific V H / V L V in pairing L The sequence is composed of structurally similar V L Sequence substitution.

[0105] Therefore, in one embodiment, the antibody or antigen-binding portion thereof of the present application comprises:

[0106] (a) a heavy chain variable region comprising the amino acid sequence listed in Table 1; and

[0107] (b) a light chain variable region comprising the amino acid sequence listed in Table 1, or a V domain of another GS linker antibody L , wherein the antibody specifically binds to a GS linker.

[0108] In another embodiment, the antibody or antigen-binding portion thereof of the present application comprises:

[0109] (a) CDR1, CDR2, and CDR3 of the heavy chain variable region listed in Table 1; and

[0110] (b) CDR1, CDR2, and CDR3 of the light chain variable region listed in Table 1, or the CDRs of another GS linker antibody, wherein the antibody specifically binds to a GS linker.

[0111] In another embodiment, the antibodies or antigen-binding portions thereof of the present application include the heavy chain variable region CDR2 of a GS linker antibody and the CDRs of another antibody that binds to a GS linker, such as the heavy chain variable region CDR1 and / or CDR3, and / or the light chain variable region CDR1, CDR2 and / or CDR3 of another GS linker antibody.

[0112] Furthermore, it is well known in the art that the CDR3 domain, independent of CDR1 and / or CDR2, can independently determine the binding specificity of an antibody to the same antigen, and it can be predicted that multiple antibodies with the same binding specificity can be generated based on the CDR3 sequence. See, eg, Klimka et al., British J. of Cancer 83(2):252-260 (2000); Beiboer et al., J. Mol. Biol. 296:833-849 (2000); Rader et al., Proc. Natl. Acad. Sci. USA 95: 8910-8915 (1998); Barbas et al. al., J. Am. Chem. Soc. 116: 2161-2162 (1994); Barbas et al., Proc. Natl. Acad. Sci. US. A. 92: 2529-2533 (1995); Ditzel et al., J. Immunol. 157: 739-749 (1996).

[0113] In another embodiment, the antibodies or antigen-binding portions thereof of the present application comprise heavy and / or light chain variable region sequences or CDR1, CDR2, and CDR3 sequences that exhibit one or more conservative modifications to the GS linker antibodies or antigen-binding portions thereof of the present application. It is known in the art that some conservative sequence modifications do not abolish antigen binding. See, for example, Brummell et al., (1993) Biochem 32:1180-8.

[0114] Thus, in one embodiment, the antibody or antigen-binding portion thereof comprises a heavy chain variable region and / or a light chain variable region, the heavy chain variable region and the light chain variable region comprising CDR1, CDR2 and CDR3, respectively, wherein:

[0115] (a) the heavy chain variable region CDR1 comprises the sequence listed in Table 1, and / or conservative modifications thereof; and / or

[0116] (b) the heavy chain variable region CDR2 comprises the sequence listed in Table 1, and / or conservative modifications thereof; and / or

[0117] (c) the heavy chain variable region CDR3 comprises the sequence listed in Table 1, and / or conservative modifications thereof; and / or

[0118] (d) the light chain variable region CDR1, and / or CDR2, and / or CDR3 comprise the sequences listed in Table 1, and / or conservative modifications thereof; and

[0119] (e) The antibody, or antigen-binding portion thereof, specifically binds to a GS linker.

[0120] The term "conservative sequence modification" as used herein refers to amino acid modifications that do not significantly affect or change the binding properties of the antibody. Such conservative modifications include amino acid replacements, additions, and deletions. Modifications can be introduced into the present application's antibodies or their antigen-binding portion thereof by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid replacements are amino acid residues that are replaced with amino acid residues having similar side chains. Groups of amino acid residues with similar side chains are known in the art. These groups of amino acid residues include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR region of the antibodies of the present application, or antigen-binding portions thereof, can be replaced with other amino acid residues from the same side chain group, and the resulting antibodies can be tested for retention of function (i.e., the function described above) using the functional assays described herein.

[0121] The antibody or antigen binding portion thereof of the present application can be used with one or more V H / V L The antibody sequence is used as the starting material to prepare the genetically modified antibody. The antibody can be prepared by modifying one or two variable regions (i.e., V H and / or V L ) (e.g., in one or more CDR regions and / or one or more framework regions) to improve binding affinity.

[0122] The variable region modification can be to change V H and / or V L The amino acid residues in the CDR1, CDR2 and / or CDR3 regions are mutated to improve one or more binding properties (e.g., affinity) of the target antibody. Point mutations or PCR-mediated mutations can be used to introduce mutations, and their effects on antibody binding or other functional properties can be evaluated in vitro or in vivo assays known in the art. Preferably, conservative modifications known in the art are introduced. The mutations can be amino acid replacements, additions, or deletions, but are preferably replacements. In addition, no more than one, two, three, four, or five residues in the CDR regions are typically changed.

[0123] In another embodiment, the present application provides an isolated GS linker monoclonal antibody or an antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, comprising: (a) V H CDR1 region, comprising the sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acid substitutions, deletions or additions; (b) V H CDR2 region, comprising the sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acid substitutions, deletions or additions; (c) V H CDR3 region, comprising the sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acids substituted, deleted or added; (d) V L CDR1 region, comprising the sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acids substituted, deleted or added; (e) V L CDR2 region, comprising the sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acid substitutions, deletions or additions; and (f) V L The CDR3 region comprises the sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acids substituted, deleted or added.

[0124] Antibody C H1 The hinge region of the hinge region can be modified, such as by increasing or decreasing the number of cysteine ​​residues in the hinge region. This method is further described in U.S. Patent No. 5,677,425. H1 The cysteine ​​residues in the hinge region can be used to, for example, facilitate the assembly of the heavy and light chains or to increase / decrease the stability of the antibody.

[0125] The glycosylation of the antibody can be modified. For example, a deglycosylated antibody (i.e., the antibody lacks glycosylation) can be prepared. Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen. Such glycosylation modification can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate one or more variable region backbone glycosylation sites, thereby eliminating glycosylation at that position. Such deglycosylation can increase the affinity of the antibody for the antigen. See, for example, U.S. Patents 5,714,350 and 6,350,861. Glycosylation is known to occur in motifs containing the NXS / T sequence. In some cases, it is preferred that the GS linker antibody or its antigen-binding portion does not contain variable region glycosylation. This can be achieved by selecting an antibody that does not contain a glycosylation motif in the variable region or by mutating residues in the glycosylation region.

[0126] In a preferred embodiment, the antibody or antigen-binding portion thereof does not contain an asparagine isomerization site. Deamidation of asparagine may occur in NG or DG sequences, creating an isoaspartic acid residue that introduces a kink into the polypeptide chain and reduces its stability (isoaspartic acid effect).

[0127] The monoclonal antibody of the present application can be prepared using phage display technology. Phage display technology is to insert the gene of exogenous coded polypeptide or protein (such as antibody in the form of scFv) into the appropriate position of phage coat protein structural gene by genetic engineering technology, correctly express in reading frame, make exogenous polypeptide or protein (such as scFv) form fusion protein on the capsid protein of phage, and be presented on phage surface with the reassembly of progeny phage.Then utilize target molecule (such as GS linker), adopt suitable elutriation method, wash away phage that does not specifically bind target molecule. Use acid and alkali or competitive molecule to elute the bound phage again, the phage infection Escherichia coli after neutralization is amplified, and through 3-5 rounds of enrichment, progressively improve the phage ratio that can specifically recognize target molecule, finally obtain the polypeptide or protein that recognizes target molecule.

[0128] Other methods for preparing monoclonal antibodies include somatic cell hybridization (hybridoma), viral or oncogenic transformation of B lymphocytes, etc. The methods for preparing chimeric antibodies are also well known in the art. The antibodies or antigen-binding portions thereof of the present application can also be generated in host cell transfectomas using, for example, recombinant DNA technology in combination with gene transfection methods (e.g., Morrison, S. (1985) Science 229: 1202). In one embodiment, the DNA encoding partial or full-length light and heavy chains obtained by standard molecular biological techniques is inserted into one or more expression vectors so that the gene is operably linked to the transcription and translation regulatory sequences. In this case, the term "operably linked" refers to that the antibody gene is connected to the vector so that the transcription and translation control sequences in the vector exercise their established functions of regulating antibody gene transcription and translation.

[0129] The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody gene. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers from cytomegalovirus (CMV), simian virus 40 (SV40), adenoviruses, such as the adenovirus major late promoter (AdMLP), and polyomavirus. Alternatively, non-viral regulatory sequences such as the ubiquitin promoter or the β-globin promoter may be used. In addition, regulatory elements are composed of sequences from different sources, such as the SRα promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1. The expression vector and expression control sequences are selected to be compatible with the expression host cell used.

[0130] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or different expression vectors. In a preferred embodiment, the variable region is inserted into an expression vector encoding the heavy chain constant region and the light chain constant region of the desired subtype to construct a full-length antibody gene, so that V H With the C in the carrier H Operatively connected, V L With the C in the carrier L Operably connected. Alternatively, the recombinant expression vector can encode a signal peptide that promotes secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into a vector so that the signal peptide is connected to the amino terminus of the antibody chain gene in the reading frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin).

[0131] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the present application may carry other sequences, such as sequences (e.g., replication origins) and selectable marker genes that regulate the replication of the vector in the host cell. Selectable marker genes can be used to select the host cell into which the vector has been introduced. For example, conventional selectable marker genes confer drug resistance, such as G418, hygromycin, or methotrexate resistance, to the host cell into which the vector has been introduced. Preferred selectable marker genes include dihydrofolate reductase (DHFR) genes (for methotrexate selection / amplification of dhfr host cells) and neo genes (for G418 selection).

[0132] For the expression of light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The term "transfection" in its various forms includes a variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextrose transfection, and the like. Although it is theoretically feasible to express the present antibody or its antigen-binding portion in prokaryotic or eukaryotic host cells, it is preferred that the antibody be expressed in eukaryotic cells, most preferably in mammalian host cells, because eukaryotic cells, particularly mammalian cells, are more likely to assemble and secrete properly folded and immunologically active antibodies than prokaryotic cells.

[0133] Preferred mammalian host cells for expressing the recombinant antibodies of the present application include Slc35C1 gene knockout cell lines, FUT8 knockout cell lines, variant CHO cell lines Lec13, rat hybridoma cell lines YB2 / 0, cell lines containing small interfering RNA specifically for the FUT8 gene, cell lines that co-express β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II, Chinese hamster ovary (CHO cells) (including dhfr-CHO cells administered with a DHFR selectable marker), NSO myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is prepared by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or preferably sufficient to allow secretion of the antibody into the culture medium in which the host cell is grown. The antibody or its antigen-binding portion can be recovered from the culture medium using a protein purification method.

[0134] In certain embodiments, CDR regions are implanted into the variable regions of genetically modified antibodies. Antibodies interact with target antigens primarily through amino acid residues located in the three heavy chain complementary determining regions (CDRs) and the six light chain CDRs. For this reason, the amino acid residues within the CDRs are more diverse between individual antibodies than the sequences outside the CDRs. Because CDR sequences are responsible for the primary antibody-antigen interaction, recombinant antibodies that mimic the properties of a specific natural antibody can be expressed by constructing an expression vector containing the CDR sequence of a specific natural antibody implanted into the framework sequence of a different antibody with different properties (Riechmann et al., (1998) Nature 332:323-327; Jones et al., (1986) Nature 321:522-525; Queen et al., (1989) Proc. Natl. Acad; USA 86:10029-10033; US Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0135] Therefore, another embodiment of the present application relates to an isolated monoclonal antibody or antigen-binding fragment thereof, and / or a bispecific antibody, comprising a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3 having the sequence of the present application, and the light chain variable region comprises CDR1, CDR2 and CDR3 having the sequence of the present application. Although these antibodies comprise the V H and V L CDR sequences, which may contain different framework sequences.

[0136] Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the Vbase human germline sequence database (www.mrc-cpe.cam.ac.uk / vbase) and in Kabat et al., (1991), supra; Tomlinson et al., (1992) J. Mol. Biol. 227:776-798; and Cox et al., (1994) Eur. J. Immunol. 24:827-836. As another embodiment, germline DNA sequences for human heavy and light chain variable region genes can be found in the Genbank database.

[0137] The antibody protein sequence is compared to protein sequence databases by using one of the sequence similarity searching methods known in the art as gapped BLAST (Altschul et al., (1997)).

[0138] Preferred framework sequences for use in the antibodies of the present application are those that are structurally similar to the framework sequences used in the antibodies of the present application. H The CDR1, CDR2, and CDR3 sequences can be implanted into a framework region having the same sequence as the germline immunoglobulin gene from which the framework sequence is derived, or the CDR sequences can be implanted into a framework region that contains one or more mutations compared to the germline sequence. For example, in some cases, it is beneficial to mutate residues in the framework region to maintain or enhance the antigen binding of the antibody (see, e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0139] On the other hand, the present application provides nucleic acid molecules encoding the heavy chain / light chain variable region or CDR of the antibody or antigen-binding portion thereof of the present application. The nucleic acid can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques. The nucleic acid of the present application can be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0140] The nucleic acid of the present application can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared by transgenic mice carrying human immunoglobulin genes), cDNA encoding the light chain and heavy chain of the antibody prepared by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene repositories (e.g., using phage display technology), nucleic acids encoding such antibodies can be collected from gene banks.

[0141] The preferred nucleic acid molecules of the present application include V encoding the GS linker monoclonal antibody H and V L Once the sequences encoding V H and V L These DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. H or V L The term "operably linked" refers to a DNA fragment that is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" means that the two DNA fragments are linked together so that the amino acid sequences encoded by the two DNA fragments are in the reading frame.

[0142] Code V H The isolated DNA of the region can be operably linked to the V H The DNA encoding the heavy chain constant region (C H1 、C H2 and C H3 The heavy chain constant region can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD, but is preferably IgG1. For Fab fragment heavy chain genes, the V H The DNA of the heavy chain C region can be operably linked to theH1 The constant region of another DNA molecule is attached.

[0143] Code V L The isolated DNA of the region can be operably linked to the V L DNA encoding the light chain constant region C L The light chain constant region is converted into a full-length light chain gene by using another DNA molecule from the human light chain constant region. The sequences of human light chain constant region genes are known in the art, and DNA fragments comprising these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a kappa or lambda constant region.

[0144] To create scF v gene encoding V H and V L The DNA fragment can be operably linked to another fragment encoding a flexible linker, such that V H and V L The sequence can be expressed as a continuous single-chain protein, where V H and V L The regions are connected by this flexible linker.

[0145] On the other hand, the present application relates to a bispecific molecule comprising an antibody or antigen-binding portion thereof of the present application linked to at least one other functional molecule, such as another peptide or protein (e.g., another antibody or receptor ligand), to generate a bispecific molecule that binds to at least two different binding sites or targeting molecules. The term "bispecific molecule" includes molecules with three or more specificities. In some embodiments, the bispecific molecule of the present application may comprise i) an antibody or antigen-binding portion thereof that specifically binds to a GS linker of the present application, and ii) an antibody or antigen-binding portion thereof that targets a disease-associated antigen. i) and ii) may be linked together, for example, by fusion through recombinant expression, or bound together via, for example, a disulfide bond. The disease-associated antigen may be, for example, a tumor-associated antigen.

[0146] Bispecific molecules can appear in a variety of formats and sizes. At one end of the size spectrum, bispecific molecules maintain the traditional antibody format, except that they have two binding arms, each with a different specificity, instead of having two binding arms with the same specificity. At the other extreme are bispecific molecules composed of two single-chain antibody fragments (scFv) linked by a peptide chain, called Bs(scFv)2 constructs. Bispecific molecules of intermediate size include two different F(ab) fragments connected by a peptide linker. These and other forms of bispecific molecules can be prepared by genetic modification, somatic cell hybridization, or chemical methods.

[0147] The present application also provides a chimeric antigen receptor, which may comprise a) an extracellular domain, which may comprise the antibody of the present application that specifically binds to the GS linker or an antigen-binding portion thereof, particularly the antibody of the present application in scFv form, b) a transmembrane region, and c) an intracellular signaling domain.

[0148] The present application also provides an immune cell, such as a T cell or a NK cell, which contains the chimeric antigen receptor of the present application.

[0149] In order to facilitate detection of the antibodies or antigen-binding portions thereof of the present application, the antibodies or antigen-binding portions thereof may comprise a detection substance. For example, the antibodies or antigen-binding portions thereof of the present application may be fused with a detection substance (e.g., via recombinant expression), or coupled with a detection substance. The antibodies or antigen-binding portions thereof of the present application may comprise a detection substance in the heavy chain constant region, the light chain constant region, the framework (FR) region of the heavy chain variable region, or the framework region of the light chain variable region. In particular, the antibodies or antigen-binding portions thereof may comprise a detection substance in the framework region of the heavy chain constant region or the light chain constant region.

[0150] The detection substance can be any substance that is easy to detect with the naked eye or an instrument, including, but not limited to, fluorescent labels, cold light labels, immunodetectable labels, radiolabels, chemical labels, nucleic acid labels, and polypeptide labels. Commonly used detection substances include fluorescent labels or chemiluminescent labels. Fluorescent labels can be, for example, fluorescein isothiocyanate (FITC), which can be used for qualitative and quantitative observations, such as by fluorescence microscopy, and can be conveniently used for, for example, flow cytometry. Chemiluminescent labels can be peroxidases (such as horseradish peroxidase (HRP)) or alkaline phosphatase (AP), which can quickly develop color through the addition of substrates corresponding to each enzyme, and then perform qualitative and quantitative observations. Substrates for horseradish peroxidase include 3,3'-diaminobenzidine (DAB) and 3,3',5,5'-tetramethylbenzidine (TMB). Substrates for alkaline phosphatase include 4-nitrophenylphosphate disodium salt hexahydrate (pNPP).

[0151] Methods for making the antibody or antigen-binding portion thereof contain a detection substance, such as coupling or recombinant expression of a fusion protein, are known to those skilled in the art.

[0152] The antibodies or antigen-binding portions thereof of the present application can also be qualitatively and quantitatively observed by adding a secondary antibody that binds to the heavy chain constant region or light chain constant region, particularly the heavy chain constant region such as the Fc region.

[0153] On the other hand, the present application provides a composition comprising the antibody or antigen-binding portion thereof, bispecific molecule, chimeric antigen receptor (CAR), CAR-carrying cell, nucleic acid molecule, expression vector, or host cell of the present application.

[0154] The antibody or its antigen-binding portion thereof of the present application may include a detection substance, such as a fluorescent marker or a chemiluminescent marker. In some embodiments, the antibody or its antigen-binding portion thereof of the present application may include a chemiluminescent marker, and the composition may further include a reagent that makes the chemiluminescent marker glow. In some embodiments, the chemiluminescent marker may be horseradish peroxidase, and the reagent that makes the chemiluminescent marker glow may be peroxide (e.g., H2O2) and 3,3'-diaminobenzidine (DAB), or peroxide peroxide (e.g., H2O2) and 3,3',5,5'-tetramethylbenzidine (TMB). In some embodiments, the chemiluminescent marker may be alkaline phosphatase, and the reagent that makes the chemiluminescent marker glow may be 4-nitrophenylphosphate disodium salt hexahydrate (pNPP). In some embodiments, the antibody or its antigen-binding portion thereof of the present application includes a heavy chain constant region (e.g., Fc region), and the composition further includes an antibody that specifically binds to the heavy chain constant region (e.g., Fc region) and contains a detection substance. In some embodiments, the antibody or antigen-binding portion thereof of the present application comprises a heavy chain constant region (such as an Fc region), and the composition further comprises an antibody that specifically binds to the heavy chain constant region (such as an Fc region) and comprises a fluorescent marker. The fluorescent marker can be fluorescein isothiocyanate. In some embodiments, the antibody or antigen-binding portion thereof of the present application comprises a heavy chain constant region (such as an Fc region), and the composition further comprises an antibody that specifically binds to the heavy chain constant region (such as an Fc region) and comprises a chemiluminescent marker, and the composition further comprises a reagent that causes the chemiluminescent marker to emit light.

[0155] The antibodies or antigen-binding portions thereof, and the compositions of the present application have various applications, such as use in detecting or quantifying proteins containing a GS linker, particularly in detecting the presence or amount of a protein containing a GS linker in a sample by, for example, Western blotting, detecting the presence or amount of cells bound by a protein containing a GS linker in a sample by, for example, flow cytometry, detecting the presence or amount of cells expressing a protein containing a GS linker in a sample by, for example, flow cytometry, or detecting the presence or amount of cells expressing a protein containing a GS linker by viral transduction in a sample by, for example, flow cytometry.

[0156] Specifically, the present application provides a method for detecting a molecule containing a GS linker in a sample, comprising:

[0157] i) contacting the sample with the antibody or antigen-binding portion thereof of the present application,

[0158] ii) detecting the presence of the antibody or antigen-binding portion thereof in the sample,

[0159] The presence of the antibody or antigen-binding portion thereof in the sample indicates the presence of a molecule containing a GS linker in the sample.

[0160] Alternatively, the present application provides a method for quantifying molecules containing a GS linker in a sample, comprising:

[0161] i) contacting the sample with the antibody or antigen-binding portion thereof of the present application,

[0162] ii) detecting the presence of the antibody or antigen-binding portion thereof in the sample,

[0163] The amount of GS linker-containing molecules in the sample is quantified based on the amount of the antibody or antigen-binding portion thereof in the sample.

[0164] Alternatively, the present application provides a method for purifying a molecule containing a GS linker in a sample, comprising:

[0165] i) contacting the sample with a solid phase carrier coupled with the antibody or antigen-binding portion thereof of the present application,

[0166] ii) separating the sample molecules bound to the solid phase carrier in i) from the sample,

[0167] iii) eluting the sample molecules bound to the solid support from the solid support,

[0168] The solid phase carrier includes magnetic beads, resin, and agarose beads; the sample molecules bound to the solid phase carrier are molecules containing GS linkers.

[0169] The GS linker that can be detected using the method of the present application can be any GS linker, in particular a GS linker with a length of more than 6 amino acids, in particular a GS linker with a length of 6-20 amino acids, in particular a GS linker with a length of 6-15 amino acids, including, but not limited to, (G2S)2, (G2S)4, (G3S)3, (G4S)2, and (G4S)3.

[0170] The molecule comprising a GS linker can be any molecule comprising a GS linker, such as a recombinant protein, a nucleic acid-protein complex, and the like, for example, an antibody comprising a GS linker, or a chimeric antigen receptor comprising a GS linker. The antibody comprising a GS linker can be a monospecific antibody, a bispecific antibody, or a multispecific antibody, and can include various antibody formats such as scFv, Fab, and nanobodies.

[0171] The sample can be any sample containing a molecule with a GS linker, such as a sample containing an antibody with a GS linker, or a sample containing a CAR with a GS linker. In some embodiments, the sample can include cells treated with an antibody containing a GS linker. In some embodiments, the sample can include cells introduced with a vector containing a sequence for expressing a chimeric antigen receptor containing a GS linker.

[0172] The antibodies or antigen-binding portions thereof of the present application have different binding preferences for various GS linkers. Therefore, if the specific information of the GS linker in the molecule is known before detection, a step of selecting an antibody or antigen-binding portion thereof based on the GS linker can be included before step i) of the method. If the type of GS linker contained in the molecule is unknown before detection, one can select and use multiple GS antibodies or antigen-binding portions thereof described in the present application, or select an antibody suitable for multiple GS linkers, for example, an antibody or antigen-binding portion thereof whose VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences set forth in NYDMY (SEQ ID NO: 2), VSYKNGRAHYASWAKG (SEQ ID NO: 8), GPL, QASQSVWNNNDLV (SEQ ID NO: 17), DASTLSS (SEQ ID NO: 21), and LGGFSCSTGDCGA (SEQ ID NO: 28), respectively.

[0173] Qualitative or quantitative detection of antibodies or their antigen-binding portions can rely on detection substances carried by the antibodies or their antigen-binding portions, or on secondary antibodies that can bind to the antibodies or their antigen-binding portions, all of which are within the capabilities of those skilled in the art.

[0174] The composition of the present application may be a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. The composition may optionally comprise one or more other pharmaceutically active ingredients, such as another anti-tumor antibody, or an immunopotentiating antibody, or a non-antibody anti-tumor agent, or an immunopotentiator. The composition of the present application may be used in combination with, for example, another anticancer agent or another immunopotentiator.

[0175] In some embodiments, the compositions of the present application may comprise i) the bispecific molecule of the present application, and ii) a T cell expressing a chimeric antigen receptor, wherein the chimeric antigen receptor comprises a) an extracellular domain comprising a GS linker, b) a transmembrane region, and c) an intracellular signaling domain. The chimeric antigen receptor may comprise a GS linker at any suitable position in the extracellular domain to activate T cells when bound to the bispecific molecule of the present application. In some embodiments, the extracellular domain of the chimeric antigen receptor may comprise a non-antigen binding single-chain antibody comprising a GS linker. In some embodiments, the extracellular domain of the chimeric antigen receptor may comprise a non-antigen binding single-chain antibody comprising a GS linker.

[0176] In some embodiments, the compositions of the present application may comprise i) T cells expressing the chimeric antigen receptor of the present application, and ii) an antibody or antigen-binding portion thereof that targets a disease-associated antigen and is linked to a GS linker. The GS linker may be linked to the antibody or antigen-binding portion thereof that targets the disease-associated antigen via a peptide, such as a peptide of 10-30 amino acids, such that when the CAR-T cell binds to the GS linker, it does not affect the specific binding of the antibody or antigen-binding portion thereof that targets the disease-associated antigen to the disease-associated antigen. Importantly, the antibody or antigen-binding portion thereof that targets the disease-associated antigen does not contain a GS linker to prevent the binding of the CAR-T cell from adversely affecting its binding to the disease-associated antigen.

[0177] The above compositions can be used to treat various diseases. The disease can be associated with a disease-associated antigen targeted by the bispecific molecule or the antibody or antigen-binding portion thereof containing a GS linker and targeting a disease-associated antigen in the composition. The disease-associated antigen can be, for example, a tumor-associated antigen.

[0178] The composition may contain any number of excipients. Useful excipients include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersing or suspending agents, solubilizing agents, colorants, flavoring agents, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003).

[0179] The composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or push injection). Based on the difference in route of administration, the active ingredient can be wrapped in a material to protect it from the influence of acid and other natural conditions that may inactivate it. "Parenteral administration" refers to a mode different from intestinal and topical, usually carried out by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, supra-dura mater and intrasternal injection and push injection. Alternatively, the antibody of the present application can be administered by non-parenteral route, such as topical, epidermal or mucosal administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical.

[0180] The compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in microemulsions, liposomes, or other ordered structures suitable for high drug concentration.

[0181] The amount of active ingredient prepared in a single dosage form together with the carrier material will vary depending on the subject being treated and the particular mode of administration, and is essentially the amount of the composition that produces a therapeutic effect. In percentage terms, this amount is about 0.01 to about 99% of the active ingredient combined with the pharmaceutically acceptable carrier.

[0182] The dosage regimen is adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a bolus may be administered, multiple divided doses may be administered over time, or the dose may be reduced or increased in proportion to the severity of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. A dosage unit form refers to physically discrete units suitable for single administration to a subject; each unit contains a predetermined amount of the active ingredient calculated to produce the desired therapeutic effect together with a pharmaceutical carrier. Alternatively, the antibody may be administered as a sustained-release formulation, in which case the required frequency of administration is reduced.

[0183] For administration of the antibody, the dosage may be about 0.001-100 mg / kg host body weight. An exemplary treatment regimen involves administration once per week.

[0184] A "therapeutically effective amount" of the compositions of the present application results in a reduction in the severity of disease symptoms and an increase in the frequency and duration of symptom-free periods. For example, for the treatment of a tumor-bearing subject, a "therapeutically effective amount" inhibits tumor growth by at least about 20%, at least about 40%, even at least about 60%, and more particularly at least about 80%, compared to an untreated subject. A therapeutically effective amount of a therapeutic antibody can reduce tumor size or alleviate symptoms in a subject, which can be a human or another mammal.

[0185] The composition can be a sustained release formulation, including implants, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. The compositions can be administered via medical devices, such as (1) needle-free subcutaneous injection devices (e.g., U.S. Patents 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) microinfusion pumps (U.S. Patent 4,487,603); (3) transdermal delivery devices (U.S. Patent 4,486,194); (4) push injection devices (U.S. Patents 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Patents 4,439,196 and 4,475,196).

[0186] In certain embodiments, the components of the compositions of the present application may be formulated to ensure appropriate distribution in vivo. For example, to ensure that the therapeutic components of the compositions of the present application cross the blood-brain barrier, the components may be formulated in liposomes, which may also additionally contain targeting functional groups to enhance selective delivery to specific cells or organs.

[0187] Various aspects and embodiments of the present application will be discussed with reference to the accompanying drawings and the following examples. Other aspects and embodiments will be clear to those skilled in the art. All documents described herein are incorporated herein by reference in their entirety. Although the present application has been described in conjunction with the exemplary embodiments, many equivalent modifications and variations will be clear to those skilled in the art when the present application is given. Thus, the exemplary embodiments of the present application are exemplary and non-restrictive. Various changes may be made to the embodiments without departing from the purpose and scope of the present application.

[0188] Example 1. Animal immunization and serum titer determination

[0189] The (G4S)3 (SEQ ID NO: 68) polypeptide was added to a maleimide-activated bovine serum albumin (BSA) carrier protein at a molar ratio of 35:1 and reacted at room temperature for 2 hours to couple the (G4S)3 polypeptide to BSA. (G4S)3-BSA was then used as an antigen for animal immunization. Specifically, three New Zealand rabbits, designated R13087#, R13088#, and R13089#, were subcutaneously immunized with an emulsion containing 200 μg of (G4S)3-BSA and 200 μl of Freund's complete adjuvant (Sigma-Aldrich, CAT#: F5881). The rabbits were boosted four times every two weeks with subcutaneous injections of an emulsion containing 200 μg of (G4S)3-BSA and Freund's incomplete adjuvant (Sigma-Aldrich, CAT#: F5506).

[0190] Four days before blood collection, the serum titer of the animals was tested by indirect ELISA. Specifically, the (G4S)3 polypeptide was added to the maleimide-activated ovalbumin (OVA) carrier protein at a molar ratio of 35:1, and the reaction was carried out at room temperature for 2 hours to couple the (G4S)3 polypeptide to OVA. 1 μg / ml of (G4S)3 polypeptide or 1 μg / ml of (G4S)3-OVA was added to the ELISA plate according to 100 μl wells and coated overnight at 4°C. After washing the ELISA plate once with PBST (PBS and 0.05% Tween by volume), PBST containing 1% BSA (mass / volume percentage) was added to 200 μl wells and blocked at 37°C for 2 hours. Then discard the blocking solution, add 100 μl of gradient diluted animal serum to each well (the first well is diluted with PBST containing 1% BSA at a ratio of 1:1000, and then diluted in a 2-fold gradient), and incubate at 37°C for 1 hour. After washing the ELISA plate four times with PBST, add 100 μl of HRP-conjugated anti-rabbit IgG Fc secondary antibody (GenScript, CAT#: A01856, diluted 1:20000 in PBST containing 1% BSA) and incubate at 37°C for 0.5 hours. After washing the ELISA plate four times with PBST, add TMB colorimetric solution and incubate at room temperature in the dark for 15 minutes. Finally, add 50 μl of 0.5M HCl stop solution to terminate the reaction. Use an enzyme reader to read the plate at 450nm. The criterion for determining the effective titer of serum is the OD value of the diluted serum. 450 The reading is greater than the blank background OD 450 2.1 times the reading.

[0191] The results showed that sera from R13087# and R13088# had higher titers for (G4S)3 peptide than R13089#, reaching 1:512,000. Sera from R13087# and R13088# also had higher titers for (G4S)3-OVA than R13089#, with titers exceeding 1:512,000, indicating the presence of antibodies specific for (G4S)3, including (G4S)3 in (G4S)3-OVA. In summary, R13087# and R13088# had higher titers for both (G4S)3 peptide and (G4S)3-OVA than R13089#, so these two animals were selected for further booster immunizations via intraperitoneal and intravenous injections of 400 μg (G4S)3-BSA (without adjuvant).

[0192] Example 2. Obtaining single B cells expressing antibodies

[0193] After the booster immunization, take fresh blood from the rabbit and perform separation within 4 hours after ex vivo. To isolate peripheral blood mononuclear cells (PBMCs), human peripheral blood lymphocyte separation medium (FICOLL) (Solarbio, CAT# P8900) was added to a centrifuge tube at a 1:1 ratio to the blood. The solution was added slowly and carefully to prevent mixing of the blood and the separation medium. The tube was then pre-cooled to 25°C and centrifuged at 400 × g for 30 minutes. The blood separation in the tube was observed and the PBMCs, floating in the turbid layer (white in color), were aspirated. The PBMCs were then added to a cell culture plate coated with 1 mg of (G4S)3-OVA to enrich for (G4S)3-specific memory B cells. The number of enriched cells was determined using a hemocytometer, and the cells were serially diluted until the cell density reached 5–15 cells / ml. 200 μl of the cell solution was pipetted into a 96-well plate at a density of 1–3 cells / well. After single B cells were cultured and expanded, positive clones that recognized (G4S)3 were selected and cultured further in 24-well plates.

[0194] Indirect ELISA was used to detect the binding ability of antibodies secreted by B cells to proteins containing (G4S)3. Specifically, 1 μg / mL of (G4S)3-OVA was added to the ELISA plate at 100 μl / well and coated overnight at 4°C. After washing the ELISA plate once with PBST (containing 0.05% volume percentage Tween), PBST containing 1% BSA (mass / volume percentage) was added at 200 μl / well and blocked at 37°C for 2 hours. The blocking solution was then discarded, and 100 μl of B cell culture supernatant was added to each well, incubated at 37°C for 1 hour, and the above-mentioned positive serum (i.e., rabbit serum after booster immunization) diluted 1000 times was used as a positive control, and blank culture medium was used as a negative control. After washing the plate four times with PBST, 100 μl / well of HRP-conjugated anti-rabbit IgG Fc secondary antibody (GenScript, CAT#: A01856, diluted 1:20,000 in PBST containing 1% BSA) was added and incubated at 37°C for 0.5 h. The plate was washed four times with PBST and then TMB colorimetric solution was added. The plate was incubated at room temperature in the dark for 15 min. Finally, 50 μl / well of 0.5 M HCl stop solution was added to terminate the reaction, and the plate was read at 450 nm using a microplate reader.

[0195] The results showed that the OD of the positive control 450 The reading was 2.784, and the OD of the negative control 450 The reading value was 0.073. Indirect ELISA detected a total of 8 positive clones, namely R177.B8, R177.B11, R177.D13, R177.E4, R177.G3, R177.G5, R177.G12 and R177.I8, with an OD of 0. 450The readings were 2.55, 0.899, 2.539, 1.668, 0.8, 1.005, 2.447, and 1.118 respectively.

[0196] Example 3. Sequencing of the variable regions of antibodies and preparation of recombinant antibodies

[0197] Total RNA was extracted from B cells using TRIzol (Ambion, CAT#: 15596-026) and PCR amplified using antibody subtype-specific primers and universal primers (Takara PrimeScript TM The cDNA was reverse transcribed into cDNA using a First-Strand cDNA Synthesis Kit (CAT#: 6110A). Rabbit immunoglobulin heavy and light chain variable region fragments were subsequently amplified by RACE PCR. The resulting PCR fragments were subcloned into the pMD18-T vector system (Takara, CAT#: 6011). The inserts were sequenced using vector-specific primers. The nucleotide and protein sequences of the heavy and light chain variable regions of these eight monoclonal antibodies were ultimately obtained and are listed in Tables 1 and 3.

[0198] Table 1. Sequences or SEQ ID NOs of the variable regions and CDRs of the antibodies of the present application

[0199] DNA fragments encoding the light chain variable region (nucleotide sequence set forth in SEQ ID NO: 56, 57, 58, 59, 60, 61, 62, or 63) plus the light chain constant region (nucleotide sequence set forth in SEQ ID NO: 67), as well as the heavy chain variable region (nucleotide sequence set forth in SEQ ID NO: 48, 49, 50, 51, 52, 53, 54, or 55) plus the heavy chain constant region (nucleotide sequence set forth in SEQ ID NO: 65) were synthesized and cloned into the pTT5 expression vector. After sequencing, the plasmids were co-transfected into CHO-S cells. Following transfection, the cells were cultured in a shake flask at 37°C for 6 days, and the supernatant was collected and used for antibody purification. During antibody purification, the tubing and Protein A column were first rinsed with 0.2M NaOH to remove pyrogens. The column was then equilibrated with equilibration buffer (50mM Tris, 150mM NaCl, pH 8.0). The harvested cell supernatant was diluted 1:1 with 2× equilibration buffer and filtered through a 0.22μm filter to remove insoluble particles and bacteria. After the cell supernatant was passed through the Protein A column, the column was washed with 1× equilibration buffer to remove nonspecifically bound proteins. The target antibody was then eluted using a 0.22μm filter with sterile 0.1M sodium citrate, pH 3.5. The eluate containing the target antibody was neutralized by adding one-ninth volume of sterile 1M Tris-HCl, pH 9.0, to a final pH of 7.4. The antibody was then concentrated by ultrafiltration using a 50KD ultrafiltration membrane, and the elution buffer was exchanged with PBS, pH 7.2. The antibody concentration was then measured using a Nanodrop, with an extinction coefficient of 1.43. Finally, the purified antibodies were analyzed by reducing and non-reducing polyacrylamide gel electrophoresis (SDS-PAGE) to determine their molecular weight and purity.

[0200] The molecular weight and purity of the eight antibodies were determined. Under non-reducing conditions, the molecular weight of each antibody was slightly lower than its actual molecular weight, approximately 120 kDa. Under reducing conditions, due to the destruction of disulfide bonds by the reducing agent, electrophoresis revealed molecular weights of approximately 55 kDa and 25 kDa for the heavy and light chains of the antibodies, respectively. The purity of all eight antibodies by reduced electrophoresis was greater than 90%.

[0201] Example 4. Antibody Binding Ability to Different GS Linkers

[0202] Indirect ELISA was used to evaluate the binding ability of the purified antibodies to various GS linkers.

[0203] Specifically, different GS linkers, namely (G2S)2 (SEQ ID NO: 69), (G2S)4 (SEQ ID NO: 70), (G3S)3 (SEQ ID NO: 71), G4S (SEQ ID NO: 72), (G4S)2 (SEQ ID NO: 73), and (G4S)3 (SEQ ID NO: 68), were added to an ELISA plate at a concentration of 4 μg / mL at a rate of 100 μl per well and coated overnight at 4°C. The plate was washed once with PBST (PBS containing 0.05% by volume Tween), and then 200 μl / well of PBST containing 1% BSA (mass / volume percentage) was added and blocked at 37°C for 2 hours. The blocking solution was then discarded, and 100 μl of a serially diluted antibody of the present application was added to each well and incubated at 37°C for 1 hour. After washing the plate four times with PBST, 100 μl / well of HRP-conjugated anti-rabbit IgG Fc secondary antibody (GenScript, CAT#: A01856, diluted 1:20,000 in PBST containing 1% BSA) was added and incubated at 37°C for 0.5 h. The plate was washed four times with PBST, and TMB colorimetric solution was added. The plate was incubated at room temperature in the dark for 15 min. Finally, 50 μl of 0.5 M HCl stop solution was added to terminate the reaction, and the plate was read at 450 nm using a microplate reader.

[0204] As shown in Figure 1 (AH), the eight antibodies of the present application have weak binding to the (G4S)1 linker, strong binding to (G4S)3, and varying binding strengths to the other linkers. Among them, antibody R177.B8 has strong binding to (G3S)3, (G4S)2, and (G4S)3, antibody R177.B11 has strong binding to (G2S)2, (G2S)4, (G4S)2, and (G4S)3, R177.G3 has strong binding to (G4S)2 and (G4S)3, and R177.G12 has strong binding to (G2S)4, (G4S)2, and (G4S)3. Except for the (G4S)1 linker, R177.D13, R177.E4, R177.G5, and R177.I8 have good binding effects on all tested GS linkers.

[0205] Example 5. Affinity determination of antibodies and scFv containing GS linkers

[0206] The Gator label-free bioassay system was used to test the binding ability of the eight antibodies described herein to a scFv containing a (G4S)3 linker. The linker-containing scFv was prepared based on the sequence of brolucizumab, specifically binds to VEGFA, and comprises, from N-terminus to C-terminus, a heavy chain variable region, a linker, and a light chain variable region. The amino acid sequences of the heavy and light chain variable regions are shown in SEQ ID NOs: 74 and 75, respectively.

[0207] Select protein A probe and soak it in 250μl buffer K (1×PBS+0.002% Tween 20+0.02% BSA) for 10 minutes before use. Then use buffer K to dilute the antibody of the present application to a final concentration of 5.0μg / ml, and add 200μl to each well. Dilute scFv with buffer K, with a starting concentration of 5.0μg / ml, 2-fold gradient dilution, a total of 3 concentrations, and the volume of each gradient sample is 200μl, which is added to the plate. Use 10mM glycine-hydrochloric acid buffer with pH 2.0 as the regeneration buffer and buffer K as the neutralization buffer. Open the instrument cover, lift the gasket to tilt shaker A, and put in the sample plate, put the probe plate into the position of shaker B, and cover the cover. Select the K Kinetics module, set Step 1 to Baseline, select the location where Buffer K is located, and set the duration to 120 seconds; set Step 2 to Sample Loading, select the location where the antibody of the present application is located, and set the duration to 120 seconds; set Step 3 to Baseline, select the location where Buffer K is located, and set the duration to 30 seconds; set Step 4 to Binding, select the location where the scFv is located, and set the duration to 200 seconds; set Step 5 to Dissociation, select the location where Buffer K is located, set the duration to 300 seconds, and set the number of regenerations to 3. Then run the program to test the binding affinity of the antibody of the present application to the scFv containing the GS linker.

[0208] The results are shown in Table 2. The affinities of the scFv containing (G4S)3 and 8 antibodies, namely R177.B8, R177.B11, R177.D13, R177.E4, R177.G3, R177.G5, R177.G12, and R177.18, are all at the nmol level, which are 0.334 nmol, 3.26 nmol, 2.07 nmol, 1.95 nmol, 2.79 nmol, 2.82 nmol, 2.82 nmol, and 2.80 nmol, respectively, indicating that the 8 antibodies of the present application all have high affinities for (G4S)3-scFv.

[0209] Table 2. Binding affinity of 8 antibodies to scFv containing (G4S)3

[0210] Example 6. Antibody Detection of scFv Containing a GS Linker in Western Blot

[0211] To 1×10 6 HEK-293 cells were added with 200 μL of RIPA lysis buffer (Biyuntian, CAT#: P0013B), mixed, and incubated at 4°C for 30 min. Subsequently, the cells were centrifuged at 12,000 × g for 15 min. The supernatant was collected and the protein concentration was determined using a BCA kit (Thermo, CAT#: 23225) according to the instructions. The supernatant was then diluted with RIPA lysis buffer to a protein concentration of 2 mg / mL.

[0212] Use PBS to dilute the (G4S)3-containing scFv used in Example 5 to 20 μg / mL. The obtained HEK-293 cell lysis supernatant dilution was mixed with PBS at a volume of 1:1, and recorded as sample 1; the obtained scFv liquid was mixed with PBS at a volume of 1:1, and recorded as sample 2; the obtained HEK-293 cell lysis supernatant dilution was mixed with the obtained scFv liquid at a volume of 1:1, and recorded as sample 3. Add half the volume of 4× loading buffer (GenScript, CAT#: M00676) and half the volume of PBS to samples 1, 2, and 3, mix well, and place in a 100°C metal bath for 10 minutes. Then load the sample at 10 μL / well and perform polyacrylamide gel electrophoresis. After the electrophoresis is completed, use eBlot TM The proteins were transferred to a PVDF membrane using the L1 Rapid Transfer System. Each of the eight antibodies listed in this application was used as the primary antibody at 0.5 μg / mL. The membrane was incubated at room temperature for 2 hours, followed by three 5-minute washes with PBST. The membrane was then incubated at room temperature for 1 hour with a 0.1 μg / mL HRP-conjugated anti-rabbit IgG Fc secondary antibody (GenScript, CAT#: A01856). The membrane was then washed three times with PBST for 5 minutes each. Finally, the membrane was exposed using ECL chemiluminescent solution (TANON, CAT#: 180-5001).

[0213] The results are shown in Figure 2 (AH). All eight antibodies of the present application effectively detected 50 ng of scFv containing a (G4S)3 linker, indicating that the detection sensitivity of the antibodies of the present application is high. In addition, no obvious non-specific bands were observed when the sample was HEK-293 lysate supernatant, indicating that the antibodies of the present application have high binding specificity.

[0214] Example 7. Antibody detection of proteins containing GS linkers in flow cytometry

[0215] The sequences of rituximab targeting CD20 and muromonab targeting CD3 were used to construct a bispecific antibody. The specific structure is rituximab VL-(G4S)3 linker-VH-(G4S) linker-muromonab VH-(G4S)3 linker-VL-His tag. The amino acid sequences of the heavy chain and light chain variable regions of rituximab and the heavy chain and light chain variable regions of muromonab are shown in SEQ ID NOs: 76, 77, 78, and 79, respectively.

[0216] Take 5×10 5 Raji cells were washed once with 500 μl FACS buffer (PBS + 1% BSA), centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in 200 μl FACS buffer and 2 μg of the above-mentioned bispecific antibody was added. The cells were incubated at room temperature for 20 minutes, and then the cells were washed once with 500 μl FACS buffer and centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in 200 μl FACS buffer and 2 μg of the antibodies R177.B8, R177.D13, R177.G12 of the present application or FITC-labeled mouse anti-His positive control antibody (GenScript, CAT#: A01620) were added and incubated at room temperature for 20 minutes. The cells were washed once with 500 μl FACS buffer and centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. 200 μl of FACS buffer was added to resuspend the cells, and 2 μg of FITC-labeled anti-rabbit IgG Fc secondary antibody (Jackson, CAT#: 111-095-008) was added to the cells incubated with the antibody of the present application. The reaction was carried out at room temperature for 20 minutes, and the proportion of fluorescent cells in the total cells was detected by flow cytometry.

[0217] As shown in Figure 3, the proportion of positive cells detected by the antibodies R177.B8, R177.D13, and R177.G12 of the present application was 100%, 99.9%, and 99.6%, respectively, which is consistent with the positive control anti-his antibody. It can be seen that the antibodies of the present application can detect proteins containing GS linkers in flow cytometry.

[0218] Example 8. Antibody detection of lentiviral transduction titer

[0219] A lentivirus carrying a gene encoding a chimeric antigen receptor (CAR) containing a scFv was serially diluted and used to infect HEK-293 cells to obtain CAR-expressing HEK-293 cells. The scFv contained in the CAR contains a (G4S)3 linker between the heavy chain variable region and the light chain variable region.

[0220] Specifically, HEK-293 cells were evenly seeded at a density of 40,000 cells / well in a 24-well plate, with 480 μL of culture medium per well. The concentrated lentivirus was serially diluted using serum-free DMEM medium at a ratio of 1:81, 1:243, 1:729, and 1:2187. After vortexing and mixing, 20 μL was added to the 24-well plate seeded with HEK-293 cells. After 12 hours of infection, the culture medium was replaced with complete culture medium. After infection for another 48 hours, the cells were washed once with 500 μL of FACS buffer (PBS + 1% BSA), centrifuged at 1000 rpm for 5 minutes, and the supernatant discarded. The cells were resuspended in 100 μL of FACS buffer and 1 μg of FITC-labeled R177.B8, R177.D13, or R177.G12 antibodies were added and incubated at room temperature for 20 minutes. The cells were washed once with 500 μL of FACS buffer, centrifuged at 1000 rpm for 5 minutes, and the supernatant discarded. The cells were resuspended in 100 μl FACS buffer and the CAR positivity rate (i.e., the percentage of fluorescent cells in total cells) was detected by flow cytometry to calculate the viral transduction titer.

[0221] Among them, the antibody was FITC labeled according to the following operation. FITC (SIGMA-ALDRICH, CAT#: F7250-100MG) was dissolved in DMF to 1 mg / mL, and then FITC was added to the antibody R177.B8, R177.D13, or R177.G12 of the present application at a mass ratio of FITC to antibody of 0.2:1, and the reaction was carried out at room temperature for 2 hours. The reaction solution was then placed in an ultrafiltration tube and centrifuged at 4°C and 12000×g for 10 minutes. After the centrifugation, the liquid in the outer tube was removed, and the inner tube was filled with PBS. The centrifugation-addition of PBS process was repeated three times, and the antibody-FITC reaction buffer was replaced with PBS to complete the labeling of FITC on the antibody.

[0222] As shown in Figure 4, after HEK-293 cells were combined and stained with the antibodies of the present application, HEK-293 cells infected with different numbers of viruses were clearly divided into groups. As the virus dilution factor increased, the proportion of fluorescent cells gradually decreased, which is consistent with the theoretical decrease in the number of cells infected by the virus or the gradual decrease in CAR expression on the cells. When cells were infected with viruses of the same dilution factor, the CAR positivity rates detected by the three antibodies of the present application were basically the same. According to the titer calculation formula: virus titer = (CAR-positive cell ratio × starting cell number × virus dilution multiple) / (virus volume), for example, the virus titer of a 1:2187-fold dilution detected using R177.B8 = (5.55% × 40,000 × 2187 times) / (0.02 mL) = 2.43E+08TU / mL, and the virus titer of a 1:729-fold dilution detected using R177.B8 = (13.3% × 40,000 × 729 times) / (0.02 mL) = 1.94E+08TU / mL. After taking the average value, the virus transduction titer detected using R177.B8 was calculated to be 2.19E+08TU / mL. Similarly, the viral transduction titers detected by R177.D13 and R177.G12 antibodies were calculated to be 2.09E+08TU / mL and 1.97E+08TU / mL, respectively, showing high consistency, indicating that the screened antibodies can be used to detect lentiviral transduction titers.

[0223] Example 9. Antibody Detection of CAR-T Cells

[0224] Using the same procedure as in Example 8, part of the antibody was labeled with iFluor 647 (AAT Bioquest, CAT#: 1031).

[0225] The same steps as in Example 8 were used to label part of the antibody with FITC.

[0226] In addition, some antibodies were PE-labeled. The antibody reduced with TECP (Thermo, CAT#: 20490) was added to phycoerythrin (PE) activated with SMCC (Thermo, CAT#: 22322) at a mass ratio of 0.5:1 and reacted overnight at 4°C. After the reaction, the reaction solution was placed in an ultrafiltration tube and centrifuged at 4°C and 12,000 × g for 10 minutes. After centrifugation, the liquid in the outer tube was removed and the inner tube was filled with PBS. The centrifugation-PBS refilling process was repeated three times to replace the antibody-PE reaction buffer with PBS to complete the PE labeling on the antibody.

[0227] Take 1×10 6T cells, comprising 40% CAR-positive T cells and 60% CAR-negative T cells (CAR scFv with a (G4S)3 linker), were washed once with 500 μl FACS buffer (PBS + 1% BSA), centrifuged at 500 × g for 5 minutes, and the supernatant discarded. The cells were resuspended in 200 μl FACS buffer and 2 μg of iFluor647-labeled R177.G5, PE-labeled R177.I8, FITC-labeled protein L (Acro, CAT#: RPL-PF141), or iFluor 647-labeled anti-Fab antibody (Jackson, CAT#: 109-605-006) were added and incubated at room temperature for 20 minutes. The cells were then washed once with 500 μl FACS buffer, centrifuged at 500 × g for 5 minutes, and the supernatant discarded. The cells were resuspended in 200 μl FACS buffer and the proportion of positive cells (i.e., the proportion of fluorescent cells to total cells) was determined by flow cytometry.

[0228] As shown in the results of Figure 5, the antibodies, anti-Fab antibodies and protein L of the present application can all characterize CAR-T cells. However, the CAR-T cell positivity rate detected by protein L was 52.75%, which is quite different from 40%, with low accuracy, and problems such as poor clustering effect, high background value, and high non-specific adsorption. The positive rates detected by anti-Fab antibodies, R177.G5, and R177.I8 were 38.71%, 39.26%, and 38.22%, respectively, which are all close to 40%. It can be seen that these antibodies can accurately detect the positive rate of CAR-T cells. In contrast, the cell clustering effect after staining with the antibody of the present application is significantly better than that of the anti-Fab antibody, which is conducive to the judgment of the positive cell population.

[0229] In summary, the GS linker antibody of the present application is superior to protein L and anti-Fab antibodies in the characterization of CAR-T cells.

[0230] Example 10. Antibody Purification of scFv Containing a GS Linker

[0231] The antibody of the present invention is used to purify a scFv containing a (G4S)3 linker. The linker-containing scFv is prepared based on the sequence of FMC63, which targets CD19. From the N-terminus to the C-terminus, it comprises a light chain variable region, a linker, and a heavy chain variable region. The amino acid sequences of the light chain variable region and the heavy chain variable region are shown in SEQ ID NOs: 80 and 81, respectively.

[0232] The antibody R177.B8 of the present invention was coupled to the purification medium CNBr Focurose 4FF (Huiyan Bio, CAT#: HQ030301025M) at different mass-to-volume ratios (antibody: medium = 10:1; 15:1; 20:1) to obtain an antibody-coupled purification medium. 100 μL of the antibody-coupled purification medium was added to the gravity column. After the liquid was drained, 1 mL of Escherichia coli lysate containing a (G4S)3 linker scFv was added to the gravity column. After the liquid was drained, 1 mL of PBS was added to wash the purification medium twice. 0.1 M glycine (pH 3.0) was then used for elution, the eluate was collected, and neutralized with 1 M Tris-HCl (pH 9.0). The protein concentration was quantified using a BCA kit (Thermo, CAT#: 23225) according to the operating procedures in the instructions.

[0233] Using 4-20% precast gel (GenScript, CAT#: M00656), the purified scFv containing G4S)3 linker at different coupling ratios (antibody: medium = 10:1; 15:1; 20:1) were analyzed by SDS-PAGE electrophoresis. The antibodies were stained using an L1 protein stainer (GenScript, CaT#: L00657C), and molecular size and purity were estimated by comparing the stained bands with a protein marker (GenScript, CaT#: M00624). As shown in Figure 6, the molecular weight of the antibodies purified at different conjugation ratios was approximately 28 kD, consistent with the expected molecular weight, and the protein purity was ≥95%, indicating that the screened antibodies can be used to purify scFvs containing a GS linker. Lane 1 shows a protein marker, Lane 2 shows a (G4S)3 linker-containing scFv purified using a conjugation medium with a 10:1 antibody: medium ratio; Lane 3 shows a (G4S)3 linker-containing scFv purified using a 15:1 antibody: medium ratio; and Lane 4 shows a (G4S)3 linker-containing scFv purified using a 20:1 antibody: medium ratio.

[0234] Table 3. Some amino acid and nucleotide sequences mentioned in the article

[0235] Although the present application has been described in conjunction with one or more embodiments, it should be understood that the present application is not limited to these embodiments. The description in this application is intended to cover all variants and equivalents, all of which are included in the subject matter and scope of the appended claims. All documents cited in this article are incorporated herein by reference in their entirety.

Claims

1. An isolated monoclonal antibody or antigen-binding portion thereof, which is capable of specifically binding to a GS linker, comprising i) a heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3, and ii) a light chain variable region comprising VL CDR1, VL CDR2 and VL CDR3, wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively comprise: (1) NYAIM (SEQ ID NO: 1), VIYANGDPYCASWAKG (SEQ ID NO: 5), GGF, QSSQSIYNKNDLV (SEQ ID NO: 14), GISTLDS (SEQ ID NO: 20), and LGGSCSSSGDCGA (SEQ ID NO: 25); (2) NYDMY (SEQ ID NO: 2), VSYKNGRAHYASWAKG (SEQ ID NO: 8), GPL, QASQSVWNNNDLV (SEQ ID NO: 17), DASTLSS (SEQ ID NO: 21), and LGGFSCSTGDCGA (SEQ ID NO: 28); (3) NYDMY (SEQ ID NO: 2), VSYASGRTYSVRWAKG (SEQ ID NO: 10), GPL, QASQTVWKNNDLV (SEQ ID NO: 18), DASTLSS (SEQ ID NO: 21), and LGGFSCSSGDCGA (SEQ ID NO: 25); (4) NYDMY (SEQ ID NO: 2), VIYKNGNAHSASWARG (SEQ ID NO: 12), GPF, QASQSVWNNNDLV (SEQ ID NO: 17), DASTLSS (SEQ ID NO: 21), and LGGFSCSRGDCGS (SEQ ID NO: 31); (5) NHAIM (SEQ ID NO: 3), VIYSNGNPYCARWVKG (SEQ ID NO: 7), GGF, KTSQSTYNNNDLV (SEQ ID NO: 16), GVSTLDS (SEQ ID NO: 22), and LGGFRCSSGDCGA (SEQ ID NO: 27); (6) NYDMY (SEQ ID NO: 2), VIYVNGNTHYASWAKG (SEQ ID NO: 6), GPF, QASQSVWKNKDLV (SEQ ID NO: 15), DASTLSS (SEQ ID NO: 21), and LGGFSCSRGDCGA (SEQ ID NO: 26); Or (8) The amino acid sequences represented by TNAMT (SEQ ID NO: 4), TITISGNKYYASWAKG (SEQ ID NO: 11), GVVQSLVL (SEQ ID NO: 13), RSSQNVYNNNGLG (SEQ ID NO: 19), DAADLAS (SEQ ID NO: 24), and AGGYSSGSIDNT (SEQ ID NO: 30), or amino acid sequences comprising 1 to 3 amino acid substitutions in each CDR compared to the above amino acid sequences.

2. The antibody or antigen-binding portion thereof of claim 1, wherein the heavy chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 32, 35, 37, 39, 34, 33, 36, or 38.

3. The antibody or antigen binding portion thereof of claim 1, wherein the light chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 40, 43, 45, 47, 42, 41, 44, or 46.

4. The antibody or antigen-binding portion thereof of claim 1, wherein the heavy chain variable region and the light chain variable region comprise an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to (1) SEQ ID NOs: 32 and 40; (2) SEQ ID NOs: 35 and 43; (3) SEQ ID NOs: 37 and 45; (4) SEQ ID NOs: 39 and 47; (5) SEQ ID NOs: 34 and 42; (6) SEQ ID NOs: 33 and 41; (7) SEQ ID NOs: 36 and 44; or (8) SEQ ID NOs: 38 and 46.

5. The antibody or antigen-binding portion thereof according to claim 1, further comprising a heavy chain constant region and / or a light chain constant region. The antibody or antigen-binding portion thereof according to claim 1 , further comprising a detection substance.

7. The antibody or antigen-binding portion thereof according to claim 6, wherein the detection substance is a fluorescent marker, biotin or a chemiluminescent marker.

8. The antibody or antigen-binding portion thereof according to claim 1, which is a rabbit-derived antibody, a chimeric antibody, or a humanized antibody.

9. An isolated bispecific molecule comprising the antibody or antigen binding portion thereof of any one of claims 1-8.

10. The bispecific molecule of claim 9, further comprising an antibody or antigen-binding portion thereof that targets a disease-associated antigen.

11. A chimeric antigen receptor comprising: a) an extracellular domain comprising an antibody in the form of a single-chain antibody according to any one of claims 1 to 4 and 8, b) transmembrane region, and c) Intracellular signaling domain.

12. A nucleic acid molecule encoding the antibody or antigen binding portion thereof of any one of claims 1-8, the bispecific molecule of claim 9 or 10, or the chimeric antigen receptor of claim 11.

13. The nucleic acid molecule according to claim 12, comprising the nucleotide sequence shown in any one of SEQ ID NOs: 48-63.

14. An expression vector comprising the nucleic acid molecule according to claim 12 or 13.

15. A host cell comprising the expression vector according to claim 14, or having the nucleic acid molecule according to claim 12 or 13 integrated into its genome.

16. A composition comprising the antibody or antigen binding portion thereof of any one of claims 1-8, the bispecific molecule of claim 9 or 10, the chimeric antigen receptor of claim 11, the nucleic acid molecule of claim 12 or 13, the expression vector of claim 14, or the host cell of claim 15.

17. The composition of claim 16, wherein the antibody or antigen-binding portion thereof comprises a chemiluminescent label, and the composition further comprises a reagent that causes the chemiluminescent label to emit light.

18. The composition of claim 16, wherein the antibody or antigen-binding portion thereof comprises an Fc region, and the composition further comprises an antibody that specifically binds to the Fc region and contains a detection substance.

19. The composition of claim 18, wherein i) the detection substance contained in or coupled to the antibody that specifically binds to the Fc region is a fluorescent marker; or ii) the detection substance contained in the antibody that specifically binds to the Fc region is a chemiluminescent marker, and the composition further comprises a reagent that makes the chemiluminescent marker emit light.

20. The composition of claim 16, comprising: i) the bispecific molecule of claim 9 or 10, and ii) a T cell expressing a chimeric antigen receptor, wherein the chimeric antigen receptor comprises a) an extracellular domain comprising a non-antigen binding single chain antibody comprising a GS linker, b) transmembrane region, and c) an intracellular signaling domain; or i) a T cell expressing the chimeric antigen receptor of claim 11, and ii) an antibody or antigen-binding portion thereof linked to a GS linker and targeting a disease-associated antigen.

21. A method for detecting a molecule containing a GS linker in a sample, comprising: i) contacting the sample with the antibody or antigen-binding portion thereof according to any one of claims 1 to 8, ii) detecting the presence of the antibody or antigen-binding portion thereof in a sample, The presence of the antibody or antigen-binding portion thereof in the sample indicates the presence of a molecule containing a GS linker in the sample.

22. A method for purifying a molecule containing a GS linker in a sample, comprising: i) contacting the sample with a solid phase carrier coupled to the antibody or antigen-binding portion thereof according to any one of claims 1 to 8, ii) separating the molecules in the sample that are bound to the solid phase carrier in i) from the sample, iii) eluting the molecules bound to the solid phase support from the solid phase support, The solid phase carrier comprises magnetic beads, resins, and agarose beads; and the molecules bound to the solid phase carrier are molecules containing GS linkers.

23. The method of claim 21 or 22, wherein the GS linker is (G2S)2 (SEQ ID NO: 69), (G2S)4 (SEQ ID NO: 70), (G3S)3 (SEQ ID NO: 71), (G4S)2 (SEQ ID NO: 73), or (G4S)3 (SEQ ID NO: 68).

24. The method of claim 21 or 22, wherein the molecule containing a GS linker is an antibody containing a GS linker or a cell containing a GS linker.

25. The method of claim 24, wherein the sample comprises cells treated with an antibody containing a GS linker, or comprises cells introduced with a vector, wherein the vector comprises a sequence for expressing a chimeric antigen receptor containing a GS linker.

26. The method of claim 21 or 22, wherein the GS linker-containing molecules in the sample are quantified based on the amount of the antibody or antigen-binding portion thereof in the sample.

27. Use of the composition of claim 20 in the preparation of a medicament for treating a disease associated with a disease-associated antigen targeted by the bispecific molecule or the antibody or antigen-binding portion thereof containing a GS linker and targeting a disease-associated antigen in the composition.

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