WO2025119330A1 - Molecule binding to amino acid linker and use thereof - Google Patents

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

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese patent application number CN202311692347.2 and filing date December 8, 2023, the entire contents of which are incorporated herein by reference.

Field of the Invention

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

In recombinant biomacromolecules, two or more functional modules are often connected by a linker to form an overall structure. For example, a linker can be used to connect proteins, nucleic acids, and/or polypeptides to achieve a specific functional combination or enhancement. In some cases, a linker also serves to separate two or more functional modules so that each module can perform its own function without interfering with each other.

The choice of linker in recombinant biomacromolecules depends mainly on the type of molecules to be connected, the stability requirements of the connection, and the specific needs of subsequent applications. Amino acid linkers, such as glycine-serine linkers (i.e., GS linkers), are one of the most commonly used linkers, which can be designed into different sequence combinations and lengths in the form of (GnS)m, where n and m can be integers with a minimum of 1. ( _G2S ) ₂ , ( _G2S ) ₄ , ( _G3S ) ₃ , ( _G4S ) ₁ , ( _G4S ) ₂ , ( _G4S ) ₃ are several commonly used GS linkers, which are often used to connect different functional domains of proteins or polypeptides and have the characteristics of flexibility and ductility. These linker sequences are widely used in many research fields, such as protein engineering, drug delivery systems, etc.

GS linkers are often used to connect the heavy chain variable region (VH) and light chain variable region (VL) in single-chain antibodies (scFv). Compared with the classic IgG antibody molecule, scFv has the advantages of small molecular weight, strong penetration, low immunogenicity, and high specificity. Therefore, it has received more and more attention in the application of targeted therapy, imaging diagnosis, cell therapy, etc. In targeted therapy, drugs or toxins can be connected to scFv, and the drugs can be delivered to the target tissue with the help of the specificity and low immunogenicity of scFv, thereby achieving specific killing of target cells. In imaging diagnosis, due to the strong penetration and short half-life of scFv, its distribution index in tumor tissue is high compared with full-length antibodies during radiological imaging, and it is less harmful to the body. In cell therapy, scFv, as the extracellular domain of CAR in chimeric antigen receptor T cells (CAR-T), determines the targeting and efficacy of CAR-T cells. In biological monitoring, due to the high affinity and easy preparation of scFv, it is often used to monitor harmful substances such as water sources and food. In addition, due to its simple structure, scFv is often used in the construction of bispecific antibodies.

GS linkers are also often 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.

Many recombinant proteins containing GS linkers, such as the above-mentioned scFv, or CARs or multispecific antibodies containing two or more nanobodies, do not contain Fc regions, so the most commonly used universal Fc targeting antibodies cannot be used to detect the presence of these recombinant antibodies or quantify them. At present, protein L, anti-Fab antibodies, etc. are usually used for the detection of proteins containing scFv, but they all have certain limitations. For example, protein L mainly targets the light chain of the κ subtype, but cannot detect other subtypes, and has problems such as high nonspecificity and low detection rate. Anti-Fab antibodies are mostly polyclonal antibodies, with large batch differences, and have problems such as low versatility and poor specificity. Therefore, other means with high specificity and good versatility are needed to detect scFv antibodies, etc.

Summary of the invention

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 with the traditional means of using protein L, anti-Fab antibodies, target antigens, antibodies targeting idiotypic antibodies, etc., the antibodies of the present application i) have a certain versatility, that is, they are suitable for 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 suitable for most GS connectors except (G ₄ S) ₁ , ii) have a higher binding specificity for proteins containing GS connectors, the detection accuracy is higher than the protein L detection method, and the ability to group CAR-T cells based on scFv containing GS connectors is stronger than anti-Fab antibodies, iii) have a higher binding sensitivity to proteins containing GS connectors, and can detect proteins containing 2pmol of GS connectors.

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 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 (referred to as disease-targeted antibody) with a GS connector and targeting disease-related antigens can be constructed, so that T cells expressing anti-GS-CAR can recognize cells expressing disease-related antigens through disease-targeted antibodies, and trigger attacks on cells expressing disease-related antigens. Alternatively, a GS connector (referred to as GS connector-CAR) can be included in the extracellular domain of CAR, and a bispecific molecule (referred to as GS connector + disease dual-targeted antibody) comprising the GS antibody of the present application or its antigen binding portion and an antibody or its antigen binding portion targeting disease-related antigens can be constructed, so that T cells expressing GS connector-CAR can recognize cells expressing disease-related antigens through GS connector + disease dual-targeted antibodies, and trigger attacks on cells expressing disease-related antigens. Constructing the above-mentioned universal CAR-T cells, and matching corresponding disease-targeted antibodies or GS connectors + disease dual-targeted antibodies, can reduce the cost of cell therapy to a certain extent.

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, 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 a GS linker with a 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.

Thus, in a first aspect, the present application provides an isolated monoclonal antibody or an antigen-binding portion thereof, which is capable of specifically binding to a GS linker. The antibody or antigen-binding portion thereof of the present application may be rabbit-derived, chimeric, or humanized.

The antibody or its antigen-binding portion of the present application may include i) a heavy chain variable region, which may contain VH CDR1, VH CDR2 and VH CDR3, wherein VH CDR1, VH CDR2 and VH CDR3 may respectively include (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), 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) Q SSQSTYNKNDLV (SEQ ID NO: 14), GISTLDS (SEQ ID NO: 20), and LGGFSCSSGDCGA (SEQ ID NO: 25); (2) Q ASQSVWNNNDLV (SEQ ID NO: 17), DASTLSS (SEQ ID NO: 21), and LGGFSCSTGDCGA (SEQ ID NO: 28); (3) QA SQTVWKNNDLV (SEQ ID NO: 18), DASTLSS (SEQ ID NO: 21), and LGGSCSSSGDCGA (SEQ ID NO: 25); (4) QA SQSVWNNNDLV (SEQ ID NO: 17), DASTLSS (SEQ ID NO: 21), and LGGFSCSRGDCGS (SEQ ID NO: 31); (5) KTS The amino acid sequence shown is (SEQ ID NO: 14), (SEQ ID NO: 15), (SEQ ID NO: 21), and (SEQ ID NO: 26); (7) QASQSVWNNNDLV (SEQ ID NO: 17), (SEQ ID NO: 23), and (SEQ ID NO: 29); or (8) RSSQNVYNNNGLG (SEQ ID NO: 19), (SEQ ID NO: 24), and (SEQ ID NO: 30). Also provided are variants of the above antibodies or antigen-binding portions comprising up to about 3 amino acid residue substitutions, such as 1, 2, or 3 amino acid residue substitutions, in each CDR compared to the above antibodies or antigen-binding portions thereof.

The isolated monoclonal antibody or its antigen-binding portion of the present application may 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 may 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: 10), GFSQSIYNKNDLV (SEQ ID NO: 11), GISTLDS (SEQ ID NO: 12), and LGGFSCSSGDCGA (SEQ ID NO: 13); 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), DAST LSS (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: 2) 1), 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 LGGFRCSSGDC GA (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) ): (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) the amino acid sequence shown in 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 comprising up to about 3 amino acid residue substitutions, such as 1, 2, or 3 amino acid residue substitutions, in each CDR compared to the above antibodies or antigen-binding portions thereof.

The heavy chain variable region of the antibody or antigen-binding portion thereof of the present application may 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 may be encoded by the nucleotide sequences shown in SEQ ID NO: 48, 51, 53, 55, 50, 49, 53 and 54, respectively.

The light chain variable region of the antibody or antigen-binding portion thereof of the present application may 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 may be encoded by the nucleotide sequences shown in SEQ ID NO: 56, 59, 61, 63, 58, 57, 60 or 62, respectively.

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 with (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.

In some embodiments, the isolated monoclonal antibody or antigen-binding portion thereof of the present application may 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 may respectively comprise the amino acid sequences shown 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). The heavy chain variable region may 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 may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 40.

In some embodiments, the isolated monoclonal antibody or antigen-binding portion thereof of the present application may 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 may respectively comprise the amino acid sequences shown 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). The heavy chain variable region may 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 may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 43.

In some embodiments, the isolated monoclonal antibody or antigen-binding portion thereof of the present application may 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 may respectively comprise the amino acid sequences shown 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). The heavy chain variable region may 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 may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 45.

In some embodiments, the isolated monoclonal antibody or antigen-binding portion thereof of the present application may 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 may respectively comprise the amino acid sequences shown 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). The heavy chain variable region may 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 may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 47.

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 connected to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is connected 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, CH ₂ and CH ₃ of the heavy chain constant region. In one embodiment, the heavy chain constant region may comprise the amino acid sequence shown 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 shown in SEQ ID NO: 66. The amino acid sequences shown in SEQ ID NOs: 64 and 66 may be encoded by the nucleotide sequences shown in SEQ ID NOs: 65 and 67, respectively.

The antibody or antigen-binding portion thereof of the present application comprises two heavy chains and two light chains in some embodiments, or is composed of two heavy chains and two light chains, wherein each heavy chain comprises the above-mentioned heavy chain constant region sequence, heavy chain variable region sequence and/or CDR sequence, and each light chain comprises the above-mentioned light chain constant region sequence, light chain variable region sequence and/or CDR sequence. In some embodiments, the antibody or antigen-binding portion thereof of the present application can be Fab, F(ab′) ₂ fragment, Fv, scFv or (scFv) ₂ , etc.

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).

The antibody or antigen-binding portion thereof of the present application may contain a detection substance. 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 contain 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 (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 may contain 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.

The present application also provides a bispecific molecule, which comprises the antibody or antigen binding portion thereof that specifically binds to the GS linker of the present application. For example, the bispecific molecule of the present application may comprise i) the antibody or antigen binding portion thereof that specifically binds to the 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, fused together by recombinant expression, or combined 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 of the present application, the antibody or antigen binding portion thereof that specifically binds to the GS linker of the present application may be humanized. In particular, in the bispecific molecule of the present application, the antibody or antigen binding portion thereof that targets a disease-associated antigen may be humanized or humanized.

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

The present application also includes a nucleic acid molecule encoding the antibody or its antigen-binding portion, bispecific molecule, or chimeric antigen receptor of the present application. The nucleic acid molecule of the present application may include a nucleotide sequence shown in any one of SEQ ID NO: 48-63. The present application may also provide an expression vector and a host cell. The expression vector may include the nucleic acid molecule of the present application. The host cell may include the expression vector of the present application, or the nucleic acid molecule of the present application may be integrated into its genome.

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

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.

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., recombinantly expressed), 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 a heavy chain constant region, a light chain constant region, a framework region of a heavy chain variable region, or a framework region of a light chain variable region. In particular, the antibody or its antigen binding portion may include a detection substance in the framework region of a heavy chain constant region or a light chain constant region.

In some embodiments, the antibody or 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., H ₂ O ₂ ) and 3,3'-diaminobenzidine (DAB), or peroxide peroxide (e.g., H ₂ O ₂ ) 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 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 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 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.

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

The composition 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. In some embodiments, the extracellular domain of the chimeric antigen receptor may comprise a non-antigen binding single-chain antibody, which comprises a GS linker. In some embodiments, the extracellular domain of the chimeric antigen receptor may comprise a non-antigen binding single-chain antibody, which may comprise a GS linker between its heavy chain variable region and light chain variable region.

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 more than 6 amino acids 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, (G ₂ S) ₂ , (G ₂ S) ₄ , (G ₃ S) ₃ , (G ₄ S) ₂ , and (G ₄ S) ₃ . In particular, the GS linker may be (G ₄ S) ₃ or (G ₄ S) ₂ . The antibody or antigen binding portion thereof that targets a disease-associated antigen may not comprise a GS linker.

In another aspect, the present application provides 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 of the present application,

ii) detecting the presence of the antibody or antigen-binding portion thereof in the 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.

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

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

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

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.

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

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

ii) separating the sample molecules in the sample that are bound to the solid phase carrier in i) from the sample,

iii) eluting the sample molecules bound to the solid phase carrier from the solid phase carrier,

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

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

The GS linker may be any GS linker, in particular a GS linker of more than 6 amino acids 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, (G ₂ S) ₂ , (G ₂ S) ₄ , (G ₃ S) ₃ , (G ₄ S) ₂ , and (G ₄ S) ₃ . In particular, the GS linker may be (G ₄ S) ₂ or (G ₄ S) ₃ .

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

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 a GS linker.

When the GS linker is ( _G2S ) ₂ , 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); (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) antibodies or antigen-binding portions thereof having the amino acid sequences shown 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).

When the GS linker is ( _G2S ) ₄ , 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); (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) antibodies or antigen-binding portions thereof having the amino acid sequences shown 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).

When the GS linker is (G ₃ S) ₃ , the antibodies (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) comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 in the present application, respectively; 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.

When the GS linker is (G ₄ S) ₂ , any antibody or antigen-binding portion thereof described in the present application can be selected.

When the GS linker is (G ₄ S) ₃ , any antibody or antigen-binding portion thereof in the present application can be selected, in particular, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively contain (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); (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: 2 ID 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.

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.

The antibody or its antigen binding portion of the present application may include a detection substance. For example, the antibody or its antigen binding portion of the present application may be fused with a detection substance (e.g., recombinantly expressed), or coupled with a detection substance. The antibody or its antigen binding portion of the present application may not affect any position of the antigen binding of the antibody or its antigen binding portion, such as 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 framework region includes a detection substance. In particular, the antibody or its antigen binding portion 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. The chemiluminescent marker may be a peroxidase (e.g., horseradish peroxidase (HRP)) or alkaline phosphatase (AP).

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

The method of the present application can use the antibody or antigen-binding portion of the present application containing a chemiluminescent marker, and contact the antibody or antigen-binding portion of the present application with a reagent that makes the chemiluminescent marker emit light, and judge whether the antibody or antigen-binding portion of the present application is in the sample, or how much antibody or antigen-binding portion of the present application is present, by the chemiluminescent signal. In some embodiments, the method of the present application can use the antibody or antigen-binding portion of the present application containing horseradish peroxidase, and contact the antibody or antigen-binding portion of the present application with peroxide (such as H ₂ O ₂ ) and 3,3'-diaminobenzidine (DAB), or peroxide peroxide (such as H ₂ O ₂ ) and 3,3',5,5'-tetramethylbenzidine (TMB). In some embodiments, the method of the present application can use the antibody or antigen-binding portion of the present application containing alkaline phosphatase, and contact the antibody or antigen-binding portion of the present application with 4-nitrophenylphosphate disodium salt hexahydrate (pNPP).

The method 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 by viral transduction in a sample by, for example, flow cytometry.

The present application also relates to a method for treating a disease in a subject in need thereof, comprising administering the composition of the present application to the subject, wherein the composition comprises 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, or i) a T cell expressing the chimeric antigen receptor of the present application, and ii) an antibody or an 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 in the composition or the antibody or an antigen-binding portion thereof that contains a GS linker and targets a disease-associated antigen. The disease-associated antigen may be a tumor-associated antigen.

The present application also protects the use of the antibody or antigen-binding portion thereof, or the composition of the present application in detecting or quantifying a protein containing a GS linker, in particular, detecting the presence or content of a protein containing a GS linker in a sample by, for example, Western blotting, detecting the presence or content of cells bound by a protein containing a GS linker in a sample by, for example, flow cytometry, detecting the presence or content of cells expressing a protein containing a GS linker in a sample by, for example, flow cytometry, or detecting the presence or content of cells expressing a protein containing a GS linker by viral transduction by, for example, flow cytometry in a sample.

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.

It should be noted that in the present application, especially in the claims, terms such as "comprising", "including", etc. may have the meanings assigned by the Chinese Patent Law; and terms such as "essentially composed of..." have the meanings assigned 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.

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 limiting. 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

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.

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.

Figure 2 shows the binding of the 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) of the present application to proteins containing a GS linker in Western blot detection.

FIG. 3 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.

FIG. 4 shows cells transduced with lentivirus detected by the antibodies of the present application.

FIG. 5 shows cells expressing chimeric antigen receptor with a GS linker detected by the antibodies of the present application.

FIG. 6 shows the SDS-PAGE electrophoresis results of the antibody-purified scFv containing a GS linker of the present application.

DETAILED DESCRIPTION

The terms used in this document, unless otherwise specified, have the common meanings in dictionaries, textbooks, technical reference books, or the meanings commonly understood by those skilled in the art. The following descriptions of some terms are only for the purpose of facilitating the understanding of this application, and are not intended to be particularly limited to these terms, unless otherwise specified.

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

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

The term "comprising" or "including" means that the elements, integers or steps described are included, but the addition of any other elements, integers or steps is not excluded. In the text, when the term "comprising" or "including" is used, unless otherwise specified, the combination of the elements, integers or steps described is also covered.

"GS linker" refers to a peptide composed of glycine and serine in the form of (GnS)m, wherein 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, 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 .

The "detection substance" in the present 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 cold light marker, an immunodetectable marker, a radiation marker, 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 be directly colored or enzymatically colored. By including (for example, coupling) a detection substance on the antibody or its antigen-binding portion of the present application, the antibody or its antigen-binding portion of the present application can be located or quantified.

"Coupled" in the present application may refer to a state in which two organic chemical groups are connected by a certain chemical reaction, or a state in which two substances are attached to each other by any reaction or any chemical bond. For example, the detection substance may be connected to the antibody or its antigen-binding portion by reacting with a side chain group on an amino acid of the antibody or its antigen-binding portion of the present application, or the detection substance may be attached to the antibody or its antigen-binding portion of the present application by, for example, a van der Waals bond.

The "non-antigen binding single-chain antibody" herein refers to a scFv-like peptide having 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. The "non-antigen binding single-chain antibody" may comprise a heavy chain variable region-like peptide (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 variable region-like peptide (i.e., an amino acid length similar to that of an antibody light chain variable region).

The term "antibody" herein is intended to include IgG, IgA, IgD, IgE and IgM full-length antibodies and any antigen-binding fragments thereof (i.e., antigen-binding portions). Full-length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains, the heavy chains and light chains being linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as _VH or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, namely _CH1 , _CH2 and _CH3 . Each light chain consists of a light chain variable region (abbreviated as _VL or VL) and a light chain constant region. The light chain constant region consists of one domain, _CL . _{The VH} and _VL regions can also be divided into hypervariable regions called complementarity determining regions (CDRs), which are separated by more conservative framework regions (FRs). Each _VH and _VL 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 an immunoglobulin 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. The "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.

The term "antigen-binding portion" of an antibody (or simply antibody portion) as used herein 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 a fragment of a full-length antibody. Examples of binding fragments included in the "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of _VL , _VH , _CL and _CH1 ; (ii) a F(ab') ₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) a Fd fragment consisting of _VH and _CH1 ; (iv) a scFv fragment consisting of a single-arm _VL and _VH of an antibody; (v) a dAb fragment consisting of _VHH (Ward et al., (1989) Nature 341: 544-546); (vi) an isolated complementarity determining region (CDR); and (vii) a dAb- _VL , a fragment comprising a single variable domain and a heavy chain constant domain. In addition, although the two domains _VL and _VH of the scFv fragment are encoded by different genes, they can be connected by a recombinant method via a synthetic linker that makes the two become a single protein chain, wherein the _VL and _VH regions are paired to form a monovalent molecule. 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.

"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.

"Nanobody" or " _VHH " refers to a single antigen-binding polypeptide comprising a single monomeric variable region comprising three complementarity determining regions (CDRs), which is capable of binding to antigen without pairing with other corresponding CDR-containing polypeptides.

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.

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

The term "rabbit 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 antibody of the present application may contain amino acid residues 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 antibody" does not include antibodies in which CDR sequences derived from other mammalian species are inserted into the rabbit framework sequence.

The term "chimeric antibody" refers to an antibody that combines genetic material from one species with genetic material from another species. In particular, the chimeric antibody in the present application refers to an antibody obtained by combining non-human genetic material with human genetic material.

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 antibodies naturally produced in the human body.

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

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 other amino acid peptides, and the binding affinity or binding activity to the GS linker is higher than other reference molecules, such as 1 times, 5 times, 10 times, etc. Specificity determination methods include, but are not limited to, SPR, Western blotting, ELISA, RIA, ECL, IRMA testing, and peptide scanning.

"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, and if necessary, spaces are introduced in the sequence comparison to achieve the maximum percentage of sequence identity between the two sequences. Those skilled in the art can perform pairwise sequence comparisons or multiple sequence alignments to determine the percentage of sequence identity between two or more nucleic acid or amino acid sequences by a variety of methods, such as using computer software, such as ClustalOmega, T-coffee, Kalign, and MAFFT, etc.

"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 recognizing and binding 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 a 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.

The term " _EC50 ", also known as half maximal effect concentration, refers to the concentration of drug that elicits 50% of the maximal effect.

The term " _IC50 " refers to the half inhibitory concentration, which is the concentration of a drug or inhibitor required to inhibit a specified biological process by half.

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.

The term "therapeutically effective amount" refers to an amount of the present invention molecule 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 easily determine the actual effective amount.

The antibody or antigen-binding portion thereof that specifically binds to the GS linker of the present application can bind to a variety of GS linkers with high binding force, including, but not limited to, (G ₂ S) ₂ , (G ₂ S) ₄ , (G ₃ S) ₃ , (G ₄ S) ₂ , and (G ₄ S) ₃ . In particular, the antibody or antigen-binding portion thereof of the present application can specifically bind to (G ₄ S) ₂ or (G ₄ S) ₃ .

Compared with the traditional methods of using protein L, target antigen, antibodies targeting idiotypic antibodies, etc., the antibodies or antigen-binding portions thereof of the present application i) have certain 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) 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 containing or not containing GS linker scFv, is stronger than that of anti-Fab antibodies, iii) proteins containing GS linkers have high binding sensitivity, 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.

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 determined by the Kabat numbering system, and the CDR sequences or sequence numbers determined 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 determined by the IMGT, Chothia, AbM, or Contact numbering systems based on the full-length sequence of the variable region.

The _VH and/or _VL sequences (or CDR sequences) of other antibodies that bind to the GS linker can be "mixed and paired" with the _VH and/or _VL sequences (or CDR sequences) of the antibodies of the present application. Preferably, when _VH and _VL (or CDRs therein) are mixed and paired, the _VH sequence in a specific _VH / _VL pairing can be replaced by a structurally similar _VH sequence. Similarly, it is preferred that the _VL sequence in a specific _VH / _VL pairing is replaced by a structurally similar _VL sequence.

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

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

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

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

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

(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.

In another embodiment, the antibody or antigen-binding portion thereof of the present application comprises 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.

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, e.g., 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.U.S.A.95:8910-8915 (1998); Barbas et al., J.Am.Chem.Soc.116:2161-2162(1994); Barbas et al., Proc.Nat l. Acad. Sci. US. A. 92: 2529-2533 (1995); Ditzel et al., J. Immunol. 157: 739-749 (1996).

In another embodiment, the antibody or antigen-binding portion thereof of the present application comprises a heavy chain and/or light chain variable region sequence or CDR1, CDR2 and CDR3 sequence that has one or more conservative modifications with the GS linker antibody or antigen-binding portion thereof of the present application. It is known in the art that some conservative sequence modifications do not eliminate antigen binding. See, for example, Brummell et al., (1993) Biochem 32: 1180-8.

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 respectively comprising CDR1, CDR2 and CDR3, wherein:

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

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

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

(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

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

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 replacement, addition and deletion. Modifications can be introduced into the present application antibody or its antigen-binding portion by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid replacement is the replacement of amino acid residues with amino acid residues having similar side chains. Amino acid residue groups with similar side chains are known in the art. These amino acid residue groups 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 antibody or antigen-binding portion thereof of the present application can be replaced with other amino acid residues of the same side chain group, and the resulting antibody can be tested for retention of function (i.e., the function described above) using the functional assays described herein.

The antibodies or antigen-binding portions thereof of the present application can be prepared into genetically modified antibodies using antibodies having one or more _VH / _VL sequences of the GS linker antibodies or antigen-binding portions thereof of the present application as starting materials. Antibodies can be genetically modified by modifying one or more residues within one or both variable regions (i.e., _VH and/or _VL ) (e.g., in one or more CDR regions and/or one or more framework regions) to improve binding affinity.

Variable region modifications can be mutating amino acid residues in the _VH and/or _VL CDR1, CDR2 and/or CDR3 regions 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 in vitro or in vivo assays known in the art. Preferably, conservative modifications known in the art are introduced. Mutations can be amino acid substitutions, additions or deletions, but are preferably substitutions. In addition, no more than one, two, three, four or five residues in the CDR regions are typically changed.

In addition, 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, which comprises: (a) a _VH CDR1 region comprising a sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acids substituted, deleted or added; (b) a _VH CDR2 region comprising a sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acids substituted, deleted or added; (c) a _VH CDR3 region comprising a sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acids substituted, deleted or added; (d) a _VL CDR1 region comprising a sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acids substituted, deleted or added; (e) a _VL CDR2 region comprising a sequence of the present application, or an amino acid sequence with one, two, three, four or five amino acids substituted, deleted or added; and (f) a _VL 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.

The hinge region of antibody _CH1 can be modified, altered, for example, by increasing or decreasing the number of cysteine residues in the hinge region. This method is further described in U.S. Pat. No. 5,677,425. The cysteine residues in the hinge region of _CH1 can be altered, for example, to facilitate the assembly of heavy and light chains or to increase/decrease the stability of the antibody.

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, for example, to increase the affinity of the antibody for the antigen. Such glycosylation modification can be achieved, for example, by changing 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 N-X-S/T sequences. 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 the residues in the glycosylation region.

In a preferred embodiment, the antibody or antigen-binding portion thereof does not contain an asparagine isomerism site. Deamidation of asparagine may occur in the N-G or D-G sequence, creating an isoaspartic acid residue, which introduces a kink into the polypeptide chain and reduces its stability (isoaspartic acid effect).

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 the phage coat protein structural gene through genetic engineering technology, and correctly express it in the reading frame, so that the exogenous polypeptide or protein (such as scFv) forms a fusion protein on the capsid protein of the phage, and is presented on the phage surface with the reassembly of the progeny phage. Then, using the target molecule (such as GS linker), a suitable elutriation method is adopted to wash away the phage that does not specifically bind to the target molecule. Then, the bound phage is eluted with acid, alkali or competitive molecules, and the neutralized phage is infected with Escherichia coli for amplification. After 3-5 rounds of enrichment, the phage ratio that can specifically identify the target molecule is gradually increased, and the polypeptide or protein that recognizes the target molecule is finally obtained.

Other methods for preparing monoclonal antibodies include somatic cell hybridization (hybridoma), viral or oncogenic transformation of B lymphocytes, etc. 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 produced in host cell transfectomas using, for example, recombinant DNA technology combined with gene transfection methods (e.g., Morrison, S. (1985) Science 229: 1202). In one embodiment, 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 genes are operably linked to transcription and translation regulatory sequences. In this case, the term "operably linked" means that the antibody gene is linked to the vector so that the transcription and translation control sequences within the vector exercise their established functions of regulating antibody gene transcription and translation.

The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control transcription or translation of antibody genes. 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), adenovirus, such as the adenovirus major late promoter (AdMLP) and polyoma virus. Alternatively, non-viral regulatory sequences may be used, such as the ubiquitin promoter or the β-globin promoter. 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 type I virus. The expression vector and expression control sequence are selected to be compatible with the expression host cell used.

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 the expression vector of the heavy chain constant region and the light chain constant region that have encoded the desired subtype and constructs the full-length antibody gene, so that _VH is operably connected to the C _H in the vector, and _VL is operably connected to the C _L in the vector. Alternatively, the recombinant expression vector can encode a signal peptide that promotes the 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 (that is, a signal peptide from a non-immunoglobulin).

In addition to the antibody chain gene and regulatory sequence, the recombinant expression vector 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. The selectable marker gene can be used to select the host cell into which the vector has been introduced. For example, the selectable marker gene usually confers drug resistance to the host cell into which the vector has been introduced, such as G418, hygromycin, or methotrexate resistance. Preferred selectable marker genes include dihydrofolate reductase (DHFR) genes (for methotrexate selection/amplification of dhfr host cells) and neo genes (for G418 selection).

For the expression of light and heavy chains, expression vectors encoding heavy and light chains are transfected into host cells by standard techniques. Multiple forms of the term "transfection" include 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, etc. Although it is theoretically feasible to express the present application antibody or its antigen-binding portion in prokaryotic or eukaryotic host cells, it is preferred that the antibody is 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.

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, co-expressing 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 the recombinant expression vector encoding the 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 the antibody to be expressed in the host cell, or preferably sufficient to allow the antibody to be secreted 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.

In some embodiments, CDR regions are implanted into variable regions that can be used to genetically modify antibodies. Antibodies interact with target antigens primarily through amino acid residues located in three heavy chain complementary determining regions (CDRs) and six light chain CDRs. For this reason, the amino acid residues in CDRs are more diverse between individual antibodies than sequences outside CDRs. Because the CDR sequence is responsible for the main antibody-antigen interaction, recombinant antibodies that mimic the properties of specific natural antibodies can be expressed by constructing an expression vector containing the CDR sequence of a specific natural antibody implanted into the backbone 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; U.S.A. 86: 10029-10033; U.S. Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

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 _VH and _VL CDR sequences of the antibodies of the present application, they may contain different framework sequences.

Such a framework sequence can be obtained from a public DNA database or public reference that includes germline antibody gene sequences. For example, germline DNA sequences for human heavy chain and light chain variable region genes can be obtained in the Vbase human germline sequence database (www.mrc-cpe.cam.ac.uk/vbase) and 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 chain and light chain variable region genes can be obtained in the Genbank database.

The antibody protein sequence was compared to a protein sequence database by using one of the sequence similarity search methods known in the art called gap BLAST (Altschul et al., (1997)).

Preferred framework sequences for use with the antibodies of the present application are those that are structurally similar to the framework sequences used with the antibodies of the present application. _{The VH} 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., US Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

On the other hand, the 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 application. The nucleic acid can be present in whole cells, in cell lysates, or in partially purified or substantially pure form. The 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 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.

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), cDNAs encoding the light and heavy chains of antibodies prepared by hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding such antibodies can be collected from gene banks.

Preferred nucleic acid molecules of the present application include those encoding the _VH and _VL sequences or CDRs of the GS linker monoclonal antibody. Once the DNA fragments encoding _VH and _VL are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region genes into full-length antibody chain genes, Fab fragment genes or scFv genes. In these manipulations, the DNA fragment encoding _VH or _VL is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" refers to two DNA fragments being linked together so that the amino acid sequences encoded by the two DNA fragments are in the reading frame.

The isolated DNA encoding the _VH region can be converted into a full-length heavy chain gene by operably connecting the _VH encoding DNA to another DNA molecule encoding the heavy chain constant region ( _CH1 , _CH2 and _CH3 ). The sequences of human heavy chain constant region genes are known in the art, and DNA fragments comprising these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is preferably an IgG1 constant region. For Fab fragment heavy chain genes, the DNA encoding the _VH region can be operably connected to another DNA molecule encoding only the heavy chain _CH1 constant region.

The isolated DNA encoding the _VL region can be converted into a full-length light chain gene by operably connecting the _VL encoding DNA to another DNA molecule encoding the light chain constant region _CL . The sequences of human light chain constant region genes are known in the art, and DNA fragments including these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a kappa and lambda constant region.

To create an _scFv gene, the DNA fragments encoding _VH and _VL can be operably linked to another fragment encoding a flexible linker so that the _VH and _VL sequences can be expressed as a continuous single-chain protein, in which the _VH and _VL regions are connected by the flexible linker.

On the other hand, the present application relates to a bispecific molecule comprising an antibody or an antigen-binding portion thereof of the present application connected to at least one other functional molecule, such as another peptide or protein (e.g., another antibody or a 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 an antigen-binding portion thereof that specifically binds to a GS linker of the present application, and ii) an antibody or an antigen-binding portion thereof that targets a disease-associated antigen. i) and ii) may be linked together, for example, fused together by recombinant expression, or bound together via, for example, a disulfide bond. The disease-associated antigen may be, for example, a tumor-associated antigen.

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

The present application also provides a chimeric antigen receptor, which may include a) an extracellular domain, which may include 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.

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

In order to facilitate detection of the antibody or its antigen binding portion of the present application, the antibody or its antigen binding portion may include a detection substance. For example, the antibody or its antigen binding portion of the present application may be fused with a detection substance (e.g., recombinantly expressed), or coupled with a detection substance. The antibody or its antigen binding portion of the present application may include a detection substance in the framework (FR) region of the heavy chain constant region, the light chain constant region, the heavy chain variable region, or the light chain variable region. In particular, the antibody or its antigen binding portion may include a detection substance in the framework region of the heavy chain constant region or the light chain constant region.

The detection substance can be any substance that is easy to detect with the naked eye or instrument, including, but not limited to, fluorescent labels, cold light labels, immunodetectable labels, radiation labels, chemical labels, nucleic acid labels, and polypeptide labels. Commonly used detection substances include fluorescent labels or chemiluminescent labels. The fluorescent label can be, for example, fluorescein isothiocyanate (FITC), which can be qualitatively and quantitatively observed by, for example, a fluorescence microscope, and can be conveniently used in, for example, flow cytometry. The chemiluminescent label can be a peroxidase (such as horseradish peroxidase (HRP)) or alkaline phosphatase (AP), etc., which can be quickly colored by adding substrates corresponding to each enzyme, and then qualitatively and quantitatively observed. The substrates of horseradish peroxidase include 3,3'-diaminobenzidine (DAB) and 3,3',5,5'-tetramethylbenzidine (TMB). The substrates of alkaline phosphatase include 4-nitrophenylphosphate disodium salt hexahydrate (pNPP).

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

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, especially the heavy chain constant region such as the Fc region.

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

The antibody or its antigen-binding portion 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 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., H ₂ O ₂ ) and 3,3'-diaminobenzidine (DAB), or peroxide peroxide (e.g., H ₂ O ₂ ) 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 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 may 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 makes the chemiluminescent marker emit light.

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 content of proteins containing a GS linker in a sample by, for example, Western blotting, detecting the presence or content of cells bound by proteins containing a GS linker in a sample by, for example, flow cytometry, detecting the presence or content of cells expressing proteins containing a GS linker in a sample by, for example, flow cytometry, or detecting the presence or content of cells expressing proteins containing a GS linker by viral transduction by, for example, flow cytometry in a sample.

Specifically, the present application provides 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 of the present application,

ii) detecting the presence of the antibody or antigen-binding portion thereof in the 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.

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

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

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

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.

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

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

ii) separating the sample molecules in the sample that are bound to the solid phase carrier in i) from the sample,

iii) eluting the sample molecules bound to the solid phase carrier from the solid phase carrier,

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

The GS linker that can be detected by 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, (G ₂ S) ₂ , (G ₂ S) ₄ , (G ₃ S) ₃ , (G ₄ S) ₂ , and (G ₄ S) ₃ .

The molecule comprising the GS linker can be any molecule comprising the GS linker, such as a recombinant protein, a nucleic acid-protein complex, etc., such as an antibody comprising a GS linker, or a chimeric antigen receptor comprising a GS linker. The antibody comprising the GS linker can be a monospecific antibody, a bispecific antibody, or a multispecific antibody, which can include various forms of antibodies such as scFv, Fab, and nanobodies.

The sample may 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 may contain cells treated with an antibody containing a GS linker. In some embodiments, the sample may contain cells introduced with a vector, wherein the vector contains a sequence for expressing a chimeric antigen receptor containing a GS linker.

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 the antibody or antigen-binding portion thereof according to the GS linker can be included before step i) of the method. If it is not known what kind of GS linker is contained in the molecule before detection, a plurality of GS antibodies or antigen-binding portions thereof in the present application can be selected, or antibodies suitable for a plurality of GS linkers can be selected, such as antibodies or antigen-binding portions thereof whose VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 respectively contain the amino acid sequences shown 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).

The qualitative or quantitative detection of antibodies or their antigen-binding portions may 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.

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

In some embodiments, the composition of the present application may include 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 include 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 include a non-antigen binding single-chain antibody, which includes a GS linker. In some embodiments, the extracellular domain of the chimeric antigen receptor may include a non-antigen binding single-chain antibody, which includes a GS linker between its heavy chain variable region and light chain variable region.

In some embodiments, the composition of the present application may include i) T cells 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 can 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, so that when CAR-T 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 avoid the binding of CAR-T, which adversely affects its binding to the disease-associated antigen.

The above composition can be used to treat various diseases. The disease can be related to the 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 a tumor-associated antigen, etc.

The composition may contain any number of excipients. Excipients that may be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersants or suspending agents, solubilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003).

Composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or push injection). Based on the difference of administration route, active ingredient can be wrapped in material to protect it from acid and other natural conditions that may inactivate it. "Parenteral administration" refers to a mode different from intestinal and topical, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intramembranous, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, supradinium 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.

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 concentrations.

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-about 99% of the active ingredient combined with a pharmaceutically acceptable carrier.

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 to be treated; each unit contains a predetermined amount of an 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.

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.

A "therapeutically effective amount" of the composition of the present application causes a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods. For example, for the treatment of tumor-bearing subjects, 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 untreated subjects. A therapeutically effective amount of a therapeutic antibody can reduce tumor size, or alleviate symptoms in a subject, who can be a human or another mammal.

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. Pat. Nos. 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. Pat. No. 4,487,603); (3) transdermal delivery devices (U.S. Pat. No. 4,486,194); (4) push injection devices (U.S. Pat. Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Pat. Nos. 4,439,196 and 4,475,196).

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

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 are 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 exemplary embodiments, many equivalent modifications and variations are clear to those skilled in the art when the present application is given. Thus, the exemplary embodiments of the present application are exemplary, non-restrictive. Various changes may be made to the embodiments without departing from the purpose and scope of the present application.

Example 1. Animal immunization and serum titer determination

The (G ₄ S) 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 (G ₄ S) ₃ polypeptide to BSA, and then (G ₄ S) ₃ -BSA was used as an antigen for animal immunization. Specifically, three New Zealand rabbits were subcutaneously immunized with an emulsion containing 200 μg (G ₄ S) ₃ -BSA and 200 μl Freund's complete adjuvant (Sigma-Aldrich, CAT#: F5881), and the rabbits were numbered R13087#, R13088#, and R13089#, respectively. New Zealand rabbits were boosted with subcutaneous injections of an emulsion containing 200 μg (G ₄ S) ₃ -BSA and Freund's incomplete adjuvant (Sigma-Aldrich, CAT#: F5506) every 2 weeks for a total of 4 times.

Four days before blood collection, the serum titer of the animals was detected by indirect ELISA. Specifically, the (G ₄ S) 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 (G ₄ S) ₃ polypeptide to OVA. 1 μg/ml of (G ₄ S) 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 according 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 at a ratio of 2 times), 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 ELISA reader to read the plate at 450nm. The criterion for determining the effective titer of serum is that the OD ₄₅₀ reading of the diluted serum is greater than 2.1 times the OD ₄₅₀ reading of the blank background.

The results showed that the titers of sera from R13087# and R13088# for (G ₄ S) ₃ polypeptide were higher than those from R13089#, and the titers were 1:512,000; the titers of sera from R13087# and R13088# for (G ₄ S) ₃ -OVA were higher than those from R13089#, and the titers were greater than 1:512,000, indicating that there were antibodies specific to (G ₄ S) ₃ , including (G ₄ S) ₃ in (G ₄ S) ₃ -OVA, in the animal sera. In summary, the titers of R13087# and R13088# for both (G ₄ S) ₃ polypeptide and (G ₄ S) ₃ -OVA were higher than those from R13089#, so these two animals were selected for further booster immunization by intraperitoneal and intravenous injection of 400μg (G ₄ S) ₃ -BSA (without adjuvant).

Example 2. Obtaining a single B cell expressing an antibody

After the booster immunization, take fresh blood from the rabbit and perform separation within 4 hours after ex vivo. For the separation of peripheral blood mononuclear cells (PBMC), human peripheral blood lymphocyte separation solution (prepared by FICOLL) (Solarbio, CAT#: P8900) was added to the centrifuge tube, and blood was added in a 1:1 ratio. The blood was added slowly and carefully to prevent the blood and separation solution from mixing. After that, the centrifuge was precooled to 25°C and centrifuged at 400×g for 30 minutes. The blood separation in the centrifuge tube was observed, and the white PBMC floating in the turbid layer was aspirated. The PBMC was then added to a cell culture plate coated with 1mg (G ₄ S) ₃ -OVA to enrich memory B cells specific to (G ₄ S) _3. The number of enriched cells was obtained using a hemocytometer, and the cells were serially diluted until the cell density reached 5-15 cells/ml. 200μl of the cell solution was transferred to 96 wells with a pipette at a density of 1-3 cells/well. After single B cells were cultured and expanded, positive clones recognizing (G ₄ S) ₃ were selected and further cultured in 24-well plates.

Indirect ELISA was used to detect the binding ability of antibodies secreted by B cells to proteins containing (G ₄ S) _3. Specifically, 1 μg/mL of (G ₄ S) 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 the blank culture medium was used as a negative control. After washing the ELISA plate four times with PBST, add 100 μl/well of HRP-conjugated anti-rabbit IgG Fc secondary antibody (GenScript, CAT#: A01856, 1:20000 diluted 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/well of 0.5M HCl stop solution to terminate the reaction, and read the plate at 450nm using an ELISA reader.

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

Example 3. Sequencing of antibody variable regions and preparation of recombinant antibodies

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

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

Synthesize DNA fragments encoding light chain variable region (nucleotide sequence as shown in SEQ ID NO: 56, 57, 58, 59, 60, 61, 62 or 63) + light chain constant region (nucleotide sequence as shown in SEQ ID NO: 67), and heavy chain variable region (nucleotide sequence as shown in SEQ ID NO: 48, 49, 50, 51, 52, 53, 54, or 55) + heavy chain constant region (nucleotide sequence as shown in SEQ ID NO: 65), and clone them into the pTT5 expression vector. After sequencing is correct, the above plasmids are co-transfected into CHO-S cells. After transfection, the cells are cultured in a 37°C shake flask for 6 days, and the supernatant is collected and used for antibody purification. In antibody purification, 0.2M NaOH was first used to clean the pipes and Protein A column to remove pyrogens, and then the column was equilibrated with equilibration buffer (50mM Tris, 150mM NaCl (pH 8.0)). The harvested cell supernatant was diluted with 2× equilibration buffer at a ratio of 1:1 and filtered through a 0.22μm filter to remove insoluble particles and bacteria. After the cell supernatant passed through the Protein A column, the column was washed with 1× equilibration buffer to wash away nonspecific binding proteins, and then the target antibody was eluted with 0.1M sodium citrate (pH 3.5) that was sterilized by filtration with a 0.22μm filter. 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 replaced with PBS (pH 7.2). The antibody concentration was then measured using Nanodrop, with an extinction coefficient of 1.43. Finally, the purified antibody was tested for molecular weight and purity by reducing and non-reducing polyacrylamide gel electrophoresis (SDS-PAGE).

The molecular weight and purity identification results of the 8 antibodies showed that under non-reducing conditions, the molecular weight of each antibody was slightly lower than the actual molecular weight, about 120kDa; under reducing conditions, since the reducing agent destroyed the disulfide bond structure, electrophoresis showed that the heavy chain and light chain of the antibody molecule had molecular weights of about 55kDa and about 25kDa, respectively. The purity of the reduced electrophoresis of the 8 antibodies was greater than 90%.

Example 4. Antibody binding to different GS linkers

The indirect ELISA method was used to evaluate the binding ability of the purified antibodies to various GS linkers.

Specifically, different GS linkers, namely (G ₂ S) ₂ (SEQ ID NO: 69), (G ₂ S) ₄ (SEQ ID NO: 70), (G ₃ S) ₃ (SEQ ID NO: 71), G ₄ S (SEQ ID NO: 72), (G ₄ S) ₂ (SEQ ID NO: 73), and (G ₄ S) ₃ (SEQ ID NO: 68), were taken, and the concentration of each linker was 4 μg/mL, and 100 μl was added to each well of the ELISA plate, and coated overnight at 4°C. After washing the ELISA plate once with PBST (PBS 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. Subsequently, the blocking solution was discarded, and 100 μl of the gradient diluted antibody of the present application was added to each well, and incubated at 37°C for 1 hour. After washing the ELISA plate four times with PBST, add 100 μl/well of HRP-conjugated anti-rabbit IgG Fc secondary antibody (GenScript, CAT#: A01856, 1:20000 diluted 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, and read the plate at 450 nm using an ELISA reader.

As shown in Figure 1 (AH), the 8 antibodies of the present application have weak binding to the (G ₄ S) ₁ linker, strong binding to (G ₄ S) ₃ , and different binding strengths to the other linkers. Among them, antibody R177.B8 has strong binding to (G ₃ S) ₃ , (G ₄ S) ₂ and (G ₄ S) ₃ , antibody R177.B11 has strong binding to (G ₂ S) ₂ , (G ₂ S) ₄ , (G ₄ S) ₂ and (G ₄ S) ₃ , R177.G3 has strong binding to (G ₄ S) ₂ and (G ₄ S) ₃ , and R177.G12 has strong binding to (G ₂ S) ₄ , (G ₄ S) ₂ and (G ₄ S) ₃ . Except for the (G ₄ S) ₁ connector, R177.D13, R177.E4, R177.G5, and R177.I8 have good binding effects on all tested GS connectors.

Example 5. Affinity determination of antibodies and scFv containing GS linker

The Gator label-free bioanalysis system was used to detect the binding ability of the 8 antibodies of the present application to the scFv containing the (G ₄ S) ₃ linker. Among them, the scFv containing the linker was prepared according to the sequence of Brolucizumab, specifically binds to VEGFA, and comprises a heavy chain variable region, a linker, and a light chain variable region from the N-terminus to the C-terminus, and the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NOs: 74 and 75, respectively.

Select the 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 this application to a final concentration of 5.0μg/ml, and add 200μl to each well. Dilute the 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-HCl buffer with a pH of 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 as baseline, select the position where buffer K is located, and set the time to 120s; set step 2 as loading, select the position where the antibody of the present application is located, and set the time to 120s; set step 3 as baseline, select the position where buffer K is located, and set the time to 30s; set step 4 as binding, select the position where scFv is located, and set the time to 200s; set step 5 as dissociation, select the position where buffer K is located, set the time to 300s, and set the number of regenerations to 3 times. Then run the program to detect the binding affinity of the antibody of the present application to the scFv containing the GS linker.

The results are shown in Table 2. The affinities of the scFv containing (G ₄ S) 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 (G ₄ S) ₃ -scFv.

Table 2. Binding affinity of 8 antibodies to scFv containing (G ₄ S) ₃

Example 6. Antibody Detection of scFv Containing GS Linker in Western Blot

200 μL RIPA lysis buffer (Biyuntian, CAT#: P0013B) was added to 1×10 ⁶ HEK-293 cells, mixed and reacted at 4°C for 30 min. Subsequently, centrifuged at 12000×g for 15 min, the supernatant was collected, and the protein concentration in the supernatant was detected using a BCA kit (Thermo, CAT#: 23225) according to the instructions, and the supernatant was diluted with RIPA lysis buffer to a protein concentration of 2 mg/mL.

The scFv containing (G ₄ S) ₃ used in Example 5 was diluted to 20 μg/mL using PBS. The obtained HEK-293 cell lysis supernatant dilution was mixed with PBS at a volume ratio of 1:1, which was recorded as sample 1; the obtained scFv solution was mixed with PBS at a volume ratio of 1:1, which was recorded as sample 2; the obtained HEK-293 cell lysis supernatant dilution was mixed with the obtained scFv solution at a volume ratio of 1:1, which was recorded as sample 3. 1/2 volume of 4× loading buffer (GenScript, CAT#: M00676) and 1/2 volume of PBS were added to samples 1, 2, and 3, mixed well, and placed in a 100°C metal bath for 10 minutes. Then, 10 μL/well was loaded for polyacrylamide gel electrophoresis. After electrophoresis, the protein was transferred to a PVDF membrane using the eBlot ^TM L1 rapid transfer system, and 0.5 μg/mL of the eight antibodies of the present application were used as primary antibodies, incubated at room temperature for 2 hours, then washed three times with PBST for five minutes each, added with 0.1 μg/mL of HRP-conjugated anti-rabbit IgG Fc secondary antibody (GenScript, CAT#: A01856), incubated at room temperature for 1 hour, washed three times with PBST for five minutes each, and finally exposed using ECL chemiluminescent solution (TANON, CAT#: 180-5001).

The results are shown in Figure 2 (AH). All eight antibodies of the present application effectively detected 50 ng of scFv containing a (G ₄ S) 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 lysis supernatant, indicating that the antibodies of the present application have high binding specificity.

Example 7. Antibodies for detection of proteins containing GS linkers in flow cytometry

The sequences of rituximab targeting CD20 and muromonab targeting CD3 were used to construct a bispecific antibody. The specific structure was 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, as well as the heavy chain and light chain variable regions of muromonab are shown in SEQ ID NOs: 76, 77, 78 and 79, respectively.

Take 5×10 ⁵ Raji cells, wash once with 500 μl FACS buffer (PBS + 1% BSA), centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Use 200 μl FACS buffer to resuspend the cells, add 2 μg of the above-mentioned bispecific antibody, incubate at room temperature for 20 minutes, then use 500 μl FACS buffer to wash the cells once, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Add 200 μl FACS buffer to resuspend the cells, and add 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), and incubate at room temperature for 20 minutes. Wash the cells once with 500 μl FACS buffer, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. 200 μl FACS buffer was added to resuspend the cells, and 2 μg 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 ratio of fluorescent cells to total cells was detected by flow cytometry.

As shown in the results of Figure 3, the positive cell ratios detected by the antibodies R177.B8, R177.D13 and R177.G12 of the present application were 100%, 99.9% and 99.6%, respectively, which were 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.

Example 8. Antibody detection of lentiviral transduction titer

The lentivirus carrying the gene encoding the chimeric antigen receptor (CAR) containing scFv was graded and used to infect HEK-293 cells to obtain HEK-293 cells expressing CAR. Among them, the scFv contained in the CAR contains a (G4S) ₃ linker between the heavy chain variable region and the light chain variable region.

Specifically, HEK-293 cells were uniformly inoculated at a cell density of 40,000 cells/well in a 24-well plate, with 480 μL of culture medium per well. The concentrated lentivirus was diluted in a serum-free DMEM medium at 1:81, 1:243, 1:729, and 1:2187. After oscillation and mixing, 20 μL was added to a 24-well plate inoculated with HEK-293 cells. After 12 hours of infection, the culture medium was replaced with complete culture medium. After 48 hours of infection, the 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 100 μl 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 FACS buffer, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. Resuspend the cells in 100 μl FACS buffer and detect the CAR positivity rate (i.e., the percentage of fluorescent cells in total cells) by flow cytometry to calculate the viral transduction titer.

Among them, the antibody was FITC labeled according to the following operation. FITC (SIGMA-ALDRICH, CAT#: F7250-100MG) was dissolved into 1 mg/mL using DMF, 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 reacted 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, the inner tube was filled with PBS, and the centrifugation-adding PBS process was repeated three times to replace the antibody-FITC reaction buffer with PBS to complete the FITC labeling on the antibody.

As shown in Figure 4, after the HEK-293 cells were combined and stained with the antibodies of the present application, the 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 the expression of CAR on the cells. When the cells were infected with the virus at 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, using R177.B8 to detect a 1:2187 times diluted virus titer = (5.55% × 40,000 × 2187 times) / (0.02 mL) = 2.43E + 08 TU / mL, using R177.B8 to detect a 1:729 times diluted virus titer = (13.3% × 40,000 × 729 times) / (0.02 mL) = 1.94E + 08 TU / mL, the average value is calculated to obtain the result of using R177.B8 to detect virus transduction titer of 2.19E + 08 TU / mL. Similarly, it can be calculated that the viral transduction titers detected by R177.D13 and R177.G12 antibodies are 2.09E+08TU/mL and 1.97E+08TU/mL, respectively, showing a high consistency, indicating that the screened antibodies can be used to detect lentiviral transduction titers.

Example 9. Antibody detection of CAR-T cells

Using the same steps as in Example 8, some antibodies were labeled with iFluor 647 (AAT Bioquest, CAT#: 1031).

The same procedure as in Example 8 was used to label part of the antibody with FITC.

In addition, some antibodies were PE-labeled. The antibody reduced by TECP (Thermo, CAT#: 20490) was added to phycoerythrin (PE) activated by 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 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-adding PBS process was repeated three times, and the antibody-PE reaction buffer was replaced with PBS to complete the PE labeling on the antibody.

Take 1×10 ⁶ T cells, including 40% CAR-positive T cells and 60% CAR-negative T cells (scFv on CAR with (G ₄ S) ₃ linker), wash the cells once with 500μl FACS buffer (PBS+1% BSA), centrifuge at 500×g for 5 minutes, and discard the supernatant. Resuspend the cells with 200μl FACS buffer, and add 2μg 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), and incubate at room temperature for 20 minutes. Then wash the cells once with 500μl FACS buffer, centrifuge at 500×g for 5 minutes, and discard the supernatant. 200 μl of FACS buffer was added to resuspend the cells, and the proportion of positive cells (i.e., the proportion of fluorescent cells to total cells) was detected using flow cytometry.

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 is 52.75%, which is quite different from 40%, with low accuracy, poor clustering effect, high background value, and high nonspecific 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 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.

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.

Example 10. Antibody Purification of scFv Containing GS Linker

The antibody of the present invention is used to purify a scFv containing a (G ₄ S) ₃ linker. The scFv containing a linker is prepared according to the sequence of FMC63 targeting CD19, and comprises a light chain variable region, a linker and a heavy chain variable region from the N-terminus to the C-terminus, and 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.

The antibody R177.B8 of the present invention was coupled to the purification medium CNBr Focurose 4FF (Huiyan Bio, CAT#: HQ030301025M) at different mass 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 (G ₄ S) ₃ linkers was added to the gravity column. After the liquid was drained, 1 mL of PBS was added to wash the purification medium twice. Then 0.1 M glycine (pH 3.0) was used for elution, the eluate was collected, and neutralized with 1 M Tris-HCl (pH 9.0). According to the operating method in the manual, the protein concentration was quantified using a BCA kit (Thermo, CAT#: 23225).

Using 4-20% precast gel (GenScript, CAT#: M00656), the purified scFv containing G ₄ S) ₃ linker at different coupling ratios (antibody: medium = 10:1; 15:1; 20:1) were analyzed by SDS-PAGE electrophoresis. The antibody was stained with L1 protein stainer (GenScript, CaT#: L00657C), and the molecular size and purity were estimated by comparing the stained band with protein marker (GenScript, CaT#: M00624). As shown in Figure 6, the molecular weight of the antibodies purified with different coupling ratios was about 28 kd, which was consistent with the expected molecular weight, and the protein purity was ≥95%, indicating that the screened antibodies can be used to purify scFv containing GS linker. Among them, lane 1 is protein marker, lane 2 is antibody: medium = 10:1 coupling medium purified scFv containing (G ₄ S) ₃ linker; lane 3 is antibody: medium = 15:1 coupling medium purified scFv containing (G ₄ S) ₃ linker; lane 4 is antibody: medium = 20:1 coupling medium purified scFv containing (G ₄ S) ₃ linker.

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

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 variant forms and equivalents, all included in the subject matter and scope of the appended claims. All documents cited in this article are fully incorporated herein by reference.

Claims (27)

An isolated monoclonal antibody or antigen-binding portion thereof, which is capable of specifically binding to a GS linker, comprises 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 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: 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 shown as 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 containing 1-3 amino acid substitutions in each CDR compared to the above amino acid sequences. An antibody or antigen-binding portion thereof according to 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 with SEQ ID NO: 32, 35, 37, 39, 34, 33, 36, or 38. An antibody or antigen-binding portion thereof according to 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 with SEQ ID NO: 40, 43, 45, 47, 42, 41, 44, or 46. An antibody or antigen-binding portion thereof according to claim 1, wherein the heavy chain variable region and the light chain variable region 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 with (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. 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. The antibody or antigen-binding portion thereof according to claim 6, wherein the detection substance is a fluorescent marker, biotin or a chemiluminescent marker. The antibody or antigen-binding portion thereof according to claim 1, which is a rabbit-derived antibody, a chimeric antibody, or a humanized antibody. An isolated bispecific molecule comprising the antibody or antigen-binding portion thereof of any one of claims 1-8. The bispecific molecule of claim 9, further comprising an antibody or antigen-binding portion thereof that targets a disease-associated antigen. 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. A nucleic acid molecule encoding the antibody or antigen-binding portion thereof of any one of claims 1 to 8, the bispecific molecule of claim 9 or 10, or the chimeric antigen receptor of claim 11. The nucleic acid molecule as described in claim 12 comprises a nucleotide sequence shown in any one of SEQ ID NOs: 48-63. An expression vector comprising the nucleic acid molecule according to claim 12 or 13. A host cell comprising the expression vector of claim 14, or having the nucleic acid molecule of claim 12 or 13 integrated into its genome. A composition comprising the antibody or antigen-binding portion thereof of any one of claims 1 to 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. 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. 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. 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. 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. 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. 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. The method of claim 21 or 22, wherein the GS linker is (G ₂ S) ₂ (SEQ ID NO: 69), (G ₂ S) ₄ (SEQ ID NO: 70), (G ₃ S) ₃ (SEQ ID NO: 71), (G ₄ S) ₂ (SEQ ID NO: 73), or (G ₄ S) ₃ (SEQ ID NO: 68). The method of claim 21 or 22, wherein the molecule containing the GS linker is an antibody containing the GS linker or a cell containing the GS linker. 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. 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. Use of the composition of claim 20 in the preparation of a medicament for treating a disease, wherein the disease is related to a disease-associated antigen targeted by the bispecific molecule in the composition or the antibody or antigen-binding portion thereof containing a GS linker and targeting a disease-associated antigen.