Antibodies that specifically bind to Strep-TagII tags and their use

Abstract

Antibodies that specifically bind to the Strep-Tag II tag are disclosed, as are the amino acid sequences of the antibodies of the invention, cloning or expression vectors, host cells, and methods for expressing or isolating the antibodies, and compositions comprising the antibodies of the invention. Methods for detecting or purifying biological samples that express the Strep-Tag II tag by means of the antibodies of the invention are also disclosed.

Description

[Technical Field]

[0001] The present invention relates to an antibody that specifically binds to the Strep-TagII tag, and discloses the amino acid sequence of the antibody, a cloning or expression vector, a host cell, a method for producing the antibody, and a method for using the antibody to purify or detect a biological sample expressing the Strep-TagII tag. [Background technology]

[0002] The development of tumor immunotherapy has brought significant attention to CAR-T (chimeric antigen receptor T cells), TCR-T (T cell receptor T cells, engineered T cell receptor T cells), and other immunotherapy strategies. The proliferation level of CAR-T or TCR-T cells in vivo is a crucial indicator of their effectiveness. Therefore, the development of reagents or methods for rapid and accurate cell detection would help effectively monitor their proliferation efficiency both in vitro and in vivo.

[0003] Currently, there are two methods for detecting CAR-T or TCR-T cells: detecting CAR or TCR gene expression in T cells using quantitative real-time PCR (qPCR), and detecting CAR or TCR protein expression in T cells. For detecting CAR protein expression in T cells, T cells are detected by flow cytometry using antibodies that specifically bind to the scFv (single-stranded variable fragment) fragment on the CAR protein, or antibodies that bind to the Fab fragment, or protein L, and similar. For detecting TCR protein expression in T cells, complexes of peptides and HLA that specifically target the TCR are commonly used in flow cytometry assays of T cells. However, the above detection methods have clear limitations. qPCR detection of gene copy number cannot accurately reflect CAR-expressing T cells because cells integrating the CAR gene usually cannot express the CAR protein, leading to false positives in qPCR detection. In methods for detecting CAR protein-positive T cells, the detection background value of Fab antibodies is high, easily leading to false positives, and protein L is only suitable for detecting CARs with a κ light chain on the scFv. Furthermore, for antibodies that target the scFv fragment on CAR proteins, and for HLA and polypeptide complexes that target TCRs, it is necessary to screen different antibodies or complexes for different CAR proteins or TCRs to establish a valid detection method, which is time-consuming, inefficient, and costly. If an accurate, highly sensitive, and universal antibody detection method for CAR-T cells can be developed, it would greatly improve the detection efficiency of transgenic cell therapy drugs and reduce detection costs. [Overview of the Initiative]

[0004] The present invention discloses isolated antibodies that specifically bind to a Strep-TagII tag comprising a heavy chain variable region (hereinafter abbreviated as VH) and a light chain variable region (hereinafter abbreviated as VL), wherein VH comprises VH-CDR1 containing the amino acid sequence of SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, and VL comprises VL-CDR1 containing the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

[0005] In some embodiments, VH includes VH-CDR1 consisting of the amino acid sequence presented in SEQ ID NO: 1, VH-CDR2 consisting of the amino acid sequence presented in SEQ ID NO: 2, and VH-CDR3 consisting of the amino acid sequence presented in SEQ ID NO: 3 or SEQ ID NO: 4, and VL includes VL-CDR1 consisting of the amino acid sequence presented in SEQ ID NO: 5 or SEQ ID NO: 6, VL-CDR2 consisting of the amino acid sequence presented in SEQ ID NO: 7 or SEQ ID NO: 8, and VL-CDR3 consisting of the amino acid sequence presented in SEQ ID NO: 9 or SEQ ID NO: 10.

[0006] In some embodiments, VH includes VH-CDR1 containing the amino acid sequence of SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 3.

[0007] In some embodiments, VH includes VH-CDR1 containing the amino acid sequence of SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 4.

[0008] In some embodiments, the VL includes VL-CDR1 containing the amino acid sequence of SEQ ID NO: 5, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 7, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 9.

[0009] In some embodiments, the VL includes VL-CDR1 containing the amino acid sequence of SEQ ID NO: 6, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 8, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 10.

[0010] In some embodiments, the antibody comprises VH and VL, where VH comprises VH-CDR1 containing the amino acid sequence of SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 3, and VL comprises VL-CDR1 containing the amino acid sequence of SEQ ID NO: 5, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 7, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 9.

[0011] In some embodiments, the antibody comprises VH and VL, where VH comprises VH-CDR1 containing the amino acid sequence of SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 2, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 4, and VL comprises VL-CDR1 containing the amino acid sequence of SEQ ID NO: 6, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 8, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 10.

[0012] In some embodiments, the antibody comprises VH and VL, where VH comprises VH-CDR1 consisting of the amino acid sequence presented in SEQ ID NO: 1, VH-CDR2 consisting of the amino acid sequence presented in SEQ ID NO: 2, and VH-CDR3 consisting of the amino acid sequence presented in SEQ ID NO: 3, and VL comprises VL-CDR1 consisting of the amino acid sequence presented in SEQ ID NO: 5, VL-CDR2 consisting of the amino acid sequence presented in SEQ ID NO: 7, and VL-CDR3 consisting of the amino acid sequence presented in SEQ ID NO: 9, or VH includes VH-CDR1 consisting of the amino acid sequence presented in SEQ ID NO: 1, VH-CDR2 consisting of the amino acid sequence presented in SEQ ID NO: 2, and VH-CDR3 consisting of the amino acid sequence presented in SEQ ID NO: 4. VL includes VL-CDR1 consisting of the amino acid sequence presented in SEQ ID NO: 6, VL-CDR2 consisting of the amino acid sequence presented in SEQ ID NO: 8, and VL-CDR3 consisting of the amino acid sequence presented in SEQ ID NO: 10.

[0013] In this invention, each CDR on VH and VL is identified by the Kabat numbering system.

[0014] In some embodiments, VH comprises, essentially consists of, or comprises the amino acid sequence of SEQ ID NO: 23 or 24, or VH comprises, essentially consists of, or comprises an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more identity with respect to SEQ ID NO: 23 or 24.

[0015] In some embodiments, VL comprises, essentially consists of, or comprises the amino acid sequence of SEQ ID NO: 25 or 26, or VL comprises, essentially consists of, or comprises an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, identity with respect to SEQ ID NO: 25 or 26.

[0016] In some embodiments, the VH or VL of the antibody further includes one or more framework regions (one or more) (hereinafter abbreviated as FWR).

[0017] In some embodiments, VH comprises one, two, three, or four FWRs, and each FWR comprises an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 11, 12, 13, 14, 15, and 16, or an amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 99% identity thereto (the amino acid sequence has one or more amino acid substitutions, insertions, or deletions as compared to any one of the amino acid sequences of SEQ ID NOs: 11, 12, 13, 14, 15, and 16). In some specific embodiments, the aforementioned VH comprises four FWRs, FWR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, FWR2 comprises the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13, FWR3 comprises the amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15, and FWR4 comprises the amino acid sequence set forth in SEQ ID NO: 16.

[0018] In some embodiments, VL comprises one, two, three, or four FWRs, and each FWR comprises an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 17, 18, 19, 20, 21, and 22, or an amino acid sequence having at least 90%, at least 95%, at least 99% identity thereto (the amino acid sequence has one or more amino acid substitutions, insertions, or deletions as compared to any one of the amino acid sequences of SEQ ID NOs: 17, 18, 19, 20, 21, and 22). In some specific embodiments, the aforementioned VL comprises four FWRs, FWR1 comprises the amino acid sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18, FWR2 comprises the amino acid sequence set forth in SEQ ID NO: 19, FWR3 comprises the amino acid sequence set forth in SEQ ID NO: 20 or SEQ ID NO: 21, and FWR4 comprises the amino acid sequence set forth in SEQ ID NO: 22.

[0019] In some embodiments, VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 23, and VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 25.

[0020] In some embodiments, VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 24, and VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 26.

[0021] In some embodiments, the heavy chain of the antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 27 or 29, or the heavy chain of the antibody comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 99% identity to SEQ ID NO: 27 or 29.

[0022] In some embodiments, the light chain of the antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 28 or 30, or the light chain of the antibody comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 99% identity to SEQ ID NO: 28 or 30.

[0023] In some embodiments, the heavy chain of the antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 27, and the light chain of the antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 28.

[0024] In some embodiments, the heavy chain of the antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 29, and the light chain of the antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 30.

[0025] In some embodiments, the aforementioned antibody further comprises at least one heavy chain constant region and a light chain constant region. The heavy chain constant region is selected from mouse IgG1, mouse IgG2a, mouse IgG2b, or mouse IgG3, and the light chain constant region is selected from a κ chain or a λ chain. Preferably, the heavy chain constant region is mouse IgG1, and the light chain constant region is the κ chain of the mouse antibody.

[0026] In some embodiments, the aforementioned antibody is a full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, monoclonal antibody, bispecific antibody, or multispecific antibody.

[0027] The exemplary antibody sequences are shown in Table 1 and the sequence listing.

[0028] [Table 1]

[0029] [Table 2]

[0030] The CDR and FWR sequences of the exemplary antibodies are shown in Tables 2 and 3.

[0031] [Table 3]

[0032] [Table 4]

[0033] The present invention also discloses isolated nucleic acid molecules encoding the aforementioned antibodies.

[0034] The present invention also discloses a cloning vector or an expression vector, the expression vector comprising the aforementioned nucleic acid molecule.

[0035] The present invention also discloses a host cell comprising one or more of the aforementioned cloning vectors or expression vectors.

[0036] The present invention also discloses a method for producing the aforementioned antibody, which comprises culturing the aforementioned host cell and isolating the antibody.

[0037] The present invention also discloses a composition or kit comprising the aforementioned antibody and one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0038] The present invention also discloses a conjugate comprising the aforementioned antibody linked to a detectable label. Those skilled in the art will understand that any detectable label can be used to construct the conjugate of the present invention. Exemplary detectable labels include, but are not limited to, fluorescent labels, enzyme substrate labels, radioisotopes, digoxigenin, biotin, or avidin, DNA molecules, or gold for detection. Here, fluorescent labels include, but are not limited to, fluorescein, rhodamine, dansyl, phycoerythrin, or Texas red. Enzyme substrate labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucoamylase, lysozyme, sugar oxidase, or β-D-galactosidase. Radioisotopes are 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 111 In, 112 In, 14 C, 64 Cu, 67 Cu, 86 Y, 88 Y, 90 Y, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi, 32P includes, but is not limited to, other lanthanides. The label can also be a luminescent label or a chromophore.

[0039] The present invention also discloses compositions or kits comprising the aforementioned conjugate and one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0040] The present invention also discloses the use of the aforementioned antibodies in the preparation of reagents or kits for detecting fusion polypeptides containing Strep-TagII tags in a sample.

[0041] The present invention also discloses a method for detecting a fusion polypeptide containing a Strep-TagII tag in a sample, comprising (i) contacting the sample containing the fusion polypeptide with an isolated antibody of the present invention in vitro under conditions that allow interaction between the antibody and the fusion polypeptide, and (ii) detecting the formation of a complex between the antibody and the fusion polypeptide. The method for detecting the complex includes, but is not limited to, at least one of flow cytometry, Western blotting, immunohistochemistry, and immunofluorescence.

[0042] In some embodiments, the sample containing the Strep-TagII tag is a CAR-T cell containing the Strep-TagII tag.

[0043] The present invention also discloses a method for purifying a fusion polypeptide containing a Strep-TagII tag in a sample, comprising: (i) contacting the sample containing the fusion polypeptide with an isolated antibody of the present invention in vitro under conditions that allow interaction between the antibody and the fusion polypeptide; and (ii) purifying the complex formed between the antibody and the fusion polypeptide. The purification method includes, but is not limited to, affinity chromatography, immunoprecipitation, and the like.

[0044] In some embodiments, the sample is a biological sample comprising at least one of blood, urine, saliva, lymph, cerebrospinal fluid, bone marrow, tissue and organs, or cells.

[0045] In specific embodiments, the blood includes at least one of serum, plasma, and whole blood.

[0046] In some embodiments, the sample includes chimeric antigen receptor cells or engineered cell receptor cells.

[0047] In some embodiments, the sample is a chimeric antigen receptor cell or a modified cell receptor cell. [Effects of the Invention]

[0048] The antibodies of the present invention have stronger affinity and can specifically bind to the Strep-TagII tag of chimeric antigen receptor cells or engineered cell receptor cells, allowing for effective monitoring of the in vitro and in vivo amplification efficiency of chimeric antigen receptor cells or engineered cell receptor cells. [Brief explanation of the drawing]

[0049] [Figure 1] Figure 1 shows the serum titer curves from mice after four immunization cycles. [Figure 2] Figure 2 shows the polyacrylamide gel electrophoresis pattern of antibody 8F8D1. Lane M represents the molecular weight standard (within 300 kD), and lane 1 represents the heavy chain (approximately 55 kD) and light chain (approximately 24 kD) of antibody 8F8D1. [Figure 3] Figure 3 shows flow cytometry graphs of antibodies 8F8D1 and 8A882. [Figure 4] Figure 4 shows the affinity test graphs for antibodies 8F8D1 and 8A882. [Figure 5] Figure 5 shows the metabolic graph of CAR-T cells in mice monitored using the antibody 8F8D1. [Modes for carrying out the invention]

[0050] The present invention is further described below by specific embodiments. Unless otherwise defined, terms used herein have the same meaning as commonly understood by those skilled in the art.

[0051] As used herein, the term “antigen” refers to a molecule that induces an immune response that may involve antibody production or activation of specific immune cells. Those skilled in the art will understand that any macromolecule, encompassing all proteins or peptides, can be used as an antigen. For example, the immunizing antigen in this invention may be Strep-TagII having the amino acid sequence of NWSHPQFEK, or Strep-TagII (NWSHPQFEK-KLH) conjugated to keyhole limpet hemocyanin (KLH). Antigens may be generated, synthesized, or derived from biological samples, including but not limited to tissue samples, tumor samples, cells, or biological fluids. As used herein, the term “detection antigen” refers to an antigen used to determine antibody titer, such as Strep-TagII (NWSHPQFEK-OVA) conjugated to ovalbumin (OVA), but those skilled in the art will understand that the antigen is not limited to NWSHPQFEK-OVA, and antigens containing NWSHPQFEK or a portion thereof can also be used as detection antigens. Therefore, the use of specific detection antigens should not be interpreted as a limitation on the method of antibody preparation.

[0052] As used herein, the terms "peptide" and "polypeptide" refer to compounds consisting of amino acid residues covalently linked by peptide bonds. As used herein, "fusion polypeptide" refers to a polypeptide to which a Strep-TagII tag is attached. For example, the Strep-TagII tag is attached to the N-terminus or C-terminus of the heavy chain variable region or light chain variable region on the extracellular domain scFv of a chimeric antigen receptor. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0053] As used herein, the term “antibody” is used in its broadest sense and encompasses, but is not limited to, various antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding moieties), as long as they exhibit desired antigen-binding activity. Antibodies include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, di-scFv, tri-scFv, Fd, and other antibody fragments that retain antigen-binding function, and can also be dimeric (diabody) or trimer (triabody) structures. Antigen-binding fragments typically include antibody light chain variable regions (VL) and antibody heavy chain variable regions (VH), which can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) and more conserved regions called framework regions (FWRs). These are scattered. The CDRs of the antibodies and antigen-binding fragments disclosed herein are defined or identified by Kabat numbering.

[0054] A full-length antibody is a protein comprising at least two heavy chains and two light chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region and a heavy chain constant region, and each light chain consists of a light chain variable region and a light chain constant region. In specific embodiments of the present invention, the heavy chain constant region may be selected from mouse-derived IgG1, IgG2a, IgG2b, or IgG3, and the light chain constant region may be selected from a κ chain or a λ chain.

[0055] As used herein, the term “chimeric antigen receptor” or “CAR” refers to an artificial receptor that has been engineered to be expressed on immune effector cells and is capable of specifically binding to an antigen. “Chimeric antigen receptor cells” refer to immune cells that express an artificial receptor capable of specifically binding to an antigen on the cell surface, and these immune cells include lymphocytes, natural killer cells, dendritic cells, monocytes / macrophages, granulocytes, mast cells, etc., and may be used in adoptive cell transfer therapy. The term “engineered cell receptor” or “TCR,” as used herein and also known as engineered T cell receptors, is a heterodimer composed of peptide chains of a TCR that has been genetically engineered in vitro to enable more effective recognition of tumor intracellular antigen peptides delivered by MHC, thereby killing and treating tumors. The fusion polypeptide in this invention comprises a CAR or TCR fused with a Strep-TagII tag.

[0056] As used herein, “isolated” means modified or removed from its natural state. For example, nucleic acids or peptides that are naturally present in living animals are not “isolated,” but the same nucleic acids or peptides that have been partially or completely separated from coexisting materials in their natural state are “isolated.” Isolated nucleic acids or proteins may exist in a substantially purified form or in a non-natural environment such as a host cell. “Isolated” antibodies are those identified and separated and / or recovered from components of their natural environment. In some embodiments, antibodies are purified to a purity much greater than 95% or 99% as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing IEF, capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). Host cells as used herein may be any prokaryotic or eukaryotic cell containing a cloning vector or expression vector, and include prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene on the chromosome or genome of the host cell. Special transgenic animals with modified immune systems may also be used to produce antibodies.

[0057] In this specification, any numerical limit or range is defined as encompassing the endpoints and specifically including all values ​​and subranges within the numerical limit or range.

[0058] Unless otherwise specified, the experimental method in the following example is the conventional method. [Examples]

[0059] Example 1. Antigen, mouse immunization, and hybridoma preparation 1. Antigen The immunizing antigen is Strep-TagII (NWSHPQFEK-KLH) conjugated to keyhole limpet hemocyanin (KLH), and the detection antigen is Strep-TagII (NWSHPQFEK-OVA) conjugated to ovalbumin (OVA). All were purchased from Chinese Peptide.

[0060] 2.Immunization An immunized antigen protein (Strep-TagII polypeptide conjugated to KLH (NWSHPQFEK-KLH)) was dissolved in physiological saline at a concentration of 0.5 mg / mL. 100 μL of the above antigen protein solution was mixed with an equal volume of complete Freund's adjuvant (Sigma; Cat#:1001646446), and Balb / c mice (female, 6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were immunized by subcutaneous multi-point injection to produce an immune response. Then, the antigen protein solution was diluted with saline to a concentration of 0.25 mg / mL, and 100 μL of the diluted antigen protein solution and Fuchs' incomplete adjuvant (Sigma; Cat#:1002036152) were mixed in a 1:1 volume on days 15, 29, and 43, respectively, and Balb / c mice were immunized by subcutaneous abdominal multi-point injection for booster immunization. For a total of four antigen immunizations, each mouse was injected with a volume of 200 μL per injection. Serum titers were determined by tail-drained blood on days 22, 36, and 50, respectively. Mouse serum titers were detected by enzyme-linked immunosorbent assay (ELISA) using the NWSHPQFEK-OVA polypeptide, and the procedure was as follows: 1 μg / mL of OVA-conjugated Strep-TagII (NWSHPQFEK-OVA) was added to a 96-well plate (purchased from Corning) at 4°C and left overnight. The ELISA plate was then washed five times with phosphate-buffered saline (PBST) containing 0.05% Tween-20. The plate was blocked at room temperature for 2 hours with 1% bovine serum albumin and washed five times with PBST. Mouse serum was then diluted four times from 1:1000 to 1:102400 and added to the ELISA plate. It was incubated at room temperature for 1 hour and washed five times with PBST. 100 μL / well of HRP-labeled goat anti-mouse secondary antibody (Invitrogen, Cat#:31430) was added, washed, and incubated at room temperature for 1 hour.Then, 50 μL / well of 3,3',5,5'-tetramethylbenzidine (TMB) was added to the plate, incubated at room temperature for 15 minutes, then stopped the reaction and read the results.

[0061] The titer results for five mice are shown according to the detection of serum titers in immunized mice (Figure 1), and the titer of mouse No. #4 was higher than that of the other mice. Mouse #4 was selected for hybridoma fusion, and three days before fusion, the mouse was intraperitoneally injected with 50 μg of immunized antigen to induce shock.

[0062] 3.Fusion Using the polyethylene glycol fusion method, the spleen of mouse #4 was excised, ground, and resuspended in Dulbecco's phosphate-buffered saline (DPBS) to extract single mouse spleen B cells. The cells were centrifuged at 500g for 5 minutes, the pellet was resuspended in DPBS, and then the cells were washed by centrifugation at 500g for 5 minutes. The spleen cells were mixed with mouse myeloma cell line sp2 / 0 (purchased from Nordic Bioscience (Beijing)) in a 3:1 ratio (cell number ratio), 1 mL of polyethylene glycol (Roche; Cat#:10783641001) was added, and the fused cells were resuspended in HAT medium (Gibco; Cat#:21060-017), plated in 96-well cell culture plates, and cultured in a carbon dioxide incubator at 37°C for 7 days. Primary screening of hybridomas was then performed by ELISA and flow cytometry. Selected hybridoma cells were cultured overnight in a carbon dioxide incubator at 37°C, and then the fused cells were seeded into 96-well plates by limiting dilution.

[0063] Example 2. Screening of positive hybridoma cells The presence of anti-Strep-TagII antibodies in the culture supernatant of hybridomas was detected by ELISA and flow cytometry, and hybridoma cells capable of unlimited proliferation and antibody secretion were screened out. Details are as follows.

[0064] 1. ELISA screening 1 μg / mL of OVA-conjugated Strep-TagII (NWSHPQFEK-OVA) was added to a 96-well plate (purchased from Corning) overnight at 4°C. After discarding the supernatant, the plate was washed five times with 0.05% Tween-20 (PBST) in phosphate buffer. The plate was blocked at room temperature for 2 hours with 1% bovine serum albumin and washed five times with PBST. 100 μL / well of hybridoma supernatant was added and incubated at room temperature for 1 hour, then washed five times with PBST. After washing, 100 μL / well of HRP-labeled goat anti-mouse secondary antibody (Invitrogen, Cat#:31430) was added and incubated at room temperature for 1 hour. Then, 50 μL / well of 3,3',5,5'-tetramethylbenzidine (TMB) was added to the plate, incubated at room temperature for 15 minutes, then stopped the reaction and read the results. Positive hybridomas were selected by ELISA screening and transferred from the 96-well culture plate to a 24-well culture plate for expansion culture. The supernatant from the 24-well culture plate was re-screened after 5 days and analyzed by flow cytometry (FACS).

[0065] 2. Flow cytometry screening Construction of a lentiviral transfer plasmid encoding a CD19-targeting CAR and preparation of a lentiviral vector: 1) A chimeric gene encoding Strep-TagII and a CD19-targeting scFv was synthesized by gene synthesis (Beijing Biomed Gene Technology). 2) Using an existing plasmid of a CD19-targeting CAR as a template (see Sequence ID No. 13 of Patent CN105177031B for the nucleotide sequence of the CD19-targeting CAR), nucleic acid fragments of the CAR molecule containing the CD8α hinge region, CD8α transmembrane region, 4-1BB intracellular region (corresponding to NP_001552.2), and CD3ζ intracellular region (corresponding to NP_000725.1) were cloned by PCR. 3) Using the chimeric gene obtained in step 1) and the nucleic acid fragment obtained in step 2) as templates, a complete nucleic acid fragment encoding Strep-TagII and a CAR targeting CD19 was cloned by PCR. 4) The complete nucleic acid fragment obtained in step 3) was inserted into the lentiviral vector pLenti6.3 / V5 (Thermo Fisher, Waltham, MA, USA) by restriction enzyme digestion and ligation to obtain a lentiviral transfer plasmid containing the genes for Strep-TagII and a CAR targeting CD19. 5) Lentiviral packaging plasmids pLP / VSVG, pLP1 / MDK, pLP2 / RSK (Thermo Fisher, Waltham, MA, USA), and the transfer plasmid obtained in step 4) were transfected into HEK293T cells using Lipofectamine 3000 (Thermo Fisher, Waltham, MA, USA). The culture medium was collected after 48 hours, centrifuged at 300 g to remove cell debris, and then ultracentrifuged at 25,000 rpm for 3 hours. The precipitate was dissolved in 1 mL of physiological saline to obtain the desired lentiviral vector.

[0066] Preparation of CAR-T cells containing Strep-TagII tags: T cells were isolated from peripheral blood mononuclear cells of healthy volunteers (Miao Tong (Shanghai) Biological Science & Technology Co., Ltd., China) using CD3 / CD28 dynabeads (Thermo Fisher). The isolated T cells (at this point, the T cells were linked to CD3 / CD28 dynabeads) were cultured for 48 hours in a fresh X-VIVO15 culture system containing IL-2 (500 IU / mL), and the T cells were infected with the lentiviral vector described above. 24 hours after viral infection of the cells, the cells were centrifuged, the culture medium was changed, and the culture was continued in the culture system described above. After 4 days of cell culture, all cells in the culture system were collected, the DynaBeads in the culture system were removed using a magnetic stand, T cells were centrifuged and counted, and the CAR-T cell content was detected by a flow cytometer (NovoCyte2060R, ACEA Biosciences, San Diego, CA, USA).

[0067] CAR-T cells containing Strep-TagII tags were collected, washed with DPBS, counted, and the cells were counted to approximately 3 × 10⁶ cells per tube. 5 Cells were dispensed into EP tubes. After collecting the supernatant by centrifugation, 100 μL / well of hybridoma supernatant was added and incubated at room temperature for 15 minutes. After two washes, 5 μL of PE-labeled rat anti-mouse IgG (BD, Cat#:550083) was added to each tube and incubated at room temperature for 15 minutes. Then, the cells were washed twice with DPBS and analyzed by flow cytometry FACS.

[0068] 3. Positive subclones Positive hybridoma clones were subcloned into 96-well plates using the limiting dilution method, and positive monoclones were identified and selected by FACS screening (same as above). RNA was extracted from the obtained monoclonal cells, reverse transcribed to DNA, and then PCR was performed using primers that specifically bind to the heavy and light chain genes of the antibody. The PCR products were then sequenced.

[0069] Example 3. Sequencing Positive monoclones were selected, and total RNA was extracted by TRIZOL. cDNA was generated by RT-PCR, and then the heavy and light chains were amplified by PCR, respectively (RT-PCR kit purchased from TransGen, Cat#:AE311-03. GXL high-fidelity DNA polymerase was used for PCR, purchased from Takara, Cat#:R050A. See product manual for specific procedures). The PCR products were then cleaned with a cleaning kit (AXYGEN, Cat#:155. See product manual for specific procedures), followed by sequencing, which was entrusted to Beijing Ruibiotech Co., Ltd. The amino acid sequences of the light chain variable region and heavy chain variable region are shown in Tables 1 and 2 and in the sequence listing. The screened antibody clone numbers were 8A882 and 8F8D1, respectively.

[0070] Example 4. Ascites preparation and antibody purification 1. Ascitic fluid preparation We ordered five Balb / c mice aged 6 to 8 weeks, injected them with 1 mL of paraffin wax (purchased from Jiangxi Yipusheng Pharmaceutical Co., Ltd.) after one week of feeding, and the following week, each mouse received 1 × 10⁶ 6 Monoclonal hybridoma cells were injected. After one week, the mice were observed to have significant abdominal distension, and the mice were punctured and aspiration every 48 hours. 3–4 mL of ascites fluid was collected from each mouse, for a total of 20 mL of ascites fluid.

[0071] 2. Antibody purification Antibodies were purified by affinity purification, and the gravity-fed column was equilibrated with binding buffer (pH 7.0) and washed with 2 to 5 times the column volume. Filtered ascites fluid was passed through the column (Protein A / G column, ordered from Thermo Fisher, Cat#: 89930); the column was washed with 5 to 10 times the column volume of binding buffer, the target protein was eluted with 0.1 M glycine at pH 3.5, and then adjusted to neutral with Tris-HCl at pH 9.0. Finally, the antibody concentration was determined using a BCA kit (Thermo; Cat#: 23227, see product manual for specific procedures). Antibody purity was identified by SDS-PAGE, and Figure 2 shows that the antibody has high purity.

[0072] Example 5. Antibody validation and affinity detection 1. Flow cytometry verification The purified, high-purity antibodies were identified by FACS. CAR-T cells containing the Strep-TagII tag prepared in Example 2.2 (manufactured by our company) were collected, washed with FACS buffer, and counted. Approximately 3 × 10 cells were counted per tube. 5Cells were dispensed into EP tubes. After centrifugation to remove the supernatant, 8A882 antibody, 8F8D1 antibody, or a positive control antibody was added at a final concentration of 1 μg / mL, and the tubes were incubated at room temperature for 15 minutes. After two washes, 5 μL of PE-labeled rat anti-mouse IgG was added to each tube, and the tubes were incubated at room temperature for 15 minutes. The cells were then washed twice, and FACS analysis was performed. The experimental results show that, under the same amount of detection cells (CAR-T) conditions, the expression rates of CAR-T cells containing the Strep-TagII tag were 48.07% and 36.41% after treatment with the 8F8D1 and 8A882 antibodies of the present invention, respectively. These are considerably higher than the expression rate of 27.63% after treatment with the positive control antibody. The above results demonstrate that the antibodies of the present invention can specifically recognize the short Strep-TagII peptide and have a higher affinity for the short Strep-TagII peptide than commercially available positive control antibodies (Figure 3). The negative control was the addition of Strep-TagII tagged antibody without any additional antibody, and the positive control was the addition of only negative control IgG, while the positive control was a commercially available anti-Strep-TagII tagged antibody (Abcam; Cat#:ab184224).

[0073] 2. Fluorescent labeling of antibodies The antibodies of this invention are labeled with two different fluorescent agents, FITC and PE, respectively, for the purpose of multi-site detection and analysis. Antibody fluorescent labeling was performed by Beijing 4A Biotech Co., Ltd.

[0074] 3. Detection of fluorescently labeled antibody affinity The antibody affinity detection method of the present invention employs a dynamic equilibrium assay method (saturation concentration method). In the presence of a small amount of antigen, the antibody is subjected to gradient dilution to detect the concentration of the antigen-antibody complex. When the concentration of the antigen-antibody complex accounts for half of the total antigen concentration, the corresponding antibody concentration value (EC50) is the KD value of the antibody against the antigen.

[0075] CAR-T cells containing Strep-TagII tags were collected, washed with DPBS, and counted. Cells were counted per tube and 3 × 10⁶ cells. 5Cells were dispensed into several EP tubes. After centrifugation to remove the supernatant, 8A882 antibody or 8F8D1 antibody and a positive control antibody (commercially available Strep-TagII antibody, Abcam; Cat#:ab184224), diluted in a continuous gradient to 10 concentrations, were added and incubated at room temperature for 15 minutes. After washing the cells twice with DPBS, PE-labeled rat anti-mouse IgG was added and incubated at room temperature for 15 minutes. After washing the cells twice, FACS analysis and EC50 values ​​were calculated using GraphPad Prism software. The EC50 values ​​of 8A882 and 8F8D1 and the positive control antibody obtained by software analysis were 0.4 nM, 0.2 nM, and 0.55 nM, respectively (Figure 4). Compared to the positive control antibody, the two antibody lines of the present invention exhibited high affinity.

[0076] Example 6. Detection of CAR-T cells in the peripheral blood of mice with tumors using antibodies. A total of 6 NCG mice (GemPharmatech, Jiangsu Province, China) were given 1.0 × 10⁶ doses. 6 Nalm-6-LAE cells (ATCC, USA) were injected into the tail vein of each mouse, and after 5 days, the mice were analyzed by luciferase in vivo imaging (Lumina-II Small Animal In vivo Imaging System, PerkinElmer, USA) to confirm that the mouse leukemia model had been successfully prepared. Then, CD19CAR-T cells were injected into the tail vein of each mouse (prepared by our company referring to the method in Example 2.2, 2 × 10⁶ cells per mouse). 6(Cells). 100 μL of peripheral blood was collected from the orbits of mice on days 2, 4, 8, 12, 21, and 28 after CAR-T cell injection for CAR-T detection. Specifically, peripheral blood was divided into two 50 μL aliquots, 3 μL of PE-labeled 8F8D1 antibody was added to each aliquot, mixed, incubated at room temperature for 20 minutes in the dark, and then added to the lysed blood sample, incubated at room temperature for 15 minutes in the dark, washed twice with DPBS, and then resuspended in 100 μL of DPBS. The CAR-T ratio was analyzed by FACS, the number of CAR-T cells per 100 μL of peripheral blood was calculated, and the metabolic profile of CAR-T cells in each mouse was plotted (Figure 5).

[0077] The data shows the distribution of CAR-T cells in mice using PE-labeled 8F8D1 antibody. This indicates that it can be effectively detected.

Claims

1. An isolated antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), which specifically binds to a Strep-TagII tag, The VH includes VH-CDR1 containing SEQ ID NO: 1, VH-CDR2 containing SEQ ID NO: 2, and VH-CDR3 containing SEQ ID NO: 3, and the VL includes VL-CDR1 containing SEQ ID NO: 5, VL-CDR2 containing SEQ ID NO: 7, and VL-CDR3 containing SEQ ID NO: 9, or The VH comprises VH-CDR1 containing SEQ ID NO: 1, VH-CDR2 containing SEQ ID NO: 2, and VH-CDR3 containing SEQ ID NO: 4, and the VL comprises VL-CDR1 containing SEQ ID NO: 6, VL-CDR2 containing SEQ ID NO: 8, and VL-CDR3 containing SEQ ID NO: 10, in an antibody.

2. The VH comprises the amino acid sequence of SEQ ID NO: 23 or 24, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto, and the VL comprises the amino acid sequence of SEQ ID NO: 25 or 26, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. The antibody according to claim 1.

3. (i) The VH comprises one or more framework regions, each of which comprises an amino acid sequence, the amino acid sequence being the sequence presented in SEQ ID NOs: 11, 12, 14, and 16, or an amino acid sequence having at least 90%, at least 95%, or at least 99% identity thereto. The VL comprises one or more framework regions, each of which comprises an amino acid sequence, the amino acid sequence being the sequence presented in SEQ ID NOs: 17, 19, 20, and 22, or an amino acid sequence having at least 90%, at least 95%, or at least 99% identity thereto, or (ii) The VH comprises one or more framework regions, each of which comprises an amino acid sequence, the amino acid sequence being the sequence presented in SEQ ID NOs: 11, 13, 15, and 16, or an amino acid sequence having at least 90%, at least 95%, or at least 99% identity thereto. The VL comprises one or more framework regions, each of which comprises an amino acid sequence, the amino acid sequence being the sequence presented in SEQ ID NOs: 18, 19, 21, and 22, or an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to it. The antibody according to claim 1.

4. The VH includes the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto, and the VL includes the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto, or The VH comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto, and the VL comprises the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. The antibody according to claim 1.

5. The antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto, and the antibody light chain comprises the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto, or The antibody heavy chain contains the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto, and the antibody light chain contains the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. The antibody according to claim 1.

6. An isolated nucleic acid encoding an antibody according to any one of claims 1 to 5.

7. A cloning or expression vector comprising the nucleic acid described in claim 6.

8. A host cell containing the nucleic acid described in claim 6.

9. An antibody conjugate comprising an antibody according to any one of claims 1 to 5, wherein the label is selected from a fluorescent label, an enzyme substrate label, a radioisotope, digoxigenin, biotin, or avidin, a DNA molecule, or gold for detection, and is linked to the label.

10. A composition or kit comprising an antibody according to any one of claims 1 to 5 or an antibody conjugate according to claim 9, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

11. A method for detecting a fusion polypeptide containing a Strip-TagII tag in a sample, (i) Contacting the sample containing the fusion polypeptide with an isolated antibody according to any one of claims 1 to 5 in vitro under conditions that allow interaction between the antibody and the fusion polypeptide; and (ii) detecting the formation of a complex between the antibody and the sample. method.

12. A method for detecting the complex comprises at least one of flow cytometry, Western blotting, immunohistochemistry, and immunofluorescence. The method according to claim 11.

13. A method for purifying a fusion polypeptide containing a Strip-TagII tag in a sample, (i) Contacting the sample with an isolated antibody according to any one of claims 1 to 5 in vitro under conditions that allow interaction between the antibody and the fusion polypeptide; and (ii) Purifying the complex formed between the antibody and the sample. method.

14. The method according to any one of claims 11 to 13, wherein the sample is a biological sample comprising at least one of blood, urine, saliva, lymph, cerebrospinal fluid, bone marrow, tissue and organs, and cells.

15. The method according to claim 14, wherein the blood comprises at least one of serum, plasma, and whole blood.

16. The method according to claim 14 or 15, wherein the sample contains chimeric antigen receptor cells or engineered cell receptor cells.

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