Antibody against CD19 antibody, antibody against CD22 antibody, and uses thereof

Abstract

Provided are an antibody against a CD19 antibody, an antibody against a CD22 antibody, and uses thereof. The two antibodies are applied to measurement of a CAR positive expression rate of a CD19 single-target CAR-T cell, measurement of a CAR positive expression rate of a CD22 single-target CAR-T cell, and measurement of a CAR positive expression rate of a CD19-CD22 double-target CAR-T cell.

Description

Antibodies against CD19 antibodies, antibodies against CD22 antibodies and their uses Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to anti-CD19 antibodies, anti-CD22 antibodies and their applications. Background Art

[0002] The chimeric antigen receptor (CAR) is the core component of CAR cell therapy drugs. It includes an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain. CAR-T cell immunotherapy is considered one of the most promising approaches to conquer tumors. CAR-T cells are genetically engineered to express CAR proteins in T cells. This CAR protein has the ability to recognize intact proteins on the membrane independently of antigen presentation, thereby triggering T cell activation and functional effects.

[0003] Currently available CAR-T cells include several targeting CD19. While CD19-targeted CAR-T cells have demonstrated promising results in the treatment of B-cell malignancies, some patients experience CD19-negative tumor recurrence after CAR-T cell therapy. This phenomenon, where tumor cells express the CD19 antigen before CAR-T therapy but no longer express it after relapse, is considered antigen-loss or escape after CAR-T cell therapy. To overcome this phenomenon, CAR-T cells have been developed that simultaneously target both CD19 and CD22, a key target for the treatment of hematologic malignancies of B lymphocyte origin. With this dual-target CAR-T product, CAR-T cells can be activated as long as one of the tumor antigen targets is recognized. The principle of CAR-T therapy demonstrates the crucial importance of measuring the CAR-positive expression rate within a CAR-T product.

[0004] When testing CAR expression rates for single-target CAR-T products, a universal method is to use anti-F(ab')2 antibodies. Commercially available anti-F(ab')2 antibodies can be combined with congenic scFv containing the Fab variable region to detect CAR expression. These antibodies can detect CARs with different targets. For example, fluorescently labeled anti-mouse F(ab')2 antibodies can be combined with mouse scFv to detect CAR expression rates. The polyclonal goat anti-mouse F(ab')2 antibody from Jackson ImmunoRearch Laboratories is currently the most commonly used. However, for dual-target CAR-T products, F(ab')2 antibodies cannot specifically distinguish the extracellular antigen recognition domains for different targets. Therefore, to more comprehensively understand dual-target products and assess their CAR expression rates, it is necessary to develop assays that can specifically distinguish the extracellular antigen recognition domains for different targets. Furthermore, developed assays for dual-target extracellular antigen recognition domains are also applicable to the corresponding single targets.

[0005] Summary of the Invention

[0006] This application provides an anti-CD19 antibody and an anti-CD22 antibody, respectively, and applies the two antibodies to the detection of the positive expression rate of CAR in CD19 single-target CAR-T cells and the detection of the positive expression rate of CAR in CD19-CD22 dual-target CAR-T cells.

[0007] An anti-CD19 antibody or an antigen-binding portion thereof, the amino acid sequence of which includes a heavy chain variable region and a light chain variable region, wherein:

[0008] The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody set forth in SEQ ID NO: 28. Optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody set forth in SEQ ID NO: 28. The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the antibody set forth in SEQ ID NO: 29. Optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the antibody set forth in SEQ ID NO: 29.

[0009] Or, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO: 30, optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO: 30, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO: 31, optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO: 31;

[0010] Or, the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region respectively include the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO: 32. Optionally, the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region are respectively the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO: 32, and the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region respectively include the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO: 33. Optionally, the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region are respectively the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO: 33.

[0011] An anti-CD19 antibody or an antigen-binding portion thereof, the amino acid sequence of which includes a heavy chain variable region and a light chain variable region, wherein:

[0012] The CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. Optionally, the CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. The CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 64, and SEQ ID NO: 5, respectively. Optionally, the CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 64, and SEQ ID NO: 5, respectively.

[0013] Or, the CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. Alternatively, the CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively, and the CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 9, SEQ ID NO: 65, and SEQ ID NO: 10, respectively. Alternatively, the CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 9, SEQ ID NO: 65, and SEQ ID NO: 10, respectively.

[0014] Or, the CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. Optionally, the CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. The CDR1, CDR2, and CDR3 of the light chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:14, SEQ ID NO:66, and SEQ ID NO:15. Optionally, the CDR1, CDR2, and CDR3 of the light chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:14, SEQ ID NO:66, and SEQ ID NO:15.

[0015] In certain embodiments of the above-mentioned anti-CD19 antibody or its antigen-binding portion, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28, optionally, the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 29, optionally, the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 29;

[0016] Or, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30, optionally, the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 30, and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 31, optionally, the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 31;

[0017] Alternatively, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 32, optionally, the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 32, and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 33, optionally, the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 33.

[0018] In certain embodiments of the above-mentioned anti-CD19 antibody or its antigen-binding portion, the CD19 antibody is a CD19 scFv, whose amino acid sequence comprises the amino acid sequence shown in SEQ ID NO: 48, optionally, its amino acid sequence is the amino acid sequence shown in SEQ ID NO: 48.

[0019] In certain embodiments, the anti-CD19 antibody or the antigen-binding portion thereof is labeled with fluorescein.

[0020] In certain embodiments of the above-mentioned anti-CD19 antibody or its antigen-binding portion, the CDR1, CDR2, and CDR3 of the heavy chain variable region of the fluorescein-labeled anti-CD19 antibody or its antigen-binding portion comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. Optionally, the CDR1, CDR2, and CDR3 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. The CDR1, CDR2, and CDR3 of the light chain variable region of the fluorescein-labeled anti-CD19 antibody or its antigen-binding portion comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 64, and SEQ ID NO: 5, respectively. Optionally, the CDR1, CDR2, and CDR3 of the light chain variable region are the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 64, and SEQ ID NO: 5, respectively.

[0021] And / or, the fluorescein is Alexa 647 Fluorescein or Alexa 405 fluorescein; optionally, the fluorescein is Alexa 647 Fluorescein.

[0022] The present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned anti-CD19 antibody or its antigen-binding site.

[0023] In certain embodiments of the isolated nucleic acid molecule, the nucleotide sequence encoding the anti-CD19 antibody or its antigen-binding portion comprises: a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 28, optionally as set forth in SEQ ID NO: 38; and a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 29, optionally as set forth in SEQ ID NO: 39;

[0024] or, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 30, optionally, as shown in SEQ ID NO: 40; and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 31, optionally, as shown in SEQ ID NO: 41;

[0025] Or, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:32, optionally, it is shown in SEQ ID NO:42; and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:33, optionally, it is shown in SEQ ID NO:43.

[0026] The present application also provides a vector comprising the above-mentioned isolated nucleic acid molecule.

[0027] The present application also provides a cell comprising any one of the above-mentioned anti-CD19 antibody or its antigen-binding site, isolated nucleic acid molecule, and vector.

[0028] The present application also provides a use of any one of the above-mentioned anti-CD19 antibody or its antigen-binding site, isolated nucleic acid molecule, vector, and cell in the preparation of a detection reagent.

[0029] The present application also provides a detection reagent, which comprises any one of the above-mentioned anti-CD19 antibody or its antigen-binding site, isolated nucleic acid molecule, vector, and cell.

[0030] In certain embodiments of the above applications or detection reagents, the detection reagents are used to detect one or more of the following samples:

[0031] A sample comprising the CD19 scFv amino acid sequence shown in SEQ ID NO: 48;

[0032] A sample comprising the CD19-CD22 scFv amino acid sequence shown in SEQ ID NO: 50;

[0033] A sample comprising the CD19 VH amino acid sequence shown in SEQ ID NO:53 and the CD19 VL amino acid sequence shown in SEQ ID NO:54.

[0034] In certain embodiments of the above-mentioned application or detection reagent, the sample is selected from the following samples: cell sample, blood sample.

[0035] In certain embodiments of the above applications or detection reagents, the detection reagents are used for one or more of the following purposes:

[0036] a. Used to detect the positive expression rate of CAR expressing the target extracellular antigen recognition domain in CAR-T cells;

[0037] b. Used to detect the concentration of CAR-T positive cells expressing the target extracellular antigen recognition domain in the patient's blood after the patient is reinfused with CAR-T cells;

[0038] c. After the patient is reinfused with CAR-T cells, it is used as a standard reference when detecting anti-drug antibodies against the target extracellular antigen recognition domain produced in the patient's blood;

[0039] The target extracellular antigen recognition domain comprises:

[0040] The CD19 scFv amino acid sequence shown in SEQ ID NO: 48;

[0041] Or, the CD19-CD22 scFv amino acid sequence as shown in SEQ ID NO: 50;

[0042] Or, the CD19 VH amino acid sequence as shown in SEQ ID NO:53 and the CD19 VL amino acid sequence as shown in SEQ ID NO:54.

[0043] In certain embodiments of the above-mentioned application or detection reagent, the sample is from a patient with a non-solid tumor.

[0044] In certain embodiments of the above-mentioned application or detection reagent, the sample is from a leukemia patient; optionally, the sample is from a lymphocytic leukemia patient; further optionally, the sample is from an acute lymphocytic leukemia patient; further optionally, the sample is from an adult acute lymphocytic leukemia patient or a childhood acute lymphocytic leukemia patient.

[0045] In certain embodiments of the above-mentioned application or detection reagent, the sample is from a lymphoma patient; alternatively, the sample is from a B-cell lymphoma patient; further alternatively, the sample is from a non-Hodgkin's lymphoma patient.

[0046] In certain embodiments of the above-mentioned application or detection reagent, the sample is from a patient with an autoimmune disease; optionally, the patient with the autoimmune disease is a patient with systemic lupus erythematosus.

[0047] A method for detecting the CAR positive rate on the surface of CD19 single-target CAR-T cells, comprising the following steps:

[0048] The CAR molecules on the surface of CD19 single-target CAR-T cells were labeled using the above-mentioned anti-CD19 antibodies or their antigen-binding sites, and the proportion of CD19 CAR+T cells was detected by flow cytometry.

[0049] In certain embodiments of the above detection method, the antibody or antigen-binding site of the anti-CD19 antibody is fluorescently labeled with Alexa Fluor 647;

[0050] And / or, in the anti-CD19 antibody or the antigen-binding portion thereof, the CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. Optionally, the CDR1, CDR2, and CDR3 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. The CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 64, and SEQ ID NO: 5, respectively. Optionally, the CDR1, CDR2, and CDR3 of the light chain variable region are the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 64, and SEQ ID NO: 5, respectively.

[0051] An anti-CD22 antibody or an antigen-binding portion thereof, the amino acid sequence of which includes a heavy chain variable region and a light chain variable region, wherein:

[0052] The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody set forth in SEQ ID NO: 34. Optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody set forth in SEQ ID NO: 34. The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the antibody set forth in SEQ ID NO: 35. Optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the antibody set forth in SEQ ID NO: 35.

[0053] Or, the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region respectively include the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO: 36. Optionally, the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region are respectively the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO: 36, and the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region respectively include the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO: 37. Optionally, the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region are respectively the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO: 37.

[0054] An anti-CD22 antibody or an antigen-binding portion thereof, the amino acid sequence of which includes a heavy chain variable region and a light chain variable region, wherein:

[0055] The CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. Optionally, the CDR1, CDR2, and CDR3 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. The CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively. Optionally, the CDR1, CDR2, and CDR3 of the light chain variable region are the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively.

[0056] Or, the CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24. Optionally, the CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24. The CDR1, CDR2, and CDR3 of the light chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27. Optionally, the CDR1, CDR2, and CDR3 of the light chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.

[0057] In certain embodiments of the above-mentioned anti-CD22 antibody or its antigen-binding portion, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 34, optionally, the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 34, and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 35, optionally, the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 35;

[0058] Alternatively, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 36, optionally, the amino acid sequence of the heavy chain variable region is the amino acid sequence as shown in SEQ ID NO: 36, and the amino acid sequence of the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 37, optionally, the amino acid sequence of the light chain variable region is the amino acid sequence as shown in SEQ ID NO: 37.

[0059] In certain embodiments of the above-mentioned anti-CD22 antibody or its antigen-binding portion, the CD22 antibody is a CD22 scFv, whose amino acid sequence comprises the amino acid sequence shown in SEQ ID NO: 49, optionally, its amino acid sequence is the amino acid sequence shown in SEQ ID NO: 49.

[0060] In certain embodiments, the above-mentioned anti-CD22 antibody or its antigen-binding site is labeled with fluorescein; optionally, the fluorescein is Alexa Fluor. 647 Fluorescein or Alexa 405 fluorescein.

[0061] The present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned anti-CD22 antibody or its antigen-binding site.

[0062] In certain embodiments of the isolated nucleic acid molecule, the nucleotide sequence encoding the anti-CD22 antibody or its antigen-binding portion comprises: a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 34, optionally as SEQ ID NO: 44; and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 35, optionally as SEQ ID NO: 45.

[0063] Or, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:36, optionally, it is shown in SEQ ID NO:46; and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:37, optionally, it is shown in SEQ ID NO:47.

[0064] The present application also provides a vector comprising the above-mentioned isolated nucleic acid molecule.

[0065] The present application also provides a cell comprising any one of the above-mentioned anti-CD22 antibody or its antigen-binding site, isolated nucleic acid molecule, and vector.

[0066] The present application also provides a use of any one of the above-mentioned anti-CD22 antibodies or their antigen-binding site, isolated nucleic acid molecule, vector, and cells in the preparation of a detection reagent.

[0067] The present application also provides a detection reagent, which comprises any one of the above-mentioned anti-CD22 antibody or its antigen-binding site, isolated nucleic acid molecule, vector, and cell.

[0068] In certain embodiments of the above applications or detection reagents, the detection reagents are used to detect one or more of the following samples:

[0069] A sample comprising the CD22 scFv amino acid sequence shown in SEQ ID NO: 49;

[0070] A sample comprising the CD19-CD22 scFv amino acid sequence shown in SEQ ID NO: 50;

[0071] A sample comprising the CD22 VH amino acid sequence shown in SEQ ID NO:51 and the CD22 VL amino acid sequence shown in SEQ ID NO:52.

[0072] In certain embodiments of the above-mentioned application or detection reagent, the sample is selected from the following samples: cell sample, blood sample.

[0073] In certain embodiments of the above applications or detection reagents, the detection reagents are used for one or more of the following purposes:

[0074] a. Used to detect the positive expression rate of CAR expressing the target extracellular antigen recognition domain in CAR-T cells;

[0075] b. Used to detect the concentration of CAR-T positive cells expressing the target extracellular antigen recognition domain in the patient's blood after the patient is reinfused with CAR-T cells;

[0076] c. After the patient is reinfused with CAR-T cells, it is used as a standard reference when detecting anti-drug antibodies against the target extracellular antigen recognition domain produced in the patient's blood;

[0077] The target extracellular antigen recognition domain comprises:

[0078] The CD22 scFv amino acid sequence shown in SEQ ID NO:49;

[0079] Or, the CD19-CD22 scFv amino acid sequence as shown in SEQ ID NO: 50;

[0080] Or, the CD22 VH amino acid sequence shown in SEQ ID NO:51 and the CD22 VL amino acid sequence shown in SEQ ID NO:52.

[0081] In certain embodiments of the above-mentioned application or detection reagent, the sample is from a patient with a non-solid tumor.

[0082] In certain embodiments of the above-mentioned application or detection reagent, the sample is from a leukemia patient; optionally, the sample is from a lymphocytic leukemia patient; further optionally, the sample is from an acute lymphocytic leukemia patient; further optionally, the sample is from an adult acute lymphocytic leukemia patient or a childhood acute lymphocytic leukemia patient.

[0083] In certain embodiments of the above-mentioned application or detection reagent, the sample is from a lymphoma patient; alternatively, the sample is from a B-cell lymphoma patient; further alternatively, the sample is from a non-Hodgkin's lymphoma patient.

[0084] In certain embodiments of the above-mentioned application or detection reagent, the sample is from a patient with an autoimmune disease; optionally, the patient with the autoimmune disease is a patient with systemic lupus erythematosus.

[0085] A method for detecting the CAR positive rate on the surface of CD22 single-target CAR-T cells, comprising the following steps:

[0086] The above-mentioned anti-CD22 antibody or its antigen binding site was used to label the CAR molecules on the surface of CD22 single-target CAR-T cells, and CD22 CAR was detected by flow cytometry. + T cell ratio.

[0087] In certain embodiments of the above detection method, the anti-CD22 antibody or its antigen-binding site is fluorescently labeled with Alexa Fluor 647.

[0088] In certain embodiments of the above detection method, in the anti-CD22 antibody, CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. Alternatively, CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, and CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively. Alternatively, CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively.

[0089] Or, the CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24. Optionally, the CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24. The CDR1, CDR2, and CDR3 of the light chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27. Optionally, the CDR1, CDR2, and CDR3 of the light chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.

[0090] A method for detecting the CAR positive rate on the surface of CD19-CD22 dual-target CAR-T cells, comprising the following steps:

[0091] The above-mentioned anti-CD19 antibody or its antigen binding site, and / or the above-mentioned anti-CD22 antibody or its antigen binding site, are used to label the CAR molecules on the surface of CD19-CD22 dual-target CAR-T cells, and the CD19 CAR is detected by flow cytometry. + T cells and / or CD22 CAR + T cell ratio.

[0092] In certain embodiments of the above detection method, the anti-CD19 antibody or its antigen-binding site and / or the anti-CD22 antibody or its antigen-binding site are fluorescently labeled with Alexa Fluor 647;

[0093] In certain embodiments of the above detection method,

[0094] Among the anti-CD19 antibodies,

[0095] The CDR1, CDR2, and CDR3 of the heavy chain variable region respectively contain the amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. Optionally, the CDR1, CDR2, and CDR3 of the heavy chain variable region respectively contain the amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. The CDR1, CDR2, and CDR3 of the light chain variable region respectively contain the amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:64, and SEQ ID NO:5. Optionally, the CDR1, CDR2, and CDR3 of the light chain variable region respectively contain the amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:64, and SEQ ID NO:5.

[0096] And / or, in the anti-CD22 antibody,

[0097] The CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. Alternatively, the CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. The CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively. Alternatively, the CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively.

[0098] Or, the CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24. Optionally, the CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24. The CDR1, CDR2, and CDR3 of the light chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27. Optionally, the CDR1, CDR2, and CDR3 of the light chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] FIG1 briefly shows the screening process of anti-CD19 antibodies and anti-CD22 antibodies in the present application. DETAILED DESCRIPTION

[0100] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0101] The present application is further described below. Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those commonly used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0102] In this application, the term "chimeric antigen receptor" (CAR) is the core component of CAR cell therapy drugs, which may include an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain. CAR-T (Chimeric Antigen Receptor T) cell immunotherapy is considered one of the most promising means of conquering tumors. CAR-T cells use genetic modification methods to make T cells express CAR proteins. This CAR protein has the ability to recognize intact proteins on the membrane surface without relying on antigen presentation, thereby causing T cell activation and functional effects.

[0103] In this application, the term "extracellular antigen recognition domain" refers to the Antigen Recognition Domain (ARD). The ability of CAR cell therapy products (such as CAR-T cells) to specifically recognize and / or bind to target antigens expressed by tumor cells depends on the extracellular antigen recognition domain. So far, the antigen recognition domain has been derived from the single chain variable region (scFv) of the antibody, or from receptor-ligand interactions, TCR mimics, and variable lymphocyte receptors (VLR). So far, the most common source is the scFv antibody. The scFv antibodies targeting two or more targets include VH and VL regions targeting different targets. The different regions are directly or indirectly connected by a linker sequence, and their arrangement can be any of the following forms: target 1 VL-target 1 VH-target 2 VL-target 2 VH, target 2 VL-target 2 VH-target 1 VL-target 1 VH, target 1 VL-target 2 VL-target 2 VH-target 1 VH, target 2 VL-target 1 VL-target 1 VH-target 2 VH, the above “-” represents connection through a linker sequence.

[0104] In the present application, the term "specific recognition and / or binding" refers to the recognition and / or binding between the extracellular antigen recognition domain of CAR and a specific target, or the recognition and / or binding between the extracellular antigen recognition domain of CAR and a specific detection antibody is with greater affinity, avidity, more easily, and / or for a longer duration than CAR binds to other targets or detection antibodies.

[0105] In this application, the term "antibody" has the conventional meaning in the art and is used in the broadest sense in this application. In the field of biological sciences, by analyzing the amino acid sequences of different antibody heavy and light chains, it was found that the amino acid sequences near the N-terminus of the heavy and light chains vary greatly, while the amino acid sequences of other parts are relatively constant. Therefore, the region near the N-terminus of the antibody light and heavy chains where the amino acid sequence varies greatly is called the variable region (V), and the region near the C-terminus where the amino acid sequence is relatively stable is called the constant region (C). The V regions of the heavy and light chains are abbreviated as VH and VL, respectively, and the C regions of the heavy and light chains are abbreviated as CH and CL, respectively. Within the variable region of an antibody, there is a small number of amino acid residues that vary particularly strongly. The regions where the residue composition and arrangement of these amino acids are more prone to variation are called hypervariable regions (HVRs). There are three hypervariable regions in the V regions of the L and H chains. Because this region can form precise complementarity with the antigenic determinant in terms of spatial structure, the hypervariable region is also called the complementarity determining region (CDR). In antibodies, common CDR division rules include Kabat, AbM, Chothia, Contact, and IMGT. These rules are well known to those skilled in the art. When applying a website that implements these rules, as long as the VH and VL sequences are input and the corresponding rules are selected, CDR sequences according to different rules can be obtained. It will be understood by those skilled in the art that the scope of protection of this application covers the combination of CDR sequences obtained by adopting different rule analyses. The 6 CDR regions of an antibody jointly determine the recognition ability and specificity of the antibody to the corresponding antigen. It will be understood by those skilled in the art that when this application limits the amino acid sequences of the 6 CDR regions, the recognition ability and specificity of the antibody to the corresponding antigen are predictable.

[0106] In this application, the term "antigen binding site" has the conventional meaning in the art, and refers to the key site on an antibody that can specifically recognize and bind to an antigen, including the VH and / or VL regions.

[0107] In this application, the term "linker" generally refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links together any structure / region of an antibody or chimeric antigen receptor of the present invention. The linker can be composed of different amino acid residues (such as glycine and serine) so that adjacent protein domains are free to move relative to each other. When it is desired to ensure that two adjacent domains do not interfere with each other spatially, a longer linker can be used.

[0108] In this application, the term "isolated" generally refers to a substance obtained from its natural state by artificial means. If a substance or component is "isolated" in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolated. The term "isolated" does not exclude substances that have been artificially obtained from their natural state by artificial means or synthesized, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0109] As used herein, the term "isolated nucleic acid molecule" generally refers to nucleotides, deoxyribonucleotides, or ribonucleotides of any length in isolated form, either separated from their natural environment or artificially synthesized analogs.

[0110] The term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and protein expression can be achieved. A vector can transform, transduce, or transfect host cells, allowing the genetic material it carries to be expressed in host cells. Examples of vectors include plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage; and animal viruses. Examples of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A single vector may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication origin site. Vectors may also include components that facilitate cell entry, such as viral particles, liposomes, or protein coats, but are not limited to these. The term "transposon" refers to a discrete DNA fragment that has the ability to migrate between chromosomal loci and carry genetic information, such as the Sleeping Beauty SB system and the PB system from Lepidoptera. In some embodiments, electroporation can also be used to transduce mRNA into T cells.

[0111] In this application, the terms "comprises" and "comprising" generally mean that the features explicitly stated are included, but other elements are not excluded.

[0112] In this application, the term "about" generally refers to a fluctuation range above or below the specified value that is acceptable to those skilled in the art, such as: a variation within the range of ±0.5%-10%, for example, a variation within the range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% above or below the specified value.

[0113] In this application, CAR-T cells, such as CD19 CAR-T cells, CD19-CD22 CAR-T cells, and EGFR CAR-T cells, are prepared using existing preparation methods. We use a second-generation CAR structure, with the CD8α guide chain as the signal peptide, the corresponding scFv as the extracellular tumor antigen recognition region, the hinge region and transmembrane region adopt the CD8α structure, 4-1BB as the intracellular costimulatory signal, and CD3ζ as the T cell activation signal. The specific preparation steps are as follows:

[0114] 1. Construction of lentiviral vector: artificially synthesize the CAR structure fragment containing the corresponding scFv in the present application (the CAR structure includes, in sequence: the CD8α guide chain as shown in SEQ ID NO: 55, the corresponding scFv sequence, the CD8α hinge region as shown in SEQ ID NO: 56, the CD8α transmembrane region as shown in SEQ ID NO: 57, the 4-1BB intracellular co-stimulatory signal as shown in SEQ ID NO: 58, and the CD3ζT cell activation signal as shown in SEQ ID NO: 59, wherein: the amino acid sequence of CD19 scFv is as shown in SEQ ID NO: 48, the amino acid sequence of CD19-CD22 scFv is as shown in SEQ ID NO: 50, and the amino acid sequence of EGFR scFv is as shown in SEQ ID NO: 51 in WO2022068870A1. NO: 9), and were respectively constructed into resistance-modified empty lentiviral vectors (manufacturer: SBI, product number: CD500-CD800, as described in Example 1 of WO2021 / 121227, after resistance modification) to obtain CAR expression vectors, and then the CAR expression vectors and three packaging plasmids were transfected into 293T cells together, and functional lentiviral vectors were obtained after collection and purification. The three packaging plasmids are pMD2.0G (purchased from Biovector, product number Biovector012259), pMDLg-pRRE (purchased from Biovector, product number Biovector012251), and pRSV-Rev (purchased from Biovector, product number Biovector012253).

[0115] 2. Preparation of corresponding CAR-T cells by lentiviral transduction

[0116] The transduction experiment was performed according to conventional methods known to those skilled in the art. The transduction steps are briefly described as follows:

[0117] 1) T cell sorting: Peripheral blood mononuclear cells (PBMCs) are isolated from human single-collected blood cells, and then T cells are sorted from the PBMCs.

[0118] 2) Activation of T cells: The isolated T cells were resuspended in complete lymphocyte culture medium (X-VIVO15 medium + 5% FBS + 300 IU / ml IL-2 or X-VIVO15 medium + 5% FBS + 5 ng / ml IL-15 + 10 ng / ml IL-7) to a final concentration of (1-2) × 10 6 cells / ml, and add 5-10 μl of CD3 / CD28 magnetic beads for stimulation. After mixing, place in an incubator for culture at 37°C + 5% CO2 for at least 24 hours.

[0119] 3) Lentiviral T cell transduction: Remove the activated cultured T cells, add polybrene to a final concentration of 8 μg / ml, mix thoroughly, and slowly add the lentiviral vector at an MOI of 2. After mixing, centrifuge at 1500 rpm for 1.5 hours. Then, incubate in an incubator at 37°C + 5% CO2 for at least 24 hours.

[0120] 4) Expansion and culture of transduced T cells: Take out the transduced cells and monitor the cell density to maintain the cell density at (0.5-1)×10 6 cells / ml for use in subsequent examples.

[0121] Antibody or antigen-binding portion thereof of an anti-CD19 antibody

[0122] An anti-CD19 antibody or an antigen-binding portion thereof, the amino acid sequence of which includes a heavy chain variable region and a light chain variable region, wherein:

[0123] The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody set forth in SEQ ID NO: 28. Optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody set forth in SEQ ID NO: 28. The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the antibody set forth in SEQ ID NO: 29. Optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the antibody set forth in SEQ ID NO: 29.

[0124] Or, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO: 30, optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO: 30, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO: 31, optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO: 31;

[0125] Or, the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region respectively include the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO: 32. Optionally, the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region are respectively the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO: 32, and the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region respectively include the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO: 33. Optionally, the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region are respectively the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO: 33.

[0126] Common CDR delineation rules for antibodies include Kabat, AbM, Chothia, Contact, and IMGT. These rules are well known to those skilled in the art. When using a website that implements these rules, simply input the VH and VL sequences and select the corresponding rule to obtain CDR sequences based on different rules. Those skilled in the art will understand that the scope of protection of this application covers combinations of CDR sequences obtained by analyzing using different rules.

[0127] The present application also provides an anti-CD19 antibody or an antigen-binding portion thereof, the amino acid sequence of which includes a heavy chain variable region and a light chain variable region, wherein:

[0128] The CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. Optionally, the CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. The CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 64, and SEQ ID NO: 5, respectively. Optionally, the CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 64, and SEQ ID NO: 5, respectively.

[0129] Or, the CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. Alternatively, the CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively, and the CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 9, SEQ ID NO: 65, and SEQ ID NO: 10, respectively. Alternatively, the CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 9, SEQ ID NO: 65, and SEQ ID NO: 10, respectively.

[0130] Or, the CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. Optionally, the CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13. The CDR1, CDR2, and CDR3 of the light chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:14, SEQ ID NO:66, and SEQ ID NO:15. Optionally, the CDR1, CDR2, and CDR3 of the light chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:14, SEQ ID NO:66, and SEQ ID NO:15.

[0131] In this application, the IMGT rules are used to divide CDRs.

[0132] In certain embodiments of the above-mentioned anti-CD19 antibody or its antigen-binding portion, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28, optionally, the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 29, optionally, the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 29;

[0133] Or, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30, optionally, the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 30, and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 31, optionally, the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 31;

[0134] Alternatively, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 32, optionally, the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 32, and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 33, optionally, the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 33.

[0135] In certain embodiments of the above-mentioned anti-CD19 antibody or its antigen-binding portion, the CD19 antibody is a CD19 scFv, whose amino acid sequence comprises the amino acid sequence shown in SEQ ID NO: 48, optionally, its amino acid sequence is the amino acid sequence shown in SEQ ID NO: 48.

[0136] In certain embodiments, the anti-CD19 antibody or the antigen-binding portion thereof is labeled with fluorescein.

[0137] In certain embodiments of the above-mentioned anti-CD19 antibody or its antigen-binding portion, the amino acid sequence of the fluorescein-labeled anti-CD19 antibody or its antigen-binding portion comprises a heavy chain variable region and a light chain variable region, wherein: CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. Optionally, CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 64, and SEQ ID NO: 5, respectively. Optionally, CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 64, and SEQ ID NO: 5, respectively.

[0138] And / or, the fluorescein is Alexa 647 Fluorescein or Alexa 405 fluorescein; optionally, the fluorescein is Alexa 647 Fluorescein.

[0139] Isolated nucleic acid molecules, vectors, cells

[0140] The present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned anti-CD19 antibody or its antigen-binding site.

[0141] In certain embodiments of the isolated nucleic acid molecule, the nucleotide sequence encoding the anti-CD19 antibody or its antigen-binding portion comprises: a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 28, optionally as SEQ ID NO: 38; and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 29, optionally as SEQ ID NO: 39;

[0142] or, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 30, optionally, as shown in SEQ ID NO: 40; and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 31, optionally, as shown in SEQ ID NO: 41;

[0143] Or, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:32, optionally, it is shown in SEQ ID NO:42; and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:33, optionally, it is shown in SEQ ID NO:43.

[0144] The present application also provides a vector comprising the above-mentioned isolated nucleic acid molecule.

[0145] The present application also provides a cell comprising any one of the above-mentioned anti-CD19 antibody or its antigen-binding site, isolated nucleic acid molecule, and vector.

[0146] Use of anti-CD19 antibodies or their antigen-binding portions

[0147] The present application also provides a use of any one of the above-mentioned anti-CD19 antibody or its antigen-binding site, isolated nucleic acid molecule, vector, and cell in the preparation of a detection reagent.

[0148] The present application also provides a detection reagent, which comprises any one of the above-mentioned anti-CD19 antibody or its antigen-binding site, isolated nucleic acid molecule, vector, and cell.

[0149] In certain embodiments of the above applications or detection reagents, the detection reagents are used to detect one or more of the following samples:

[0150] A sample comprising the CD19 scFv amino acid sequence shown in SEQ ID NO: 48;

[0151] A sample comprising the CD19-CD22 scFv amino acid sequence shown in SEQ ID NO: 50;

[0152] A sample comprising the CD19 VH amino acid sequence shown in SEQ ID NO:53 and the CD19 VL amino acid sequence shown in SEQ ID NO:54.

[0153] In this application, it has been demonstrated through experiments that antibodies #113, #114, and #115 produced using CD19 scFv as a target antigen can be specifically used to detect samples containing the amino acid sequence of CD19 scFv. It has also been demonstrated through experiments that antibody #113 produced using CD19 scFv as a target antigen can be specifically used to detect samples containing the amino acid sequence of CD19-CD22 dual-target scFv. Furthermore, since the CD19-CD22 dual-target CAR-T cells whose cell surface CAR positivity was detected adopted the form of CD22 VL-CD19 VL-CD19 VH-CD22 VH, where the linker sequence between CD19 VL and CD19 VH was also different from the linker sequence in the target antigen CD19 scFv used to produce the antibody, it was necessary to confirm that the antibody produced using CD19 scFv as a target antigen was not affected by changes in the linker sequence when used for detection. Antibody #113 obtained with CD19 scFv as the target antigen, for the CAR expression rate detection of the CD19-CD22 dual-target structure arranged in the order of CD22 VL-CD19 VL-CD19 VH-CD22 VH in this application, can be accurately and effectively detected, indicating that the binding site of the corresponding antibody obtained with CD19 scFv as the target antigen is not related to the connecting sequence on the target antigen, but is bound to CD19 VH or CD19 VL. Therefore, the antibody obtained with CD19 scFv as the target antigen in this application is also applicable to the dual-target structures of CD22 VL-CD22 VH-CD19 VL-CD19 VH, CD19 VL-CD19 VH-CD22 VL-CD22 VH, and CD19 VL-CD22 VL-CD22 VH-CD19 VH.

[0154] In certain embodiments of the above-mentioned application or detection reagent, the sample is selected from the following samples: cell sample, blood sample.

[0155] In certain embodiments of the above-mentioned applications or detection reagents, the detection reagents are used for one or more of the following purposes:

[0156] a. Used to detect the positive expression rate of CAR expressing the target extracellular antigen recognition domain in CAR-T cells;

[0157] b. Used to detect the concentration of CAR-T positive cells expressing the target extracellular antigen recognition domain in the patient's blood after the patient is reinfused with CAR-T cells;

[0158] c. After the patient is reinfused with CAR-T cells, it is used as a standard reference when detecting anti-drug antibodies against the target extracellular antigen recognition domain produced in the patient's blood;

[0159] The target extracellular antigen recognition domain comprises:

[0160] The CD19 scFv amino acid sequence shown in SEQ ID NO: 48;

[0161] Or, the CD19-CD22 scFv amino acid sequence as shown in SEQ ID NO:50.

[0162] Or, the CD19 VH amino acid sequence as shown in SEQ ID NO:53 and the CD19 VL amino acid sequence as shown in SEQ ID NO:54.

[0163] In certain embodiments of the above-mentioned application or detection reagent, the sample is from a patient with a non-solid tumor.

[0164] In certain embodiments of the above-mentioned application or detection reagent, the sample is from a leukemia patient; optionally, the sample is from a lymphocytic leukemia patient; further optionally, the sample is from an acute lymphocytic leukemia patient; further optionally, the sample is from an adult acute lymphocytic leukemia patient or a childhood acute lymphocytic leukemia patient.

[0165] In certain embodiments of the above-mentioned application or detection reagent, the sample is from a lymphoma patient; alternatively, the sample is from a B-cell lymphoma patient; further alternatively, the sample is from a non-Hodgkin's lymphoma patient.

[0166] In certain embodiments of the above-mentioned application or detection reagent, the sample is from a patient with an autoimmune disease; optionally, the patient with the autoimmune disease is a patient with systemic lupus erythematosus.

[0167] Method for detecting the positive rate of CAR on the surface of CD19 single-target CAR-T cells

[0168] A method for detecting the CAR positive rate on the surface of CD19 single-target CAR-T cells, comprising the following steps:

[0169] The CAR molecules on the surface of CD19 single-target CAR-T cells were labeled using the above-mentioned anti-CD19 antibodies or their antigen-binding sites, and the proportion of CD19 CAR+T cells was detected by flow cytometry.

[0170] In certain embodiments of the above detection method, the antibody or antigen-binding site of the anti-CD19 antibody is fluorescently labeled with Alexa Fluor 647;

[0171] And / or, in the anti-CD19 antibody or the antigen-binding portion thereof, the CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. Optionally, the CDR1, CDR2, and CDR3 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. The CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 64, and SEQ ID NO: 5, respectively. Optionally, the CDR1, CDR2, and CDR3 of the light chain variable region are the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 64, and SEQ ID NO: 5, respectively.

[0172] Antibody or antigen-binding portion thereof of an anti-CD22 antibody

[0173] An anti-CD22 antibody or an antigen-binding portion thereof, the amino acid sequence of which includes a heavy chain variable region and a light chain variable region, wherein:

[0174] The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody set forth in SEQ ID NO: 34. Optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody set forth in SEQ ID NO: 34. The amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the antibody set forth in SEQ ID NO: 35. Optionally, the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are respectively the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the antibody set forth in SEQ ID NO: 35.

[0175] Or, the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region respectively include the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO: 36. Optionally, the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region are respectively the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO: 36, and the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region respectively include the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO: 37. Optionally, the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region are respectively the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO: 37.

[0176] Common CDR delineation rules for antibodies include Kabat, AbM, Chothia, Contact, and IMGT. These rules are well known to those skilled in the art. When using a website that implements these rules, simply input the VH and VL sequences and select the corresponding rule to obtain CDR sequences based on different rules. Those skilled in the art will understand that the scope of protection of this application covers combinations of CDR sequences obtained by analyzing using different rules.

[0177] An anti-CD22 antibody or an antigen-binding portion thereof, the amino acid sequence of which includes a heavy chain variable region and a light chain variable region, wherein:

[0178] The CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. Optionally, the CDR1, CDR2, and CDR3 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. The CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively. Optionally, the CDR1, CDR2, and CDR3 of the light chain variable region are the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively.

[0179] Or, the CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24. Optionally, the CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24. The CDR1, CDR2, and CDR3 of the light chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27. Optionally, the CDR1, CDR2, and CDR3 of the light chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.

[0180] In certain embodiments of the above-mentioned anti-CD22 antibody or its antigen-binding portion, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 34, optionally, the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 34, and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 35, optionally, the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 35;

[0181] Alternatively, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 36, optionally, the amino acid sequence of the heavy chain variable region is the amino acid sequence as shown in SEQ ID NO: 36, and the amino acid sequence of the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 37, optionally, the amino acid sequence of the light chain variable region is the amino acid sequence as shown in SEQ ID NO: 37.

[0182] In this application, the KABAT rule is used to divide CDRs.

[0183] In certain embodiments of the above-mentioned anti-CD22 antibody or its antigen-binding portion, the CD22 antibody is a CD22 scFv, whose amino acid sequence comprises the amino acid sequence shown in SEQ ID NO: 49, optionally, its amino acid sequence is the amino acid sequence shown in SEQ ID NO: 49.

[0184] In certain embodiments, the above-mentioned anti-CD22 antibody or its antigen-binding site is labeled with fluorescein; optionally, the fluorescein is Alexa Fluor. 647 Fluorescein or Alexa 405 fluorescein.

[0185] Isolated nucleic acid molecules, vectors, cells

[0186] The present application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned anti-CD22 antibody or its antigen-binding site.

[0187] In certain embodiments of the isolated nucleic acid molecule, the nucleotide sequence encoding the anti-CD22 antibody or its antigen-binding portion comprises: a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 34, optionally as SEQ ID NO: 44; and a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 35, optionally as SEQ ID NO: 45;

[0188] Or, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:36, optionally, it is shown in SEQ ID NO:46; and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:37, optionally, it is shown in SEQ ID NO:47.

[0189] The present application also provides a vector comprising the above-mentioned isolated nucleic acid molecule.

[0190] The present application also provides a cell comprising any one of the above-mentioned anti-CD22 antibody or its antigen-binding site, isolated nucleic acid molecule, and vector.

[0191] Use of anti-CD22 antibodies or their antigen-binding portions

[0192] The present application also provides a use of any one of the above-mentioned anti-CD22 antibodies or their antigen-binding site, isolated nucleic acid molecule, vector, and cells in the preparation of a detection reagent.

[0193] The present application also provides a detection reagent, which comprises any one of the above-mentioned anti-CD22 antibody or its antigen-binding site, isolated nucleic acid molecule, vector, and cell.

[0194] In certain embodiments of the above applications or detection reagents, the detection reagents are used to detect one or more of the following samples:

[0195] A sample comprising the CD22 scFv amino acid sequence shown in SEQ ID NO: 49;

[0196] A sample comprising the CD19-CD22 scFv amino acid sequence shown in SEQ ID NO: 50;

[0197] A sample comprising the CD22 VH amino acid sequence shown in SEQ ID NO:51 and the CD22 VL amino acid sequence shown in SEQ ID NO:52.

[0198] In certain embodiments of the above-mentioned application or detection reagent, the sample is selected from the following samples: cell sample, blood sample.

[0199] In certain embodiments of the above-mentioned applications or detection reagents, the detection reagents are used for one or more of the following purposes:

[0200] a. Used to detect the positive expression rate of CAR expressing the target extracellular antigen recognition domain in CAR-T cells;

[0201] b. Used to detect the concentration of CAR-T positive cells expressing the target extracellular antigen recognition domain in the patient's blood after the patient is reinfused with CAR-T cells;

[0202] c. After the patient is reinfused with CAR-T cells, it is used as a standard reference when detecting anti-drug antibodies against the target extracellular antigen recognition domain produced in the patient's blood;

[0203] The target extracellular antigen recognition domain comprises:

[0204] The CD22 scFv amino acid sequence shown in SEQ ID NO:49;

[0205] Or, the CD19-CD22 scFv amino acid sequence as shown in SEQ ID NO: 50;

[0206] Or, the CD22 VH amino acid sequence shown in SEQ ID NO:51 and the CD22 VL amino acid sequence shown in SEQ ID NO:52.

[0207] In this application, it is demonstrated through experiments that antibodies produced using CD22 scFv as a target antigen can be specifically used to detect samples containing the CD19-CD22 dual-target scFv amino acid sequence. Such antibodies can of course be specifically used to detect samples containing the CD22 scFv amino acid sequence. Furthermore, since the CD19-CD22 dual-target CAR-T cells whose cell surface CAR positivity is detected, the CD19-CD22 dual-target scFv adopts the form of CD22 VL-CD19 VL-CD19 VH-CD22 VH, wherein: CD22 VL and CD22 VH are located on both sides of CD19VL and CD19VH. Therefore, the antibodies produced using CD22 scFv as a target antigen must confirm that their binding to the target antigen is not affected by the linker sequence. Antibodies 23E3 and 24H10 obtained with CD22 scFv as the target antigen were detected for CAR expression rate by arranging the CD19-CD22 dual-target structure in the order of CD22 VL-CD19 VL-CD19 VH-CD22 VH in this application, and they were able to detect accurately and effectively, indicating that the binding site of the corresponding antibody obtained with CD22 scFv as the target antigen and the target antigen has nothing to do with the connecting sequence on the target antigen, but binds to CD22 VH or CD22 VL. Therefore, the antibodies obtained with CD22 scFv as the target antigen in this application are also applicable to the dual-target structures of CD22 VL-CD22 VH-CD19 VL-CD19 VH, CD19 VL-CD19 VH-CD22 VL-CD22 VH, and CD19 VL-CD22 VL-CD22 VH-CD19 VH.

[0208] In certain embodiments of the above-mentioned application or detection reagent, the sample is from a patient with a non-solid tumor.

[0209] In certain embodiments of the above-mentioned application or detection reagent, the sample is from a leukemia patient; alternatively, the sample is from a lymphocytic leukemia patient; further alternatively, the sample is from an acute lymphocytic leukemia patient; further alternatively, the sample is from an adult acute lymphocytic leukemia patient or a childhood acute lymphocytic leukemia patient;

[0210] In certain embodiments of the above-mentioned application or detection reagent, the sample is from a lymphoma patient; alternatively, the sample is from a B-cell lymphoma patient; further alternatively, the sample is from a non-Hodgkin's lymphoma patient.

[0211] In certain embodiments of the above-mentioned application or detection reagent, the sample is from a patient with an autoimmune disease; optionally, the patient with the autoimmune disease is a patient with systemic lupus erythematosus.

[0212] Method for detecting the positive rate of CAR on the surface of CD22 single-target CAR-T cells

[0213] A method for detecting the CAR positive rate on the surface of CD22 single-target CAR-T cells, comprising the following steps:

[0214] The above-mentioned anti-CD22 antibody or its antigen binding site was used to label the CAR molecules on the surface of CD22 single-target CAR-T cells, and CD22 CAR was detected by flow cytometry. + T cell ratio.

[0215] In certain embodiments of the above detection method, the anti-CD22 antibody or its antigen-binding site is fluorescently labeled with Alexa Fluor 647.

[0216] In certain embodiments of the above detection method, in the anti-CD22 antibody, CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. Alternatively, CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, and CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively. Alternatively, CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively.

[0217] Or, the CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24. Optionally, the CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24. The CDR1, CDR2, and CDR3 of the light chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27. Optionally, the CDR1, CDR2, and CDR3 of the light chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.

[0218] Method for detecting the positive rate of CAR on the surface of CD19-CD22 dual-target CAR-T cells

[0219] A method for detecting the CAR positive rate on the surface of CD19-CD22 dual-target CAR-T cells, comprising the following steps:

[0220] The above-mentioned anti-CD19 antibody or its antigen binding site, and / or the above-mentioned anti-CD22 antibody or its antigen binding site, are used to label the CAR molecules on the surface of CD19-CD22 dual-target CAR-T cells, and the CD19 CAR is detected by flow cytometry. + T cells and / or CD22 CAR + T cell ratio.

[0221] In certain embodiments of the above detection method, the anti-CD19 antibody or its antigen-binding site and / or the anti-CD22 antibody or its antigen-binding site are fluorescently labeled with Alexa Fluor 647;

[0222] In certain embodiments of the above detection method,

[0223] Among the anti-CD19 antibodies,

[0224] The CDR1, CDR2, and CDR3 of the heavy chain variable region respectively contain the amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. Optionally, the CDR1, CDR2, and CDR3 of the heavy chain variable region respectively contain the amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. The CDR1, CDR2, and CDR3 of the light chain variable region respectively contain the amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:64, and SEQ ID NO:5. Optionally, the CDR1, CDR2, and CDR3 of the light chain variable region respectively contain the amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:64, and SEQ ID NO:5.

[0225] And / or, in the anti-CD22 antibody,

[0226] The CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. Alternatively, the CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. The CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively. Alternatively, the CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively.

[0227] Or, the CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24. Optionally, the CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24. The CDR1, CDR2, and CDR3 of the light chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27. Optionally, the CDR1, CDR2, and CDR3 of the light chain variable region respectively comprise the amino acid sequences as shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.

[0228] Without intending to be bound by any theory, the following examples are merely illustrative of the chimeric antigen receptors, immune effector cells, preparation methods, and uses of the present invention and are not intended to limit the scope of the present invention. The examples do not include a detailed description of conventional methods, such as those used to construct vectors and plasmids, insert protein-encoding genes into such vectors and plasmids, or introduce plasmids into host cells. Such methods are well known to those skilled in the art and are described in numerous publications, including Sambrook, J., Fritsch, E.F., and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.

[0229] Example 1. Preparation of CD19 scFv and CD22 scFv as target antigens and animal immunization

[0230] FIG1 briefly shows the screening process of anti-CD19 antibodies and anti-CD22 antibodies in the present application.

[0231] A DNA sequence encoding the amino acid sequence of a CD19 scFv (SEQ ID NO:48) was constructed into a recombinant expression vector with a His tag. The CD19 scFv was expressed in HEK293 mammalian cells and purified to obtain the target antigen CD19 scFv. Similarly, a scFv targeting another target, CD33, was prepared using the same expression vector and expression system as a negative control antigen. The amino acid sequence of this negative control antigen, CD33 scFv, had a molecular weight similar to that of the aforementioned CD19 scFv. The target antigen CD19 scFv and the negative control antigen CD33 scFv used the same linker sequence to connect the VL and VH. The target antigen CD19 scFv and the negative control antigen CD33 scFv were each used to immunize one New Zealand rabbit, with each rabbit immunized three times.

[0232] A DNA sequence encoding the amino acid sequence of CD22 scFv was constructed into a recombinant expression vector with a His tag, wherein the amino acid sequence of CD22 scFv is shown in SEQ ID NO: 49, and was expressed using mammalian HD CHO-S cells and then purified to obtain the target antigen CD22 scFv. Since in the CD19-CD22 dual-target CAR-T cells of the present application, CD22 VL and VH were designed at the N-terminus and C-terminus of CD19 scFv, respectively, when CD22 scFv was used as the target antigen, it was not desired that the obtained antibody specifically bind to the connecting sequence of CD22 scFv. Therefore, the negative control antigen selected was different: a DNA sequence encoding the connecting sequence in the amino acid sequence of CD22 scFv was constructed into the same expression vector and expression system, expressed and then purified to obtain a negative control antigen. The target antigen CD22 scFv and negative control antigen scFv obtained above were used to immunize 4 New Zealand rabbits, respectively, for a total of three immunizations.

[0233] Example 2: ELISA and FACS detection of rabbit immune serum

[0234] After three immunizations, rabbit immune serum was collected for enzyme-linked immunosorbent assay (ELISA) and flow cytometry fluorescence sorting (FACS). ELISA was used to detect the specific binding and titer of the test sample to the target antigen at the protein level. FACS was used to determine whether the test sample effectively bound to the target antigen in its natural conformation expressed on the cell surface by measuring the binding of the test sample to the target antigen CAR-T-positive cells and the binding of the test sample to untransfected T cells (UTD cells, as a negative control).

[0235] 1. Regarding the use of CD19 scFv as a target antigen

[0236] 1. ELISA test

[0237] The experimental steps are as follows: 7 days after the third immunization of New Zealand rabbits, rabbit immune serum was collected from the ear vein and diluted at different ratios as test samples; different 96-well plates were coated with the target antigen CD19 scFv at a concentration of 0.5 mg / mL and the negative control antigen at a concentration of 0.5 mg / mL and blocked overnight; the test samples were added to the coated 96-well plates, and the 96-well plates were incubated in a 37°C incubator for 1 hour; the 96-well plates were washed four times with washing solution to wash away unbound test samples; horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (purchased from Jackson ImmunoResearch Laboratories, catalog number #111-035-045) was added and incubated in a 37°C incubator for 1 hour; the 96-well plates were washed four times with washing solution, 3,3',5,5'-tetramethylbenzidine (TMB) was added for color development, and the absorbance readings were measured at 370 nm for data processing.

[0238] The test results showed that the rabbit immune serum could recognize the target antigen well and bind specifically to the target antigen with increasing dilution gradient, with a titer of 128,000:1.

[0239] 2. FACS detection

[0240] Experimental steps: For CD19 CAR-T cell suspension and UTD cell suspension, set up test tubes, negative control tubes (cell control tube, secondary antibody control tube), and positive control tubes respectively. The details are as follows: CD19 CAR-T cells and UTD were collected and resuspended to a cell density of 1×10 7 / mL to obtain cell suspension; rabbit immune serum diluted 1:400 was incubated with CD19 CAR-T cells or UTD at 4°C for 1 hour in the test tube, and the cell pellet was collected after centrifugation of each tube, and the secondary antibody Alexa 647 fluorescently labeled anti-rabbit F(ab')2 antibody (purchased from Cell Signaling Technology, catalog number #4414) was used to detect rabbit antibodies in rabbit immune serum. The cells were incubated at room temperature in the dark for 15 minutes. After centrifugation of each tube, the cell pellets were collected, washed, and resuspended for flow cytometry. A cell control tube (only CD19 CAR-T cell suspension was added without rabbit immune serum or secondary antibody, and the operation steps were the same as those of the test tube) and a secondary antibody control tube (only secondary antibody was added to the CD19 CAR-T cell suspension without rabbit immune serum, and the operation steps were the same as those of the test tube) were set up as negative controls. A positive control tube (CD19 CAR-T cell suspension was added with Alexa Fluor 500 mAb) was set up. 647 fluorescently labeled anti-mouse F(ab')2 antibody was used to directly detect the mouse CD19 scFv expressed on the surface of CD19 CAR-T cells. The anti-mouse F(ab')2 antibody was purchased from Jackson ImmunoResearch Laboratories, catalog number #115-605-072. The difference in CAR positivity between the test tube and the positive control tube was used to determine the suitability of the test sample for the next step of validation. If the difference was within 5%, the sample was used for the next step of validation.

[0241] The test results, shown in Table 1, show that the rabbit immune serum binds well to the native conformation CD19 scFv molecules expressed by CD19 CAR-T cells and does not bind to the UTD. No binding was detected in the two negative control tubes, and the CAR positivity rate measured in the test tube and the positive control tube differed by ≤5%. These results demonstrate that the rabbit immune serum binds well to the target antigen and can be used for further development of anti-CD19 scFv monoclonal antibodies.

[0242] Table 1. Flow cytometry results of rabbit immune serum binding to CAR-T positive cells / UTD cells (CAR positive percentage)

[0243] 2. Regarding the use of CD22 scFv as a target antigen

[0244] 1. ELISA test

[0245] The experimental procedures were as follows: 7 days after the third immunization of New Zealand rabbits, rabbit immune serum was collected through the ear vein and diluted at different ratios as test samples; different 96-well plates were coated with the target antigen CD22 scFv at a concentration of 1 μg / mL and the negative control antigen at a concentration of 1 μg / mL and blocked overnight; the test samples were added to the coated 96-well plates, and the 96-well plates were incubated in a 37°C incubator for 1 hour; the 96-well plates were washed four times with washing solution to remove unbound test samples; horseradish peroxidase-labeled rabbit antibody (Anti-Rabbit IgG FC Monoclonal Secondary Antibody, GenScript, #A01856) was added and incubated in a 37°C incubator for 1 hour; the 96-well plates were washed four times with washing solution, 3,3',5,5'-tetramethylbenzidine (TMB) was added for color development, and the absorbance readings were measured at 450 nm for data processing.

[0246] The test results showed that among the four rabbit immune sera, two rabbit sera (R09403# and R09404#) were able to better recognize the target antigen and specifically bind to the target antigen with increasing dilution gradient, with titers of 32000:1 and 64000:1, respectively.

[0247] 2. FACS detection

[0248] The experimental steps are as follows: CD19-CD22 CAR-T cells were collected and resuspended to a cell density of 1×10 7 / mL to obtain cell suspension; in the test tube, two rabbit immune sera R09403# and R09404# with different dilutions of 1:200, 1:400, 1:800 and 1:1600 were incubated with CD19-CD22 CAR-T cells at 4℃ for 1 hour, and the cell pellets were collected after centrifugation. Secondary antibody Alexa 647 fluorescently labeled anti-rabbit F(ab')2 antibody (purchased from Cell Signaling Technology, catalog number #4414) was used to detect rabbit antibodies in rabbit immune serum. Incubate at room temperature in the dark for 15 minutes. After centrifugation, the cell pellets were collected, washed, and resuspended for flow cytometry. A positive control tube was also set up (Alexa Fluor 500 was added to the cell suspension). 647 fluorescently labeled anti-mouse F(ab')2 antibody was used to directly detect mouse CD22 VL or VH. The anti-mouse F(ab')2 antibody was purchased from Jackson ImmunoResearch Laboratories, catalog number #115-605-072. The difference in CAR positivity between the test tube and the positive control tube was used to determine the suitability of the test sample for the next step of validation. If the difference was within 5%, the sample was used for the next step of validation.

[0249] The test results, as shown in Table 2, show that the rabbit immune serum binds well to the CD22 VL or VH expressed by CD19-CD22 CAR-T cells. FACS analysis revealed that at a dilution of 1:200, the CAR-positive percentage of rabbit serum #R09403 was ≤5% lower than the actual CAR-positive percentage detected by the positive control, Anti-mouse F(ab')2-AF647 antibody. As the dilution increased, the flow cytometric results gradually declined, indicating insufficient antibody usage. At dilutions of 1:200, 1:400, and 1:800, the CAR-positive percentage of rabbit serum #R09404 was ≤5% higher than the actual CAR-positive percentage detected by the positive control, Anti-mouse F(ab')2-AF647 antibody. At a dilution of 1:1600, the flow cytometric results indicated insufficient antibody usage, indicating insufficient antibody usage. The above results indicate that the rabbit immune serum can be used for further development of anti-CD22 scFv monoclonal antibodies.

[0250] Table 2. Flow cytometry results of rabbit immune serum binding to CAR-T cells (CAR positive percentage)

[0251] Example 3: ELISA and FACS detection of monoclonal cell culture supernatant

[0252] 1. Regarding the use of CD19 scFv as a target antigen

[0253] After confirming that the rabbit immune serum binds to the CD19 CAR-T cells, the animals were euthanized and whole blood cells were collected. PBMCs were isolated and single B cells were sorted according to conventional methods, and then monoclonal cell culture and expansion were performed according to conventional methods. The monoclonal cell culture supernatant was collected and subjected to ELISA and FACS analysis, which performed the same functions as the corresponding experiments in Example 2.

[0254] 1. ELISA test

[0255] The experimental steps are as follows: monoclonal cell culture supernatant is collected to replace rabbit immune serum, and the other steps are the same as the corresponding ELISA detection steps in the first part of Example 2 using CD19 scFv as the target antigen.

[0256] The test results are shown in Table 3. The supernatants of 15 strains obtained from monoclonal cell culture were able to recognize the target antigen CD19 scFv well and had very low binding to the negative control antigen CD33 scFv, indicating that all were positive clones.

[0257] Table 3. ELISA test results of each monoclonal cell culture supernatant

[0258] 2. FACS screening

[0259] The experimental steps are as follows: the supernatant of the positive clone is collected to replace the rabbit immune serum. The other steps are the same as the corresponding FACS detection steps in the first part of Example 2 using CD19 scFv as the target antigen, and the positive control tube is set up using the same method.

[0260] The results are shown in Table 4. Supernatants from most positive clones bind well to the native conformation CD19 scFv expressed by CD19 CAR-T cells and do not bind to the UTD. Clones 3E10 and 4F6, which showed poor overall binding in flow cytometry, were eliminated. From the remaining three clones, 1C11, 2F2, and 4E11, the subclones with the best affinity—1C11-25A4, 2F2-25B8, and 4E11-25C9—were selected for further screening.

[0261] Table 4. Flow cytometry results of the binding of the supernatant of each positive clone to CAR-T / UTD cells (CAR positive percentage)

[0262] 2. Using CD22 scFv as a target antigen

[0263] After confirming that the two rabbit immune sera numbered R09403# and R09404# bind well to the anti-CD22 VL or VH in the CD19-CD22 CAR-T cells, the corresponding animals were euthanized to obtain whole blood cells. After isolating PBMCs according to conventional methods, the PBMCs obtained from the two rabbit immune sera were mixed and single B cells were sorted, and then cloned and expanded according to conventional methods. The culture supernatant of the monoclonal cells was collected for ELISA and FACS testing. The functions of these tests are the same as those of the corresponding experiments in Example 2.

[0264] 1. ELISA test

[0265] The experimental steps are as follows: monoclonal cell culture supernatant is collected instead of rabbit immune serum, and 96-well plates are coated with 0.5 μg / mL CD22 scFv target antigen and 0.5 μg / mL negative control antigen, respectively. The remaining steps are the same as those in the second part of Example 2 for the corresponding ELISA detection using CD22 scFv as the target antigen.

[0266] The test results are shown in Table 5. The supernatants of 45 strains obtained from monoclonal cell culture were able to recognize the target antigen well and had very low binding to the control antigen, and were all positive clones.

[0267] Table 5. ELISA test results of each monoclonal cell culture supernatant

[0268] 2. FACS detection

[0269] The experimental steps are as follows: CD19-CD22 CAR-T cells and CD19 CAR-T cells are collected, with CD19 CAR-T cells used as negative control cells, and monoclonal cell culture supernatant is collected instead of rabbit immune serum. The other steps are the same as the corresponding FACS detection steps in the second part of Example 2 using CD22 scFv as the target antigen, and the positive control tube is set up using the same method.

[0270] The binding of the positive monoclonal cell culture supernatants to CD19-CD22 CAR-T cells and CD19 CAR-T cells is shown in Table 6. Among the 45 positive monoclonal cell culture supernatants, 2 strains (23E3 and 24H10) bound well to the anti-CD22 VL or VH on CD19-CD22 CAR-T positive cells and did not bind to the CD19 scFv molecules on CD19 CAR-T positive cells. The measured CAR positive percentage was consistent with the Alexa The CAR positive percentage detected by 647 fluorescently labeled anti-mouse F(ab')2 antibodies (31.6%) differed by ≤5%.

[0271] Table 6. Flow cytometry results of binding of each clone's culture supernatant to CD22-CD19 CAR-T / CD19 CAR-T cells (positive percentage)

[0272] Furthermore, specific detection was performed on the culture supernatants of the two selected monoclonal cell lines (23E3 and 24H10). The specific steps were as follows: EGFR CAR-T positive cells and UTD cells were collected, and the culture supernatants of the two screened monoclonal cell lines were collected and incubated with EGFR CAR-T positive cells and UTD cells at 4°C for 1 hour, respectively. The other steps were the same as the corresponding FACS detection steps in the second part of Example 2 using CD22 scFv as the target antigen.

[0273] The binding of the monoclonal cell culture supernatants to EGFR CAR-T-positive cells and UTD cells is shown in Table 7. The supernatants of the two monoclonal cells did not bind to EGFR CAR-T-positive cells or untransfected T cells, indicating that the supernatants of the two selected clones have good specificity and can be used for further development.

[0274] Table 7. Flow cytometry results of the binding of culture supernatants of two monoclonal cell lines to EGFR CAR-T / UTD cells (CAR positive percentage)

[0275] Example 4: FACS validation of rabbit monoclonal antibodies

[0276] 1. Regarding the use of CD19 scFv as a target antigen

[0277] The positive clone cells (1C11-25A4, 2F2-25B8, 4E11-25C9) screened in Example 3 were collected and total RNA was extracted. The heavy chain and light chain variable region nucleotide sequences were amplified by RT-PCR and sequenced to obtain the amino acid sequences of the heavy chain and light chain variable regions. The obtained heavy chain and light chain variable region nucleotide sequences were then compared with the rabbit IgG heavy chain and light chain constant region nucleotide sequences (the rabbit IgG heavy chain constant region nucleotide sequence was derived from ncbi GenBank ACCESSION: L29172.1, specifically as shown in SEQ ID NO: 61, and its amino acid sequence was derived from ncbi PROTEIN_ID: AAB59265.1, specifically as shown in SEQ ID NO: 60; the rabbit IgG light chain constant region nucleotide sequence was derived from ncbi GenBank ACCESSION: K01358.1, specifically as shown in SEQ ID NO: 63, and its amino acid sequence was derived from PROTEIN_ID: AAB59259.1, specifically as shown in SEQ ID NO:62) was recombined into an expression vector, and then mammalian cells were used to express the recombinant rabbit monoclonal antibodies. The resulting recombinant rabbit monoclonal antibodies were named #113, #114, and #115. #113, #114, and #115 corresponded to 1C11-25A4, 2F2-25B8, and 4E11-25C9, respectively. After the aforementioned sequencing, the amino acid sequences of the heavy chain variable regions of the 1C11-25A4 and #113 monoclonal antibodies were shown in SEQ ID NO:28 (the CDR1, CDR2, and CDR3 thereof were shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively), and the amino acid sequences of the light chain variable regions were shown in SEQ ID NO:29 (the CDR1, CDR2, and CDR3 thereof were shown in SEQ ID NO:4, SEQ ID NO:64, and SEQ ID NO:5, respectively). The amino acid sequences of the heavy chain variable regions of the 2F2-25B8 and #114 monoclonal antibodies were shown in SEQ ID NO: NO:30 (whose CDR1, CDR2, and CDR3 are shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively), and the light chain variable region amino acid sequence is shown in SEQ ID NO:31 (whose CDR1, CDR2, and CDR3 are shown in SEQ ID NO:9, SEQ ID NO:65, and SEQ ID NO:10, respectively). The heavy chain variable region amino acid sequence of the 4E11-25C9 and #115 monoclonal antibodies is shown in SEQ ID NO:32 (whose CDR1, CDR2, and CDR3 are shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively), and the light chain variable region amino acid sequence is shown in SEQ ID NO:33 (whose CDR1, CDR2, and CDR3 are shown in SEQ ID NO:14, SEQ ID NO:66, and SEQ ID NO:15, respectively). The binding ability of the three purified rabbit monoclonal antibodies obtained with CD19 scFv molecules expressed on the surface of CD19 CAR-T positive cells was tested to support their application in flow cytometry detection of the positive cell rate containing CD19 CAR.

[0278] Experimental procedures: Purified rabbit monoclonal antibody was used instead of rabbit immune serum. The remaining steps were identical to those in the first section of Example 2 for the corresponding FACS assay using CD19 scFv as the target antigen. Negative control tubes (cell control tube, secondary antibody control tube) and positive control tubes were set up using the same method.

[0279] The test results, shown in Table 8, show that all three rabbit monoclonal antibodies bound well to the native conformation CD19 scFv molecules expressed by CD19 CAR-T cells, but not to UTD cells. Furthermore, no binding was detected in the negative control tubes. The CAR positivity rates measured in each test tube differed by ≤5% from those in the positive control tube. This demonstrates that each monoclonal antibody binds well to the target antigen and can meet the needs of both nonclinical and clinical testing.

[0280] Table 8. Flow cytometry results of rabbit monoclonal antibodies binding to CAR-T positive cells / UTD cells (CAR positive percentage)

[0281] The monoclonal antibody screened by the present invention is an antibody that specifically recognizes the CD19 scFv as shown in SEQ ID NO: 48, and can specifically bind to CAR-T cells containing the CD19 scFv extracellular antigen recognition domain, and does not have non-specific binding with other irrelevant antibodies and membrane proteins (excluding cross-binding with the control antigen used in Example 2). The specific applications of the specific antibodies obtained by the present invention can include but are not limited to the following three aspects: a. for detecting the expression rate of CAR-T positive cells expressing the target extracellular antigen recognition domain in CAR-T cells; b. for detecting the concentration of CAR-T positive cells expressing the target extracellular antigen recognition domain in the patient's blood after the patient is reinfused with CAR-T cells; c. as a standard reference when detecting anti-drug antibodies (ADA) against the target extracellular antigen recognition domain produced in the patient's blood after the patient is reinfused with CAR-T cells; in the above applications, the target extracellular antigen recognition domain comprises:

[0282] The CD19 scFv amino acid sequence shown in SEQ ID NO: 48;

[0283] Or, the CD19-CD22 scFv amino acid sequence as shown in SEQ ID NO: 50;

[0284] Or, the CD19 VH amino acid sequence as shown in SEQ ID NO:53 and the CD19 VL amino acid sequence as shown in SEQ ID NO:54.

[0285] 2. Using CD22 scFv as a target antigen

[0286] The positive clones (23E3, 24H10) screened in Example 3 were collected and total RNA was extracted. The heavy chain and light chain variable region nucleotide sequences were amplified by RT-PCR and sequenced to obtain the amino acid sequences of the heavy chain and light chain variable regions. The obtained heavy chain and light chain variable region nucleotide sequences and rabbit IgG heavy chain and light chain constant region nucleotide sequences (rabbit IgG heavy chain constant region nucleotide sequence is derived from ncbi ACCESSION: L29172.1, specifically as shown in SEQ ID NO: 61, and its amino acid is derived from PROTEIN_ID: AAB59265.1, specifically as shown in SEQ ID N: 60; rabbit IgG light chain constant region nucleotide sequence is derived from ncbi ACCESSION: K01358.1, specifically as shown in SEQ ID NO: 63, and its nucleotide is derived from PROTEIN_ID: AAB59259.1, specifically as shown in SEQ ID NO: 62) were recombined into an expression vector, and then mammalian cells were used to express the recombinant rabbit monoclonal antibody. After the aforementioned sequencing, the amino acid sequence of the heavy chain variable region of the 23E3-1 monoclonal antibody was as shown in SEQ ID NO:34 (its CDR1, CDR2, and CDR3 are shown in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively), and the light chain variable region amino acid sequence is shown in SEQ ID NO:35 (its CDR1, CDR2, and CDR3 are shown in SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively). The heavy chain variable region amino acid sequence of the 24H10-1 monoclonal antibody is shown in SEQ ID NO:36 (its CDR1, CDR2, and CDR3 are shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively), and the light chain variable region amino acid sequence is shown in SEQ ID NO:37 (its CDR1, CDR2, and CDR3 are shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively). The two purified rabbit monoclonal antibodies were tested for their ability to bind to anti-CD22 VL or VH expressed on the surface of CD19-CD22 CAR-T positive cells to support their application in flow cytometry detection of the positive cell rate containing CD22 CAR.

[0287] Experimental steps: Only the rabbit immune serum was replaced by purified rabbit monoclonal antibody. The ratio of rabbit monoclonal antibody to CD19-CD22 CAR-T positive cells in the test tube is shown in Table 9. The remaining steps are the same as those in the corresponding FACS test in the second part of Example 2 using CD22 scFv as the target antigen. In the test in Table 9, 1×10 6 cell.

[0288] The test results are shown in Table 9. Both rabbit monoclonal antibodies bind well to the native conformation of anti-CD22 VL and / or VH expressed by CD19-CD22 CAR-T cells.

[0289] Table 9. Flow cytometry results of rabbit monoclonal antibodies binding to CD22-CD19 CAR-T positive cells (positive percentage)

[0290] The monoclonal antibody screened by the present invention is a specific antibody that specifically recognizes the amino acid sequence of CD22 scFv as shown in SEQ ID NO:49, or CD19-CD22 scFv as shown in SEQ ID NO:50, or contains both CD22 VH as shown in SEQ ID NO:51 and CD22 VL as shown in SEQ ID NO:52, and does not non-specifically bind to other antibodies and membrane proteins. The specific applications of the specific antibodies obtained by the present invention may include but are not limited to the following three aspects: a. for detecting the expression rate of CAR-T positive cells expressing the target extracellular antigen recognition domain in CAR-T cells; b. for detecting the concentration of CAR-T positive cells expressing the target extracellular antigen recognition domain in the patient's blood after the patient is reinfused with CAR-T cells; c. as a standard reference when detecting anti-drug antibodies against the target extracellular antigen recognition domain produced in the patient's blood after the patient is reinfused with CAR-T cells; in the above applications, the target extracellular antigen recognition domain comprises:

[0291] The CD22 scFv amino acid sequence shown in SEQ ID NO:49;

[0292] Or, the CD19-CD22 scFv amino acid sequence as shown in SEQ ID NO: 50;

[0293] Or, the CD22 VH amino acid sequence shown in SEQ ID NO:51 and the CD22 VL amino acid sequence shown in SEQ ID NO:52.

[0294] Example 5: FACS detection of fluorescein-labeled rabbit monoclonal antibodies

[0295] 1. Regarding the use of CD19 scFv as a target antigen

[0296] Rabbit monoclonal antibody 1C11-25A4 (#113 monoclonal antibody) was labeled with fluorescein using existing methods. The binding ability of the obtained fluorescein-labeled rabbit monoclonal antibody to the CD19scFv molecule on the surface of CD19-CD22 CAR-T positive cells was tested to confirm whether the fluorescein-labeled rabbit monoclonal antibody would not affect its application in the detection of CD19 CAR positivity rate.

[0297] Experimental steps: CD19-CD22 CAR-T cells were collected and resuspended to a cell density of 1×10 7 The experiment was divided into 2 groups, one of which was using Alexa One group was incubated with CD19-CD22 CAR-T cells at 4°C for 1 hour using a fluorescently labeled rabbit monoclonal antibody (647) (referred to as the fluorescein-mcg group). Another group was incubated with CD19-CD22 CAR-T cells at 4°C for 1 hour using a non-fluorescein-mcg group (referred to as the no-fluorescein-mcg group). After incubation, the cell pellets were collected by centrifugation, washed, resuspended, and analyzed by flow cytometry. The FACS results of the fluorescein-mcg group and the no-fluorescein-mcg group were compared. If the difference between the two groups was ≤5%, it was considered that the fluorescein labeling did not affect the rabbit monoclonal antibody's efficacy, indicating that the fluorescein-labeled rabbit monoclonal antibody could be used to detect the positive rate of CD19 CAR.

[0298] The test results are shown in Table 10. The 647 fluorescein-labeled 1C11-25A4 rabbit monoclonal antibody binds well to the CD19 scFv molecules on the surface of CD19-CD22 CAR-T cells. The difference in CAR positivity detected by FACS between the fluorescein-labeled and non-fluorescein-labeled groups was ≤5%, indicating that this fluorescein-labeled rabbit monoclonal antibody can be used for CD19 CAR positivity testing. In Table 10, "——" indicates that no testing was performed.

[0299] Table 10. Flow cytometry results of the binding of fluorescein-labeled rabbit monoclonal antibody to CD19-CD22 CAR-T

[0300] 2. Using CD22 scFv as a target antigen

[0301] The two rabbit monoclonal antibodies 24H10-1 and 23E3-1 identified in Example 4 were fluorescein-labeled using existing methods, and the binding ability of the resulting fluorescein-labeled rabbit monoclonal antibodies to the anti-CD22 portion on the surface of CD19-CD22 CAR-T positive cells was tested to confirm whether the fluorescein-labeled rabbit monoclonal antibodies would not affect their application in detecting the positive rate of CD22 CAR.

[0302] Experimental steps: CD19-CD22 CAR-T cells were collected and resuspended to a cell density of 1×10 7 The experiment was divided into 3 groups, one of which was using Alexa Alexa Fluor 405 was used as a fluorescently labeled rabbit monoclonal antibody and incubated with CD19-CD22 CAR-T positive cells at 4°C for 1 h (this group was called Alexa Fluor 405-monoclonal antibody group). Alexa Fluor 647 was used as a fluorescein-labeled rabbit monoclonal antibody and incubated with CD19-CD22 CAR-T-positive cells at 4°C for 1 hour (this group is referred to as the Alexa Fluor 647-mAb group). A third group used an unfluorescein-labeled rabbit monoclonal antibody and incubated with CD19-CD22 CAR-T-positive cells at 4°C for 1 hour (this group is referred to as the no-fluorescein-mAb group). Each group was further divided into subgroups with different feed ratios and different monoclonal antibody strains. After incubation, the cell pellet was collected by centrifugation, washed, resuspended, and analyzed by flow cytometry. The group using the fluorescein-labeled rabbit monoclonal antibody was compared with the group using the unfluorescein-labeled rabbit monoclonal antibody. If the difference in CAR positivity detected by FACS between the two groups was ≤5%, it was considered that the fluorescein labeling did not affect the function of the rabbit monoclonal antibody, indicating that the fluorescein-labeled rabbit monoclonal antibody could be used to detect the positivity rate of CD22 CAR.

[0303] The test results are shown in Table 11. In each subgroup using clone number 24H10-1 as the antibody, the test results of the rabbit monoclonal antibody labeled with Alexa Fluor 647 and produced at a feed ratio of 1:0.4 (the feed ratio refers to the feed ratio of naked antibody to fluorescein) were the best consistency with the test results of 24H10-1 without fluorescein labeling; in each subgroup using clone number 23E3-1 as the antibody, the test results of the rabbit monoclonal antibody labeled with Alexa Fluor 647 and produced at a feed ratio of 1:0.4 were the best consistency with the test results of 23E3-1 without fluorescein labeling; the above results indicate that both rabbit monoclonal antibodies (24H10-1 and 23E3-1) labeled with Alexa Fluor 647 and produced at a feed ratio of 1:0.4 can be used for CD22 CAR positive rate detection.

[0304] Table 11. Flow cytometry results of the binding of fluorescein-labeled rabbit monoclonal antibody to CD19-CD22 CAR-T

[0305] Example 6: Anti-CD19 scFv antibody and anti-CD22 scFv antibody are used to detect the positive rate of CD19-CD22 dual-target CAR-T

[0306] Using the fluorescein-labeled anti-CD19 scFv antibody and anti-CD22 scFv antibody prepared in Example 5, the CAR expression positivity rate of CD19-CD22 dual-target CAR-T cells and UTD cells was detected for the CD19 CAR positivity rate and the CD22 CAR positivity rate to confirm whether these two fluorescein-labeled antibodies can specifically evaluate the CD19 CAR positivity rate and the CD22 CAR positivity rate.

[0307] Experimental steps: CD19-CD22 CAR-T positive cells and UTD cells were collected and resuspended to a cell density of 1×10 7 / mL; set up test tubes and negative control tubes, where test tube 1 uses Alexa The cells were labeled with 647-anti-CD19 scFv antibody (1C11-25A4, i.e., #113) and FITC-CD3 antibody, and detection tube 2 was detected using Alexa The cells were labeled with 647-anti-CD22 scFv antibody (23E3-1) and FITC-CD3 antibody. Cells and FITC fluorescently labeled IgG1 were added to the negative control tube as a negative control. Each tube was incubated in the dark at 2-8°C for 60 minutes, then washed with PBS, resuspended, and then 7-AAD (a dye used to distinguish dead cells from live cells) was added. After incubation in the dark for 10 minutes, CD3 was immediately detected by flow cytometry. + CAR + (Anti-CD19) T cells and CD3 + CAR + (Anti-CD22) T cell ratio. CD3 is a marker for distinguishing whether they are T cells.

[0308] The test results are shown in Table 12. Both anti-CD19 scFv antibodies and anti-CD22 scFv antibodies bind well to CD19 scFv molecules and CD22 scFv VL / VH molecules on CD22-CD19 CAR-T cells. The CAR expression positivity results obtained by detecting CD19 scFv and CD19 scFv VL / VH on the surface of CD19-CD22 dual-target CAR-T cells differ by ≤5%, and neither anti-CD19 scFv antibodies nor anti-CD22 scFv antibodies bind to untransfected T cells, indicating that this detection method can be used for exclusive CAR (anti-CD19) and / or CAR (anti-CD22) positivity detection.

[0309] Table 12 Flow cytometry results of binding of anti-CD19 scFv antibody, anti-CD22 scFv antibody and CD22-CD19 CAR-T

[0310] Sequence Description

[0311] SEQ ID NO: 1, amino acid sequence of the antibody heavy chain variable region CDR1 of #113 anti-CD19 scFv;

[0312] SEQ ID NO: 2, amino acid sequence of the antibody heavy chain variable region CDR2 of #113 anti-CD19 scFv;

[0313] SEQ ID NO: 3, amino acid sequence of the antibody heavy chain variable region CDR3 of #113 anti-CD19 scFv;

[0314] SEQ ID NO: 4, amino acid sequence of the antibody light chain variable region CDR1 of #113 anti-CD19 scFv;

[0315] SEQ ID NO: 5, amino acid sequence of the antibody light chain variable region CDR3 of #113 anti-CD19 scFv;

[0316] SEQ ID NO: 6, amino acid sequence of the antibody heavy chain variable region CDR1 of #114 anti-CD19 scFv;

[0317] SEQ ID NO: 7, amino acid sequence of the antibody heavy chain variable region CDR2 of #114 anti-CD19 scFv;

[0318] SEQ ID NO: 8, amino acid sequence of the antibody heavy chain variable region CDR3 of #114 anti-CD19 scFv;

[0319] SEQ ID NO: 9, amino acid sequence of the antibody light chain variable region CDR1 of #114 anti-CD19 scFv;

[0320] SEQ ID NO: 10, amino acid sequence of the antibody light chain variable region CDR3 of #114 anti-CD19 scFv;

[0321] SEQ ID NO: 11, amino acid sequence of the antibody heavy chain variable region CDR1 of #115 anti-CD19 scFv;

[0322] SEQ ID NO: 12, amino acid sequence of the antibody heavy chain variable region CDR2 of #115 anti-CD19 scFv;

[0323] SEQ ID NO: 13, amino acid sequence of the antibody heavy chain variable region CDR3 of #115 anti-CD19 scFv;

[0324] SEQ ID NO: 14, amino acid sequence of the antibody light chain variable region CDR1 of #115 anti-CD19 scFv;

[0325] SEQ ID NO: 15, amino acid sequence of the antibody light chain variable region CDR3 of #115 anti-CD19 scFv;

[0326] SEQ ID NO: 16, amino acid sequence of the antibody heavy chain variable region CDR1 of #23E3-1 anti-CD22 scFv;

[0327] SEQ ID NO: 17, amino acid sequence of the antibody heavy chain variable region CDR2 of #23E3-1 anti-CD22 scFv;

[0328] SEQ ID NO: 18, amino acid sequence of the antibody heavy chain variable region CDR3 of #23E3-1 anti-CD22 scFv;

[0329] SEQ ID NO: 19, amino acid sequence of the antibody light chain variable region CDR1 of #23E3-1 anti-CD22 scFv;

[0330] SEQ ID NO: 20, amino acid sequence of the antibody light chain variable region CDR2 of #23E3-1 anti-CD22 scFv;

[0331] SEQ ID NO: 21, the amino acid sequence of the antibody light chain variable region CDR3 of #23E3-1 anti-CD22 scFv;

[0332] SEQ ID NO: 22, amino acid sequence of the antibody heavy chain variable region CDR1 of #24H10-1 anti-CD22 scFv;

[0333] SEQ ID NO: 23, amino acid sequence of the antibody heavy chain variable region CDR2 of #24H10-1 anti-CD22 scFv;

[0334] SEQ ID NO: 24, amino acid sequence of the antibody heavy chain variable region CDR3 of #24H10-1 anti-CD22 scFv;

[0335] SEQ ID NO: 25, amino acid sequence of the antibody light chain variable region CDR1 of #24H10-1 anti-CD22 scFv;

[0336] SEQ ID NO: 26, amino acid sequence of the antibody light chain variable region CDR2 of #24H10-1 anti-CD22 scFv;

[0337] SEQ ID NO: 27, the amino acid sequence of the antibody light chain variable region CDR3 of #24H10-1 anti-CD22 scFv;

[0338] SEQ ID NO: 28, amino acid sequence of the antibody heavy chain variable region of #113 anti-CD19 scFv;

[0339] SEQ ID NO: 29, amino acid sequence of the antibody light chain variable region of #113 anti-CD19 scFv;

[0340] SEQ ID NO: 30, amino acid sequence of the antibody heavy chain variable region of #114 anti-CD19 scFv;

[0341] SEQ ID NO: 31, amino acid sequence of the antibody light chain variable region of #114 anti-CD19 scFv;

[0342] SEQ ID NO: 32, amino acid sequence of the antibody heavy chain variable region of #115 anti-CD19 scFv;

[0343] SEQ ID NO: 33, amino acid sequence of the antibody light chain variable region of #115 anti-CD19 scFv;

[0344] SEQ ID NO: 34, amino acid sequence of the antibody heavy chain variable region of #23E3-1 anti-CD22 scFv;

[0345] SEQ ID NO: 35, the amino acid sequence of the antibody light chain variable region of #23E3-1 anti-CD22 scFv;

[0346] SEQ ID NO: 36, amino acid sequence of the antibody heavy chain variable region of #24H10-1 anti-CD22 scFv;

[0347] SEQ ID NO: 37, amino acid sequence of the antibody light chain variable region of #24H10-1 anti-CD22 scFv;

[0348] SEQ ID NO: 38, nucleotide sequence encoding the antibody heavy chain variable region of #113 anti-CD19 scFv;

[0349] SEQ ID NO: 39, nucleotide sequence encoding the antibody light chain variable region of #113 anti-CD19 scFv;

[0350] SEQ ID NO: 40, nucleotide sequence encoding the heavy chain variable region of the antibody #114 anti-CD19 scFv;

[0351] SEQ ID NO: 41, nucleotide sequence encoding the antibody light chain variable region of #114 anti-CD19 scFv;

[0352] SEQ ID NO:42, nucleotide sequence encoding the antibody heavy chain variable region of #115 anti-CD19 scFv;

[0353] SEQ ID NO:43, nucleotide sequence encoding the antibody light chain variable region of #115 anti-CD19 scFv;

[0354] SEQ ID NO: 44, nucleotide sequence encoding the antibody heavy chain variable region of #23E3-1 anti-CD22 scFv;

[0355] SEQ ID NO: 45, nucleotide sequence encoding the antibody light chain variable region of #23E3-1 anti-CD22 scFv;

[0356] SEQ ID NO: 46, nucleotide sequence encoding the antibody heavy chain variable region of #24H10-1 anti-CD22 scFv;

[0357] SEQ ID NO: 47, nucleotide sequence encoding the antibody light chain variable region of #24H10-1 anti-CD22 scFv;

[0358] SEQ ID NO: 48, target antigen CD19 scFv amino acid sequence;

[0359] SEQ ID NO: 49, target antigen CD22 scFv amino acid sequence;

[0360] SEQ ID NO:50, CD19-CD22 scFv amino acid sequence;

[0361] SEQ ID NO:51, CD22 VH amino acid sequence;

[0362] SEQ ID NO:52, CD22 VL amino acid sequence;

[0363] SEQ ID NO:53, CD19 VH amino acid sequence;

[0364] SEQ ID NO: 54, CD19 VL amino acid sequence;

[0365] SEQ ID NO: 55: amino acid sequence of a leader peptide (i.e., signal peptide);

[0366] SEQ ID NO: 56: amino acid sequence of the hinge region;

[0367] SEQ ID NO: 57: amino acid sequence of the transmembrane region;

[0368] SEQ ID NO: 58: amino acid sequence of the costimulatory signaling domain;

[0369] SEQ ID NO: 59: amino acid sequence of the intracellular signaling domain;

[0370] SEQ ID NO:60: CH region amino acid sequence;

[0371] SEQ ID NO: 61: Nucleotide sequence encoding the amino acid sequence of the CH region;

[0372] SEQ ID NO:62: CL region amino acid sequence;

[0373] SEQ ID NO: 63: Nucleotide sequence encoding the amino acid sequence of the CL region;

[0374] SEQ ID NO: 64, amino acid sequence of the antibody light chain variable region CDR2 of #113 anti-CD19 scFv;

[0375] SEQ ID NO: 65, amino acid sequence of the antibody light chain variable region CDR2 of #114 anti-CD19 scFv;

[0376] SEQ ID NO: 66, amino acid sequence of the antibody light chain variable region CDR2 of #115 anti-CD19 scFv;

Claims

1. An antibody or antigen-binding portion thereof of an anti-CD19 antibody, the amino acid sequence of which includes a heavy chain variable region and a light chain variable region, wherein: The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region respectively include the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO: 28, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region respectively include the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO: 29; Or, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO: 30, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO: 31; Or, the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region respectively include the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO:32, and the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region respectively include the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO:

33.

2. The antibody or antigen-binding portion thereof of the anti-CD19 antibody according to claim 1, wherein: The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO: 28, respectively; the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO: 29, respectively; Or, the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO: 30, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO: 31, respectively; Or, the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region are the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO:32, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region are the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO:33, respectively.

3. An antibody or antigen-binding portion thereof of an anti-CD19 antibody, the amino acid sequence of which includes a heavy chain variable region and a light chain variable region, wherein: The CDR1, CDR2, and CDR3 of the heavy chain variable region comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the CDR1, CDR2, and CDR3 of the light chain variable region comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 64, and SEQ ID NO: 5, respectively; Or, CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, and CDR1, CDR2, and CDR3 of the light chain variable region respectively comprise the amino acid sequences shown in SEQ ID NO:9, SEQ ID NO:65, and SEQ ID NO:10; Or, the CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, and the CDR1, CDR2, and CDR3 of the light chain variable region respectively comprise the amino acid sequences shown in SEQ ID NO:14, SEQ ID NO:66, and SEQ ID NO:

15.

4. The antibody or antigen-binding portion thereof of the anti-CD19 antibody according to claim 3, wherein: The CDR1, CDR2, and CDR3 of the heavy chain variable region are amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the CDR1, CDR2, and CDR3 of the light chain variable region are amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 64, and SEQ ID NO: 5, respectively; Or, the CDR1, CDR2, and CDR3 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively, and the CDR1, CDR2, and CDR3 of the light chain variable region are the amino acid sequences shown in SEQ ID NO:9, SEQ ID NO:65, and SEQ ID NO:10, respectively; Or, the CDR1, CDR2, and CDR3 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively, and the CDR1, CDR2, and CDR3 of the light chain variable region are the amino acid sequences shown in SEQ ID NO:14, SEQ ID NO:66, and SEQ ID NO:15, respectively.

5. The antibody or antigen-binding portion thereof of the anti-CD19 antibody according to any one of claims 1 to 4, wherein the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 29; Or, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30, and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 31; Alternatively, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:32, and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

33.

6. The antibody or antigen-binding portion thereof of the anti-CD19 antibody according to claim 5, wherein the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 29; Or, the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 30, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 31; Or, the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:32, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:

33.

7. The antibody or antigen-binding portion thereof of the anti-CD19 antibody according to claim 6, wherein the CD19 antibody is a CD19 scFv, and its amino acid sequence comprises the amino acid sequence shown in SEQ ID NO:

48.

8. The antibody or antigen-binding portion thereof of the anti-CD19 antibody according to claim 7, wherein the amino acid sequence of the CD19 antibody is the amino acid sequence shown in SEQ ID NO:

48.

9. The antibody or antigen binding site thereof of the anti-CD19 antibody according to any one of claims 1 to 8, wherein the antibody or antigen binding site thereof of the anti-CD19 antibody is labeled with fluorescein.

10. The antibody or antigen-binding portion thereof of the anti-CD19 antibody according to claim 9, wherein the fluorescein is Alexa 647 Fluorescein or Alexa 405 Fluorescein.

11. An isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or an antigen binding site thereof of an anti-CD19 antibody according to any one of claims 1 to 10.

12. The isolated nucleic acid molecule of claim 11, wherein the nucleotide sequence encoding the antibody or antigen binding site thereof of the anti-CD19 antibody of any one of claims 1-10 comprises: A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:28 and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:29; Or, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:30 and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:31; Or, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:32 and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:

33.

13. The isolated nucleic acid molecule of claim 12, wherein: The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:28 is shown in SEQ ID NO:38, and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:29 is shown in SEQ ID NO:39; Or, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:30 is shown in SEQ ID NO:40, and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:31 is shown in SEQ ID NO:41; Or, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:32 is shown in SEQ ID NO:42, and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:33 is shown in SEQ ID NO:

43.

14. A vector comprising the isolated nucleic acid molecule according to any one of claims 11-13.

15. A cell comprising any one of the anti-CD19 antibody or the antigen binding site thereof according to any one of claims 1 to 10, the isolated nucleic acid molecule according to any one of claims 11 to 13, and the vector according to claim 14.

16. Use of any one of the anti-CD19 antibody or its antigen binding site according to any one of claims 1-10, the isolated nucleic acid molecule according to any one of claims 11-13, the vector according to claim 14, and the cell according to claim 15 in preparing a detection reagent.

17. A detection reagent comprising any one of the anti-CD19 antibody or its antigen binding site according to any one of claims 1-10, the isolated nucleic acid molecule according to any one of claims 11-13, the vector according to claim 14, and the cell according to claim 15.

18. The use according to claim 16 or the detection reagent according to claim 17, which is used to detect one or more of the following samples: A sample comprising the CD19 scFv amino acid sequence as shown in SEQ ID NO:48; A sample comprising the CD19-CD22 scFv amino acid sequence as shown in SEQ ID NO:50; A sample comprising the CD19 VH amino acid sequence shown in SEQ ID NO:53 and the CD19 VL amino acid sequence shown in SEQ ID NO:

54.

19. The use or detection reagent according to claim 18, wherein the sample is selected from the following samples: cell sample, blood sample.

20. The use according to claim 16 or the detection reagent according to claim 17, wherein the detection reagent is used for one or more of the following purposes: a. Used to detect the positive expression rate of CAR expressing the target extracellular antigen recognition domain in CAR-T cells; b. Used to detect the concentration of CAR-T positive cells expressing the target extracellular antigen recognition domain in the patient's blood after the patient is reinfused with CAR-T cells; c. After the patient is reinfused with CAR-T cells, it is used as a standard reference when detecting anti-drug antibodies against the target extracellular antigen recognition domain produced in the patient's blood.

21. The use or detection reagent according to claim 20, wherein the target extracellular antigen recognition domain comprises: The CD19 scFv amino acid sequence as shown in SEQ ID NO:48; Or, the CD19-CD22 scFv amino acid sequence as shown in SEQ ID NO:50; Or, the CD19 VH amino acid sequence as shown in SEQ ID NO:53 and the CD19 VL amino acid sequence as shown in SEQ ID NO:

54.

22. The use or detection reagent according to claim 18, wherein the sample is from a patient with a non-solid tumor.

23. The use or detection reagent according to claim 22, wherein the non-solid tumor patient comprises a leukemia patient.

24. The use or detection reagent according to claim 23, wherein the leukemia patient comprises a lymphocytic leukemia patient.

25. The use or detection reagent according to claim 24, wherein the lymphocytic leukemia patient comprises an acute lymphocytic leukemia patient.

26. The use or detection reagent according to claim 25, wherein the acute lymphoblastic leukemia patients include adult acute lymphoblastic leukemia patients and childhood acute lymphoblastic leukemia patients.

27. The use or detection reagent according to claim 22, wherein the non-solid tumor patient comprises a lymphoma patient.

28. The use or detection reagent according to claim 27, wherein the lymphoma patient comprises a B-cell lymphoma patient.

29. The use or detection reagent according to claim 28, wherein the B-cell lymphoma patient comprises a non-Hodgkin's lymphoma patient.

30. The use or detection reagent according to claim 18, wherein the sample is from a patient with an autoimmune disease.

31. The use or detection reagent according to claim 30, wherein the patient with the autoimmune disease is a patient with systemic lupus erythematosus.

32. A method for detecting the CAR positive rate on the surface of CD19 single-target CAR-T cells, comprising the following steps: The anti-CD19 antibody or its antigen binding site according to any one of claims 1 to 10 is used to label the CAR molecules on the surface of CD19 single-target CAR-T cells, and the proportion of CD19 CAR+T cells is detected by flow cytometry.

33. An antibody or antigen-binding portion thereof of an anti-CD22 antibody, the amino acid sequence of which includes a heavy chain variable region and a light chain variable region, wherein: The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region respectively include the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO: 34, and the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region respectively include the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO: 35; Or, the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise SEQ ID NO: 36 The amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO: 37, and the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO: 37, respectively.

34. The antibody or antigen binding portion thereof of the anti-CD22 antibody of claim 33, wherein: The amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are the amino acid sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO: 34, respectively; the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region are the amino acid sequences of CDR1, CDR2, and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO: 35, respectively; Or, the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region are the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO:36, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region are the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO:37, respectively.

35. An antibody or antigen-binding portion thereof of an anti-CD22 antibody, the amino acid sequence of which includes a heavy chain variable region and a light chain variable region, wherein: The CDR1, CDR2, and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, and the CDR1, CDR2, and CDR3 of the light chain variable region respectively comprise the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21; Or, the CDR1, CDR2, and CDR3 of the heavy chain variable region respectively contain the amino acid sequences shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, and the CDR1, CDR2, and CDR3 of the light chain variable region respectively contain the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:

27.

36. The antibody or antigen binding portion thereof of the anti-CD22 antibody of claim 35, wherein: The CDR1, CDR2, and CDR3 of the heavy chain variable region are amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, and the CDR1, CDR2, and CDR3 of the light chain variable region are amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively; Or, the CDR1, CDR2, and CDR3 of the heavy chain variable region are the amino acid sequences shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively, and the CDR1, CDR2, and CDR3 of the light chain variable region are the amino acid sequences shown in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively.

37. The antibody or antigen-binding portion thereof of the anti-CD22 antibody according to any one of claims 33 to 36, wherein the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 34, and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 35; Alternatively, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:36, and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

37.

38. The antibody or antigen-binding portion thereof of the anti-CD22 antibody according to claim 37, wherein the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 34, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO: 35; Or, the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:36, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO:

37.

39. The antibody or antigen-binding portion thereof of the anti-CD22 antibody according to claim 38, wherein the CD22 antibody is a CD22 scFv, and its amino acid sequence comprises the amino acid sequence shown in SEQ ID NO:

49.

40. The antibody or antigen-binding portion thereof of the anti-CD22 antibody according to claim 39, wherein the amino acid sequence of the CD22 antibody is the amino acid sequence shown in SEQ ID NO:

49.

41. The anti-CD22 antibody or the antigen binding portion thereof according to any one of claims 33 to 40, wherein the anti-CD22 antibody or the antigen binding portion thereof is labeled with fluorescein.

42. The antibody or antigen binding portion thereof of the anti-CD22 antibody according to claim 41, wherein the fluorescein is Alexa 647 Fluorescein or Alexa 405 Fluorescein.

43. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen binding portion thereof of the anti-CD22 antibody of any one of claims 33-42.

44. The isolated nucleic acid molecule of claim 43, wherein the nucleotide sequence encoding the antibody or antigen binding portion thereof of the anti-CD22 antibody of any one of claims 33-42 comprises: A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:34 and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:35; Or, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:36 and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:

37.

45. The isolated nucleic acid molecule of claim 44, wherein: The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:34 is shown in SEQ ID NO:44, and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:35 is shown in SEQ ID NO:45; Or, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:36 is shown in SEQ ID NO:46, and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:37 is shown in SEQ ID NO:

47.

46. ​​A vector comprising the isolated nucleic acid molecule according to any one of claims 43-45.

47. A cell comprising any one of the anti-CD22 antibody or antigen binding site thereof according to any one of claims 33-42, the isolated nucleic acid molecule according to any one of claims 43-45, and the vector according to claim 46.

48. Use of any one of the anti-CD22 antibody or its antigen binding site according to any one of claims 33-42, the isolated nucleic acid molecule according to any one of claims 43-45, the vector according to claim 46, and the cell according to claim 47 in the preparation of a detection reagent.

49. A detection reagent comprising any one of the anti-CD22 antibody or its antigen binding site according to any one of claims 33-42, the isolated nucleic acid molecule according to any one of claims 43-45, the vector according to claim 46, and the cell according to claim 47.

50. The use according to claim 48 or the detection reagent according to claim 49, wherein the detection reagent is used to detect one or more of the following samples: A sample comprising the CD22 scFv amino acid sequence as shown in SEQ ID NO:49; A sample comprising the CD19-CD22 scFv amino acid sequence as shown in SEQ ID NO:50; A sample comprising the CD22 VH amino acid sequence shown in SEQ ID NO:51 and the CD22 VL amino acid sequence shown in SEQ ID NO:

52.

51. The use detection reagent according to claim 50, wherein the sample is selected from the following samples: cell sample, blood sample.

52. The use according to claim 48 or the detection reagent according to claim 49, wherein the detection reagent is used for one or more of the following purposes: a. Used to detect the positive expression rate of CAR expressing the target extracellular antigen recognition domain in CAR-T cells; b. Used to detect the concentration of CAR-T positive cells expressing the target extracellular antigen recognition domain in the patient's blood after the patient is reinfused with CAR-T cells; c. After the patient is reinfused with CAR-T cells, it is used as a standard reference when detecting anti-drug antibodies against the target extracellular antigen recognition domain produced in the patient's blood.

53. The use or detection reagent according to claim 52, wherein the target extracellular antigen recognition domain comprises: The CD22 scFv amino acid sequence as shown in SEQ ID NO:49; Or, the CD19-CD22 scFv amino acid sequence as shown in SEQ ID NO:50; Or, the CD22 VH amino acid sequence as shown in SEQ ID NO:51 and the CD22 VL amino acid sequence as shown in SEQ ID NO:

52.

54. The use or detection reagent according to claim 50, wherein the sample is from a patient with a non-solid tumor.

55. The use or detection reagent according to claim 54, wherein the non-solid tumor patient comprises a leukemia patient.

56. The use or detection reagent according to claim 55, wherein the leukemia patient comprises a lymphocytic leukemia patient.

57. The use or detection reagent according to claim 56, wherein the lymphocytic leukemia patient comprises an acute lymphocytic leukemia patient.

58. The use or detection reagent according to claim 57, wherein the acute lymphoblastic leukemia patients include adult acute lymphoblastic leukemia patients and childhood acute lymphoblastic leukemia patients.

59. The use or detection agent according to claim 54, wherein the non-solid tumor patient comprises a lymphoma patient.

60. The use or detection reagent according to claim 59, wherein the lymphoma patient comprises a B-cell lymphoma patient.

61. The use or detection reagent according to claim 60, wherein the B-cell lymphoma patient comprises a non-Hodgkin's lymphoma patient.

62. The use or detection reagent according to claim 50, wherein the sample is from a patient with an autoimmune disease.

63. The use or detection reagent according to claim 62, wherein the autoimmune disease patient is a systemic lupus erythematosus patient.

64. A method for detecting the CAR positive rate on the surface of CD22 single-target CAR-T cells, comprising the following steps: The CAR molecules on the surface of CD19 single-target CAR-T cells are labeled using the anti-CD19 antibody or its antigen binding site as described in any one of claims 33-42, and the proportion of CD19 CAR+T cells is detected by flow cytometry.

65. A method for detecting the CAR positive rate on the surface of CD19-CD22 dual-target CAR-T cells, comprising the following steps: The CAR molecules on the surface of CD19-CD22 dual-target CAR-T cells are labeled using an anti-CD19 antibody or its antigen binding site according to any one of claims 1-10 fluorescently labeled with Alexa Fluor 647, and / or an anti-CD22 antibody or its antigen binding site according to any one of claims 33-42 fluorescently labeled with Alexa Fluor 647, and the proportion of CD19 CAR+T cells and / or CD22 CAR+T cells is detected by flow cytometry.

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