An isolated antibody and uses thereof

Abstract

The application discloses an isolated antibody and application thereof.The isolated antibody comprises a heavy chain variable region and a light chain variable region, and comprises one or more of the following technical features: the heavy chain variable region comprises CDR-H1 with an amino acid sequence as shown in SEQ ID No.1; the heavy chain variable region comprises CDR-H2 with an amino acid sequence as shown in SEQ ID No.2; the heavy chain variable region comprises CDR-H3 with an amino acid sequence as shown in SEQ ID No.3; the light chain variable region comprises CDR-L1 with an amino acid sequence as shown in SEQ ID No.4; the light chain variable region comprises CDR-L2 with an amino acid sequence of VAS; and the light chain variable region comprises CDR-L3 with an amino acid sequence as shown in SEQ ID No.5.The isolated antibody of the application can efficiently target CD4 antigens, efficiently enrich CD4 T cells, and the CAR-T cells prepared from the enriched CD4 T cells can be activated by antigens.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an isolated antibody and its uses. Background Technology

[0002] CD4, short for Cluster of Differentiation 4, is a glycoprotein expressed on the surface of cells including helper T cells, regulatory T cells, monocytes, macrophages, and dendritic cells. It is one of the surface markers of helper T cells and an important receptor for their function, participating in the regulation of T cell proliferation, lymphokine release, and immune cell interactions, as well as regulating antibody production. It is also a receptor glycoprotein for many pathogens (including human immunodeficiency virus) infecting CD4+ cells.

[0003] The functional diversity of CD4 T cells is well-known. CD8 T cells exert cytotoxic effects in lymphocytic choriomeningitis virus infection, Listeria monocytogenes infection, and graft rejection; while CD4 T cells are thought to help B cells produce antibodies, enhance and maintain CD8 T cell immune responses, coordinate a range of anti-pathogen immune responses, and regulate / suppress immune responses to control the intensity and duration of autoimmune diseases and inflammatory responses. CD4 T cells are crucial participants in immune memory; when their numbers are significantly reduced or their function is impaired, the host becomes susceptible to many pathogens. For example, in HIV infection, when the number of CD4 T cells in the patient's blood decreases to 200 / mm³, the host becomes more susceptible to these pathogens. 3 At that time, a large number of opportunistic infections are very likely to occur.

[0004] Adoptive transfer of chimeric antigen receptor T cells (CAR-T) modified to express tumor cell surface antigens is a revolutionary cancer immunotherapy. CARs are synthetic receptors that can redirect lymphocytes, most commonly T cells, to recognize and kill target cells expressing specific antigens. In recent years, rapidly developing clinical trials of CAR-T cell therapy have actively explored its potential applications. Due to its enormous potential for continuous optimization, such as better targets, better CAR structures, and more efficient manufacturing processes, CAR-T cell therapy has attracted more attention than traditional drugs.

[0005] The production of CAR-T cells involves multiple steps and requires quality control testing throughout the process. In simple terms, the process begins with removing blood from the patient / healthy individual using leukoablation to separate white blood cells. The remaining blood is then returned to the circulatory system. Next, the white blood cells are sorted for CD4 T cells and CD8 T cells to enrich T cells. These enriched T cells are then activated and genetically engineered to express specific CARs, allowing for further expansion to reach an appropriate number before finally being reinfused into the patient.

[0006] Antibodies are essential for cell sorting. Therefore, it is of great significance to develop new antibodies for sorting and enriching isolated cells. Summary of the Invention

[0007] In view of the shortcomings of the prior art described above, the object of the present invention is to provide an isolated antibody and its use.

[0008] One objective of this invention is to provide an isolated antibody comprising a heavy chain variable region and a light chain variable region, wherein the isolated antibody has one or more of the following technical features:

[0009] The heavy chain variable region includes the amino acid sequence CDR-H1 as shown in SEQ ID No. 1;

[0010] The heavy chain variable region includes the amino acid sequence CDR-H2 as shown in SEQ ID No. 2;

[0011] The heavy chain variable region includes the amino acid sequence CDR-H3 as shown in SEQ ID No. 3;

[0012] The light chain variable region includes the amino acid sequence CDR-L1 as shown in SEQ ID No. 4;

[0013] The light chain variable region includes CDR-L2 with the amino acid sequence VAS;

[0014] The light chain variable region includes the amino acid sequence CDR-L3 as shown in SEQ ID No. 5.

[0015] GYTFTDYV (SEQ ID No. 1)

[0016] IYPGSGSA (SEQ ID No. 2)

[0017] ARRGNGTGFAY (SEQ ID No. 3)

[0018] QSVDYDGDSY (SEQ ID No. 4)

[0019] QQSYKDPLT (SEQ ID No. 5)

[0020] Preferably, the amino acid sequence of the heavy chain variable region includes A1) or A2):

[0021] A1) The amino acid sequence as shown in SEQ ID No. 6;

[0022] A2) An amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID No. 6 and having the function of the amino acid sequence defined in A1). Preferably, the amino acid sequence shown in SEQ ID No. 6 may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more homology with it.

[0023] QVQLVQSGPELKKPGASVKVSCKASGYTFTDYVIHWVKQATGQGLEWIGEIYPGSGSAYSNAKFKDRVTMTADKSSNTAYMELSSLTSDDTAVYFCARRGNGTGFAYWGQGTLVTVSS(SEQ ID No. 6)

[0024] Preferably, the amino acid sequence of the light chain variable region includes B1) or B2):

[0025] B1) The amino acid sequence as shown in SEQ ID No. 7;

[0026] B2) An amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID No. 7 and having the function of the amino acid sequence defined in B1). Preferably, the amino acid sequence shown in SEQ ID No. 7 may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more homology.

[0027] DIVLTQSPASLAVSLGQRATITCKAGQSVDYDGDSYMNWYQQKPGQPPKLLIYVASNLESGIPARFSGSGSGTDFTLNIHPVEENDAATYYCQQSYKDPLTFGQGTKLEIK(SEQ ID No.7)

[0028] Preferably, the isolated antibody further includes a framework region (FR). The framework region (FR) sequence is a human monoclonal antibody variable region or a mouse monoclonal antibody variable region framework region sequence obtained by substitution, deletion, or addition of one or more amino acids (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3). The framework region sequence can have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more homology with the framework region sequence of the human monoclonal antibody variable region sequence.

[0029] More preferably, the heavy chain variable region further includes framework regions FR1-FR4: the amino acid sequence of framework region FR1 is as shown in SEQ ID No. 8, the amino acid sequence of framework region FR2 is as shown in SEQ ID No. 9, the amino acid sequence of framework region FR3 is as shown in SEQ ID No. 10, and the amino acid sequence of framework region FR4 is as shown in SEQ ID No. 11.

[0030] QVQLVQSGPELKKPGASVKVSCKAS(SEQ ID No.8)

[0031] IHWVKQATGQGLEWIGE(SEQ ID No.9)

[0032] YSNAKFKDRVTMTADKSSNTAYMELSSLTSDDTAVYFC(SEQ ID No.10)

[0033] WGQGTLVTVSS (SEQ ID No. 11)

[0034] More preferably, the light chain variable region further includes framework regions FR1-FR4: the amino acid sequence of FR1 is as shown in SEQ ID No. 12, the amino acid sequence of FR2 is as shown in SEQ ID No. 13, the amino acid sequence of FR3 is as shown in SEQ ID No. 14, and the amino acid sequence of FR4 is as shown in SEQ ID No. 15.

[0035] DIVLTQSPASLAVSLGQRATITCKAG(SEQ ID No.12)

[0036] MNWYQQKPGQPPKLLIY(SEQ ID No.13)

[0037] NLESGIPARFSGSGSGTDFTLNIHPVEENDAATYYC(SEQ ID No.14)

[0038] FGQGTKLEIK (SEQ ID No. 15)

[0039] Preferably, the isolated antibody further includes a heavy chain constant region and a light chain constant region.

[0040] More preferably, the sequence of the heavy chain constant region includes that shown in SEQ ID No. 18.

[0041] gctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccccgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa(SEQ ID No.18).

[0042] More preferably, the nucleotide sequence of the light chain constant region includes, for example, SEQ ID No. 19.

[0043] cgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggag agtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagttcgcccgtcacaaagagcttcaacaggggagagtgt(SEQ ID No.19)

[0044] A second objective of this invention is to provide an isolated polynucleotide encoding the antibody as described above.

[0045] Preferably, the nucleotide sequence encoding the heavy chain variable region of the isolated antibody includes the sequence shown in SEQ ID No. 16: caggtccaactcgtccaaagcggccccgagctcaaaaaacccggcgcctccgtcaaagtgtcatgcaaggcaagcggctacaccttcacagactacgtcatccactgggtcaaacaagcaaccggccagggcctcgaatggatcggagaaatctaccccggctccggctccgcctactccaacgctaaattcaaggaccgcgtgaccatgaccgccgacaaaagctcaaacaccgcttacatggagctgagcagcctgacctctgacgacaccgccgtttacttctgtgcccggaggggcaatggcaccggatttgcctattggccagggaacactggtgaccgtcagcagc (SEQ ID No. 16)

[0046] Preferably, the nucleotide sequence encoding the light chain variable region of the isolated antibody includes the sequence shown in SEQ ID No. 17.

[0047] gacattgtgctgacccagagccccgcctccctggctgtgagcctgggacagagagccaccataacctgcaaggccggacagagcgtggactacgacggcgactcctatatgaactggtatcagcagaagcccggccagccccccaagctgctgatttacgtggcaagc aacctggagagcggcatccccgccagattctcaggaagcggcagcggcaccgacttcaccctgaacatccaccccgtggaggagaacgacgccgccacctactactgccagcagagctacaaagaccccctcaccttcggccaaggcaccaaactggaaatcaaa(SEQ ID No.17)

[0048] A third objective of this invention is to provide a construct containing the polynucleotides described above.

[0049] Preferably, the construct can be constructed by inserting the polynucleotides described above into the multiple cloning site of the expression vector. The construct can be transformed, transduced, or transfected into host cells, allowing the genetic material elements it carries to be expressed within the host cells.

[0050] More preferably, the construct is a viral vector or a non-viral vector. For example, non-viral vectors include: plasmids, phagemids, Cos plasmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses, etc. Viral vectors include: retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). The vector may contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain a replication initiation site. The vector may also include components that facilitate its entry into the cell, including but not limited to viral particles, liposomes, or protein coats.

[0051] The fourth objective of this invention is to provide an antibody expression system, wherein the expression system contains an exogenous polynucleotide as described above integrated into the construct or genome as described above.

[0052] Preferably, the expression system can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.

[0053] More preferably, the expression system includes many cell types, such as prokaryotic cells like Escherichia coli or Bacillus subtilis, fungal cells like yeast or Aspergillus, insect cells like S2 ​​Drosophila or Sf9, or animal cells like fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0054] A fifth objective of this invention is to provide the use of the antibody isolated as described above, or the construct as described above, or the expression system as described above, in at least one of the following:

[0055] Enrich CD4 T cells;

[0056] Detection of CD4 antigen;

[0057] Preparation of products enriched with CD4 T cells;

[0058] Prepare products for detecting CD4 antigen;

[0059] To prepare or screen drugs for CD4-related diseases.

[0060] Preferably, the method for enriching CD4 T cells includes the following steps: adding the isolated antibody and / or antibody-drug conjugate containing the antibody as described above to peripheral blood mononuclear cells, so that CD4 T cells bind to the antibody and / or antibody-drug conjugate, and then separating them to obtain the CD4 T cells.

[0061] More preferably, the method includes the following:

[0062] 1) The antibody described above or an antibody conjugate containing the antibody described above is incubated with PBMC in flow cytometry buffer to obtain an incubation solution;

[0063] 2) The incubation solution was over-selected on the column, the cells that flowed down were discarded, the column was washed with flow cytometry buffer, and the liquid that flowed down was collected to obtain the CD4 T cells.

[0064] More preferably, the ratio of peripheral blood mononuclear cells to isolated antibodies is 16 × 10⁻⁶. 6 Cells: (0.5~1.5)μg.

[0065] More preferably, the flow cytometry buffer is a 1×PBS solution containing 1.8%–2.2% FBS and 1.8 mM–2.2 mM EDTA. Specifically, the flow cytometry buffer is a 1×PBS solution containing 2% FBS and 2 mM EDTA.

[0066] Preferably, the method for detecting CD4 antigen includes contacting the sample to be tested with the antibody described above and determining the presence or amount of CD4 antigen in the sample to be tested.

[0067] Preferably, the CD4-related disease or condition can be cancer, adaptive immune disease, autoimmune disease, inflammatory disease, or infectious disease.

[0068] More preferably, the cancer is selected from the group consisting of: lung cancer, bronchial cancer, bone cancer, hepatobiliary cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, stomach cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, adenocarcinoma, leukemia (e.g., chronic lymphocytic leukemia, relapsed or refractory B-cell precursor acute lymphoblastic leukemia), myeloma, and lymphoma.

[0069] The sixth objective of this invention is to provide an antibody conjugate comprising the isolated antibody and label or separation aid as described above.

[0070] Preferably, the marker is selected from one or more of biotin, fluorescein, and horseradish peroxidase.

[0071] Preferably, the separation aid is selected from magnetic beads and agarose beads.

[0072] More preferably, the separation aid is a magnetic bead.

[0073] More preferably, the separated antibody-conjugated magnetic beads form an antibody-conjugate, and the preparation method of the antibody-conjugate includes the following steps:

[0074] (1) Carboxyl magnetic beads and the isolated antibody as described above were coupled in a buffer solution;

[0075] (2) Centrifuge, remove the supernatant, add BSA aqueous solution to seal the unreacted activated carboxyl groups on the surface of the magnetic beads;

[0076] (3) Centrifuge, collect the precipitate, wash, and resuspend in PBS preservation solution to obtain.

[0077] Preferably, the mass ratio of the carboxyl magnetic beads to the separated antibody is (1-3):1.

[0078] Preferably, the concentration of the BSA aqueous solution is 10–30 mg / mL. More preferably, in a preferred embodiment, it is 20 mg / mL.

[0079] The seventh objective of this invention is to provide a product comprising the antibody isolated as described above or the antibody conjugate as described above.

[0080] Preferably, the product is selected from reagents and kits.

[0081] More preferably, the product is typically targeted at the CD4 antigen, using the CD4 antigen as a biomarker for diagnosis. The product may also include an antibody-drug conjugate comprising isolated antibodies and a label or separation aid, the label or separation aid typically being used for conjugation with the isolated antibody. Optional label types include, but are not limited to, one or more of biotin, fluorescein, and horseradish peroxidase. Optional separation aid types include, but are not limited to, one or more of magnetic beads and agarose beads.

[0082] An eighth objective of this invention is to provide a pharmaceutical composition comprising the isolated antibody as described above, and a pharmaceutically acceptable carrier.

[0083] Preferably, the pharmaceutically acceptable carrier refers to a carrier that will not cause allergic reactions or other adverse effects in the patient to whom it is administered. Pharmaceutically acceptable carriers include, for example, liquid, gel, or solid carriers, aqueous mediators, non-aqueous mediators, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0084] Preferably, the pharmaceutically acceptable carrier may further include trace amounts of excipients, such as humectants or emulsifiers, preservatives or buffers, that can improve the shelf life or efficacy of the antibody.

[0085] As described above, the present invention has the following beneficial effects:

[0086] 1) The isolated antibody of this invention can efficiently target the CD4 antigen and is easy to prepare;

[0087] 2) The antibodies isolated by the present invention can be used to bind with agarose and magnetic beads to enrich and separate biological samples. When enriching CD4 T cells in PBMCs, more than 90% of CD4 T cells are enriched, and less than 2.5% of CD4 T cells remain in the cell waste after enrichment.

[0088] 3) The CD22-CAR-T cells prepared from the CD4 T cells obtained by the present invention can be specifically activated by antigens. Attached Figure Description

[0089] Figure 1 This is a graph showing the affinity determination results of the antibody isolated in Example 1 of the present invention.

[0090] Figure 2 This is a diagram showing the results of the antibody isolated in Example 1 of the present invention recognizing the CD4 antigen on the cell surface.

[0091] Figure 3 This is a diagram showing the results of the anti-CD4-3 antibody recognizing the CD4 antigen on the cell surface in Example 1 of the present invention.

[0092] Figure 4 This is a diagram showing the results of anti-CD4-2 antibody, anti-CD4-4 antibody, and anti-CD4-8 antibody recognizing the CD4 antigen on the cell surface in Example 1 of the present invention.

[0093] Figure 5 This is a diagram showing the results of anti-CD4-6 antibody, anti-CD4-7 antibody, anti-CD4-9 antibody, anti-CD4-10 antibody, and anti-CD4-13 recognizing the CD4 antigen on the cell surface in Example 1 of the present invention.

[0094] Figure 6A This is a graph showing the proportion of CD4T cells in PBMCs before and after antibody-conjugate sorting and enrichment in Example 4 of the present invention.

[0095] Figure 6B This is a diagram showing the proportion of CD4 T cells in the waste liquid after sorting and enrichment using antibody conjugates in Example 4 of the present invention.

[0096] Figure 7A This is a graph showing the proportion of CD4T cells in PBMCs before and after sorting and enrichment using anti-CD4-3 conjugates in Example 4 of the present invention.

[0097] Figure 7B This is a graph showing the proportion of CD4T cells in the cell waste fluid after sorting and enrichment using anti-CD4-3 conjugates in Example 4 of the present invention.

[0098] Figure 8A The figure shows the proportion of CD4T cells in PBMC cells before and after sorting and enrichment using anti-CD4-2 conjugate, anti-CD4-4 conjugate, and anti-CD4-8 conjugate, respectively, in Example 4 of the present invention.

[0099] Figure 8BThe figure shows the proportion of CD4T cells in the cell waste fluid after sorting and enrichment using anti-CD4-2 conjugate, anti-CD4-4 conjugate, and anti-CD4-8 conjugate, respectively, in Example 4 of the present invention.

[0100] Figure 9A The figure shows the proportion of CD4 T cells in PBMC cells before and after sorting and enrichment using anti-CD4-6 conjugate, anti-CD4-7 conjugate, anti-CD4-9 conjugate, anti-CD4-10 conjugate, and anti-CD4-13 conjugate in Example 4 of the present invention.

[0101] Figure 9B The figure shows the proportion of CD4T cells in the cell waste fluid after sorting and enrichment using anti-CD4-6 conjugate, anti-CD4-7 conjugate, anti-CD4-9 conjugate, anti-CD4-10 conjugate, and anti-CD4-13 conjugate, respectively, in Example 4 of the present invention.

[0102] Figure 10 This is a graph showing the total cell count of CD4 T cells on days 5, 8, 11, and 14 after viral infection in Example 6 of the present invention.

[0103] Figure 11A This is a graph showing the expression rate of chimeric antigen receptor in CAR-T cells 5 days after CD4 T cells are infected with the virus, as described in Example 6 of the present invention.

[0104] Figure 11B This is a graph showing the expression rate of chimeric antigen receptor in CAR-T cells 8 days after CD4 T cells were infected with the virus, as shown in Example 6 of the present invention.

[0105] Figure 11C This is a graph showing the expression rate of chimeric antigen receptors in CAR-T cells 11 days after CD4 T cells were infected with the virus, as described in Example 6 of the present invention.

[0106] Figure 11D This is a graph showing the expression rate of chimeric antigen receptor in CAR-T cells 14 days after CD4 T cells were infected with the virus, as described in Example 6 of the present invention.

[0107] Figure 12A The CAR-T cells (CD4+) in Example 7 of the present invention + Image showing the killing effect of CD22-CAR-T on K562-luci cells.

[0108] Figure 12B The CAR-T cells (CD4+) in Example 7 of the present invention + Image showing the killing effect of CD22-CAR-T on K562-CD22-luci cells.

[0109] Figure 13 The CAR-T cells (CD4+) in Example 7 of the present invention + IFN-γ cytokine secretion levels after CD22-CAR-T (CD22-CAR-T) acts on K562-luci and K562-CD22-luci. Detailed Implementation

[0110] Through in-depth research, the inventors have provided an antibody that specifically binds to CD4, as well as an immunoconjugate coupled to magnetic nanoparticles. Furthermore, CD4 T cells were enriched and sorted from human peripheral blood mononuclear cells, and then the CD22-targeting chimeric antigen receptor gene was introduced into these CD4 T cells to obtain CD4 T cells. + CD22-CAR-T cells can effectively kill tumor cells expressing CD22 and stimulate tumor cells to release cytokine IFN-γ to exert their killing function. Furthermore, the cells of this invention have high specificity, and this invention was completed based on these characteristics.

[0111] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0112] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0113] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0114] In the following embodiments of this application:

[0115] For the HD SIN03 CD22(V29)41BBz(ka) lentiviral vector and K562-luci and K562-CD22-luci target cells, please refer to application number CN 114149506 A.

[0116] The flow cytometry buffer was a 1×PBS solution containing 2% FBS and 2mM EDTA.

[0117] Example 1: Construction, isolation, and purification of the isolated antibody expression vector

[0118] In this embodiment, an expression vector for the isolated antibody was constructed, and the antibody was isolated and purified to obtain the isolated antibody (labeled CD4-14). This includes the following: the amino acid sequence of the heavy chain variable region of the isolated antibody includes the sequence shown in SEQ ID No. 6.

[0119] (SEQ ID No. 6, the bold and underlined parts are the heavy chain variable regions, namely CDR-H1, CDR-H2 and CDR-H3 in sequence)

[0120] The amino acid sequence of the light chain variable region of the isolated antibody includes the sequence shown in SEQ ID No. 7.

[0121] (SEQ ID No. 7, the bold and underlined parts are the heavy chain variable regions, namely CDR-L1, CDR-L2 and CDR-L3 in sequence)

[0122] 1.1 Obtaining the target fragment of the isolated antibody

[0123] The target fragment for the isolated antibody was obtained through two rounds of PCR amplification. The expression vector for the isolated antibody was developed by Shanghai Baiying Biotechnology Co., Ltd.

[0124] The first-round PCR products were obtained according to the first-round PCR reaction system and conditions.

[0125] The first round of PCR reaction system includes: 0.5 μL upstream primer, 0.5 μL downstream primer, 0.3 μL template, 0.5 μL DNA polymerase, 10 μL 5× buffer, 1 μL 10 mM dNTP, and ddH2O to make up to 50 μL.

[0126] The conditions for the first round of PCR reaction were: 95℃ for 3 min, 95℃ for 25 s, 60℃ for 20 s, 72℃ for 40 s, 72℃ for 1 min, 10℃ for ∞, with a cycle number of 25.

[0127] Heavy chain nucleotide sequence:

[0128] CAGGTCCAACTCGTCCAAAGCGGCCCCGAGCTCAAAAAACCCGGCGCCTCCGTCAAAGTGTCATGCAAGGCAAGCGGCTACACCTTCACAGACTACGTCATCCACTGGGTCAAACAAGCAACCGGCCAGGGCCTCGAATGGATCGGAGAAATCTAACCCCGGCTCCGGCTCCGCCTACTC CAACGCTAAATTCAAGGACCGCGTGACCATGACCGCCGACAAAAGCTCAAACACCGCTTACATGGAGCTGAGCAGCCTGACCTCTGACGACACCGCCGTTTACTTCTGTGCCCGGAGGGGCAATGGCACCGGATTTGCCTATTGGGGCCAGGGAACACTGGTGACCGTCAGCAGC(SEQ ID No.16)

[0129] Light chain nucleotide sequence:

[0130] GACATTTGTGCTGACCCAGAGCCCCGCCTCCCTGGCTGTGAGCCTGGGACAGAGAGCCACCATAACCTGCAAGGCCGGACAGAGCGTGGACTACGACGGCGACTCCTATATGAACTGGTATCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATTTACGTGGCAAGC AACCTGGAGAGCGGCATCCCCGCCAGATTCTCAGGAAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGGAGAACGACGCCGCCACCTACTACTGCCAGCAGAGCTACAAAGACCCCCTCACCTTCGGCCAAGGCACCAAACTGGAAATCAAA(SEQ ID No.17)

[0131] The isolated antibody was obtained according to the second round PCR reaction system and the second round PCR reaction conditions.

[0132] The second round of PCR reaction system includes: 0.5 μL upstream primer, 0.5 μL downstream primer, 0.3 μL first round PCR product, 0.5 μL DNA polymerase, 10 μL 5× buffer, 1 μL 10 mM dNTP, and ddH2O to make up to 50 μL.

[0133] The conditions for the second round of PCR reaction were the same as those for the first round of PCR reaction.

[0134] 1.2 Construction of recombinant expression vectors

[0135] The isolated antibody target fragment from step 1.1 (refer to the Axygen product manual) was recovered via gel extraction and subjected to homologous recombination. 4 μL of the gel-recovered isolated antibody target fragment, 3.5 μL of linearized vector, and 2.5 μL of recombinase were ligated. The mixture was incubated in a 0.50°C water bath for 25 min, then allowed to cool for 2-3 min before transformation and bacterial plating experiments. The mixture was incubated overnight at 37°C to obtain the recombinant expression vector.

[0136] 1.3 Colony Screening Experiment

[0137] 1.3.1 Pick a single colony from the overnight plate from step 1.2;

[0138] 1.3.2 The colonies obtained in step 1.3.1 were amplified by PCR using primers Bl-CMV-F and Bl-SEQ-R;

[0139] Bl-CMV-F: CGCAAATGGGCGGTAGGCGTG (SEQ ID No. 20)

[0140] Bl-SEQ-R: AGCGTAAAAGGAGCAACATAGT (SEQ ID No. 21)

[0141] 1.3.3 Electrophoresis identification of positive clones;

[0142] 1.3.4 Four positive bacteria were randomly selected, and the positive bacterial solutions were sequenced using Bl-CMV-F / Bl-SEQ-R primers.

[0143] 1.3.5 Select positive bacterial cultures with correctly sequenced clones, expand the culture, extract low endotoxin plasmids, and verify with sequencing.

[0144] 1.4 Prokaryotic expression of isolated antibodies

[0145] 1.4.1 Culturing CHO cells

[0146] CHO cell lines were placed in RPMI 1640 medium containing 10% inactivated fetal bovine serum, 100 IU / mL penicillin, and 100 IU / mL streptomycin. The medium was then incubated at 37°C in a 5% CO2 incubator, with the medium being changed every 48 hours.

[0147] 1.4.2 Transfection and Expression

[0148] 1) Take 145M of the cells obtained in step 1.4.1 of this embodiment, centrifuge to remove the supernatant, and retain the precipitate.

[0149] 2) Add about 0.5 mL of electroporation buffer to the precipitate obtained in 1), mix well, and then add an appropriate amount of the recombinant expression vector obtained in 1.3 (concentration 500 ng / μL) to obtain a cell plasmid suspension.

[0150] 3) After thoroughly mixing the above cell plasmid suspension, take 1 mL and add it to a 1 mL electroporation tube. Place the electroporation tube into the electroporation apparatus for electroporation.

[0151] 4) After electroporation, aliquot the cells from the electroporation tube into shake flasks containing 20 mL of culture medium and incubate statically for 40 min.

[0152] 5) After incubation, place the shake flasks in a 37°C, 270 rpm, 8% CO2 incubator. After 24 hours, add feed / sodium butyrate / double antibiotics and continue incubation for 3–7 days. On day 5, take samples for ELISA testing.

[0153] 1.5 Antibody purification

[0154] Experimental method: Protein A affinity chromatography column purification

[0155] (1) Equilibration chromatography column: l×PBS, flow rate l mL / min, 20 mL;

[0156] (2) Sample loading: flow rate 1 mL / min;

[0157] (3) Washing: 1×PBS, flow rate 1 mL / min, 20 mL;

[0158] (4) Elution: Citrate buffer (pH 3.4), 1 mL / min, collected in aliquots, approximately 500 μL per tube. A total of 10 tubes were collected, and the absorbance at 280 nm was read using a NanoDrop instrument;

[0159] (5) Dialysis: The high concentration of protein was aspirated into a dialysis bag and placed in a beaker containing 1×PBS for dialysis to obtain the purified isolated antibody.

[0160] 1.6 Basic Antibody Quality Control

[0161] 1) Concentration detection;

[0162] 2) Purity detection (SEC-HPLC);

[0163] Experimental materials: High performance liquid chromatograph, gel chromatography column, deionized water, mobile phase (Na2HPO4·12H2O, NaH2PO4·2H2O, NaCl);

[0164] Experimental methods:

[0165] SEC experiments were performed using a high-performance liquid chromatograph (LC-20AT) and a gel chromatography column. The experimental conditions were as follows:

[0166] Flow rate: 1 mL / min; Injection volume: 20 μL

[0167] Column temperature: 35℃

[0168] Detection wavelengths: 214nm, 280nm

[0169] Data collection time: 15 min

[0170] Replace the water with the mobile phase, and slowly increase the flow rate to 1,000 mL / min until the baseline stabilizes. Transfer 50 μL of antibody to the corresponding numbered vial, place the vial in the appropriate position on the instrument, and inject the sample for 15 min. Analyze, process, and save the data. Replace the mobile phase with deionized water and rinse for 1.5 h.

[0171] The results showed that the antibody purity was >90%.

[0172] Example 2: Affinity determination of isolated antibodies and detection of CD4 antigen

[0173] In this embodiment, the purified isolated antibody obtained in Example 1 was subjected to affinity assays and cell surface CD4 antigen recognition experiments, including the following steps:

[0174] 2.1 Affinity determination of isolated antibodies

[0175] Take 3×10 5 Nalm6-CD4 cells (a Nalm6 cell line overexpressing CD4) were centrifuged at 500×g for 5 min at 4°C, the supernatant was discarded, and the cells were washed once with flow cytometry buffer. The primary antibody was diluted to 4 μg / mL with flow cytometry buffer, and then serially diluted 4-fold to obtain 10 concentrations. 100 μL of each serially diluted antibody was used to resuspend the cells, and the cells were incubated on ice for 30 min. After washing twice with flow cytometry buffer, 100 μL of flow cytometry buffer was added to resuspend the cells, and 1 μg of APC-labeled goat anti-mouse IgG secondary antibody (purchased from BioLegend) was added. The cells were incubated on ice for 30 min, and after washing twice with flow cytometry buffer, 300 μL of flow cytometry buffer was added to resuspend the cells. The cells were detected by flow cytometry, and the EC50 was calculated using GraphPad.

[0176] Figure 1 This is a graph showing the affinity assay results of the isolated antibody in this embodiment.

[0177] from Figure 1 It can be seen that the EC50 of the affinity of the isolated antibody to the CD4 antigen is 0.1518 μg / mL.

[0178] 2.2 Experiment to isolate antibodies that recognize CD4 antigen on cell surface

[0179] 2.2.1 Isolate antibodies that recognize CD4 antigens on the cell surface

[0180] Mix K562-CD4 cells and K562 cells, and take 3×10 5 Centrifuge the mixed cells at 500×g for 5 min at 4℃, discard the supernatant, and wash once with flow cytometry buffer. Resuspend the cells in 100 μL of flow cytometry buffer, add 1 μg of separation antibody, incubate on ice for 30 min, wash twice with flow cytometry buffer, resuspend the cells in 100 μL of flow cytometry buffer, add 1 μg of APC-labeled goat anti-mouse IgG secondary antibody (purchased from BioLegend, corresponding to the separation antibody), incubate on ice for 30 min, wash twice with flow cytometry buffer, resuspend the cells in 300 μL of flow cytometry buffer, and analyze using flow cytometry.

[0181] Meanwhile, a blank control group was used without the addition of isolated antibodies.

[0182] Figure 2 This is a diagram showing the results of antibody isolation and recognition of CD4 antigen on the cell surface in this embodiment.

[0183] 2.2.2 Anti-CD4-3 recognizes CD4 antigen on the cell surface

[0184] Take 3×10 5 K562-CD4 cells (a K562 cell line overexpressing CD4) were centrifuged at 500×g for 5 min at 4℃, the supernatant was discarded, and the cells were washed once with flow cytometry buffer. The cells were resuspended in 100 μL of flow cytometry buffer, 1 μg of CD4-3 antibody was added, and the cells were incubated on ice for 30 min. After washing twice with flow cytometry buffer, the cells were resuspended in 100 μL of flow cytometry buffer, 1 μg of APC-labeled goat anti-mouse IgG secondary antibody (purchased from BioLegend) was added, and the cells were incubated on ice for 30 min. After washing twice with flow cytometry buffer, the cells were resuspended in 300 μL of flow cytometry buffer and analyzed by flow cytometry.

[0185] Meanwhile, a blank control group was used without anti-CD4-3 antibody.

[0186] CD4-3 was purchased from BioLegend, item number: 317402.

[0187] Figure 3 This is a diagram showing the results of the anti-CD4-3 antibody recognizing the CD4 antigen on the cell surface in this embodiment.

[0188] 2.2.3 Anti-CD4-2 and anti-CD4-4 respectively recognize the CD4 antigen on the cell surface.

[0189] K562-CD4 cells and K562 cells (purchased from ATCC) were mixed, and 3 × 10⁻⁶ cells were taken. 5 After mixing, the cells were centrifuged at 500×g for 5 min at 4℃, the supernatant was discarded, and the cells were washed once with flow cytometry buffer. The cells were resuspended in 100 μL of flow cytometry buffer, and 1 μg of anti-CD4-2 and anti-CD4-4 were added respectively. The cells were incubated on ice for 30 min, washed twice with flow cytometry buffer, and then resuspended in 100 μL of flow cytometry buffer. Then, 1 μg of APC-labeled goat anti-mouse IgG secondary antibody (purchased from BioLegend, corresponding to CD4-2 and anti-CD4-4 respectively) was added to the cells resuspended in the buffers obtained from anti-CD4-2 antibody and anti-CD4-4 antibody, and the cells were incubated on ice for another 30 min. After washing twice with flow cytometry buffer, the cells were resuspended in 300 μL of flow cytometry buffer and detected by flow cytometry.

[0190] Meanwhile, a control group without CD4 antibody was used.

[0191] CD4-2 amino acid sequence:

[0192] Heavy chain:

[0193] EVKLQESGPELVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCAREKDNYATGAWFAYWGQGTTVTVSS(SEQ ID No. 22)

[0194] Light chain:

[0195] DIVMTQSPSSLAVSVGEKVTMICKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYRTFGGGTKLEIK(SEQ ID No. 23)

[0196] CD4-4 amino acid sequence:

[0197] Heavy chain:

[0198] QVLLQQSGPELVKPGASVKMSCKASGYFFTDYVINWVKQRTGQGLEWIGEIYPGSGSSYYSEKFKGKATLTADKSSNTAYMQLSSLTSADSAVYFCARRGTGGLGFAYWGQGTLVTVSS(SEQ ID No.24)

[0199] Light chain:

[0200] DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYVASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPPTFGGGTKLEIK(SEQ ID No. 25)

[0201] Figure 4 This is a diagram showing the results of anti-CD4-2, anti-CD4-4, and anti-CD4-8 recognizing the CD4 antigen on the cell surface in this embodiment.

[0202] 2.2.4 CD4-6, anti-CD4-7, anti-CD4-9, anti-CD4-10, and anti-CD4-13 respectively recognize the CD4 antigen on the cell surface.

[0203] The difference from step 2.2.3 in this embodiment is that APC-labeled goat anti-human IgG secondary antibodies (purchased from BioLegend, corresponding to CD4-6, anti-CD4-7, anti-CD4-9, anti-CD4-10 and anti-CD4-13 respectively) are added, and the rest of the steps are the same.

[0204] CD4-6 amino acid sequence:

[0205] Heavy chain:

[0206] QVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSS(SEQ ID No.26)

[0207] Light chain:

[0208] DIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIK(SEQ ID No. 27)

[0209] CD4-7 amino acid sequence:

[0210] Heavy chain:

[0211] QVQLQQSGAEVARPRASVKLSCKASGYTFTSNGINWVKYRTGQGLEWIGEIYPRSGNIFYNERFEGKATLTADKSSTTAYMELRSLTSEDSAVYFCARRVPYFDHWGQGTTLTVSS(SEQ ID No.28)

[0212] Light chain:

[0213] DVVMTQNLRFLPVSAGDRVAMTCKASQSVGNNVAWYQRKPGQSPKLLIYYASNRYTGVPDRFTGSGSGTDFTFTISSVQVEDLAVYFCQQHYSSPFTFGTGTKLEIK(SEQ ID No.29)

[0214] CD4-8 amino acid sequence:

[0215] Heavy chain:

[0216] QVQLQEAGPGLVKPSETLSLTCSVSGGSISGDYYWFWIRQSPGKGLEWIGYIYGSGGGTNYNPSLNNRVSISIDTSKNLFSLKLRSVTAADTAVYYCASNILKYLHWLLYWGQGVLVTVSS(SEQ ID No.30)

[0217] Light chain:

[0218] SYELSQPRSVSVSPGQTAGFTCGGDNVGRKSVQWYQQKPPQAPVLVIYADSERPSGIPARFSGSNSGNTATLTISGVEAGDEADYYCQVWDSTADHWVFGGGTRLTVL(SEQ ID No.31)

[0219] CD4-9 amino acid sequence:

[0220] Heavy chain:

[0221] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVINWFDPWGQGTLVTVSS(SEQ ID No.32)

[0222] Light chain:

[0223] DIQMTQSPSSVSASVGDRVTITCRASQDISSWLAWYQHKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPYTFGQGTKLEIK(SEQ ID No.33)

[0224] CD4 - 10 amino acid sequence:

[0225] Heavy chain:

[0226] EEQLVESGGGLVKPGGSLRLSCAASGFSFSDCRMYWVRQAPGKGLEWIGVISVKSENYGANYAESVRGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCSASYYRYDVGAWFAYWGQGTLVTVSS(SEQ ID No.34)

[0227] Light chain:

[0228] DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYIYWYQQKPGQPPKLLIYLASILESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRELPWTFGQGTKVEIK(SEQ ID No.35)

[0229] CD4 - 13 amino acid sequence:

[0230] Heavy chain:

[0231] QVQLVQSGAEVKKPGASVKVSCKASGYTFTAYVISWVRQAPGQGLEWMGEIYPGSGSSYYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGDGSRFVYWGQGTLVTVSS(SEQ ID No.36)

[0232] Light chain:

[0233] DIVMTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYVASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSLQDPPTFGGGTKVEIK(SEQ ID No.37)

[0234] Figure 5This diagram shows the results of anti-CD4-6, anti-CD4-7, anti-CD4-9, anti-CD4-10, and anti-CD4-13 recognizing the CD4 antigen on the cell surface in this embodiment.

[0235] from Figure 2 , 3 As can be seen from points 4 and 5, the isolated antibody of the present invention can specifically recognize the CD4 antigen on the cell surface.

[0236] Example 3: Isolation of Antibody and Conjugation with Magnetic Beads

[0237] In this embodiment, the purified and isolated antibody from Example 1 is conjugated to magnetic nanoparticles to obtain an antibody conjugate. The experimental steps are as follows:

[0238] 1) Take 1 mg of nano magnetic particles, add 1 mg of EDC and NHS (MES dissolved at pH 5.5), and shake at 37℃ for 0.5 h; centrifuge at 20000g for 30 min to remove NHS and EDC, and obtain the precipitate.

[0239] 2) In this embodiment, the precipitate obtained in step 1) is mixed with nanoparticles and antibodies at a mass ratio of 2:1, and then 500 μL of pH 8.0 borate buffer is added and shaken at room temperature for 2.5 h.

[0240] 3) Then centrifuge at 20000g for 30min, retain the supernatant (centrifuge the supernatant together with the previous one to remove interference), and wait for BCA detection.

[0241] 4) Block the reaction with an aqueous solution containing 20 mg / mL BSA for 1 h, shake the reaction at 37 °C, then centrifuge at 20000 g for 30 min, remove the supernatant, and obtain the precipitate.

[0242] 5) Wash the precipitate obtained in step 4) of this embodiment with DEPC, resuspend it in 1 mL, take 20 μL into 1 mL of pure water, and wait for DLS detection.

[0243] 6) Centrifuge at 20000g for 30 min, remove the supernatant, resuspend in PBS preservation solution to 500 μL, filter through a 0.22 μm filter membrane to obtain the separated antibody conjugate.

[0244] Meanwhile, using anti-CD4-2, anti-CD4-4, anti-CD4-6, anti-CD4-7, anti-CD4-8, anti-CD4-9, anti-CD4-10, and anti-CD4-13 antibodies as controls, anti-CD4-2 conjugates, anti-CD4-4 conjugates, anti-CD4-8 conjugates, anti-CD4-6 conjugates, anti-CD4-7 conjugates, anti-CD4-9 conjugates, anti-CD4-10 conjugates, and anti-CD4-13 conjugates were prepared using the same method as the isolated antibodies.

[0245] Example 4: Sorting and enrichment of PBMCs using antibody conjugates

[0246] In this embodiment, the antibody-drug conjugate obtained in Example 3 was used to sort and enrich PBMCs, and the efficiency and purity of the antibody-drug conjugate in enriching and sorting CD4 T cells were detected. The experimental steps are as follows:

[0247] 4.1 Sorting and enrichment of PBMCs using antibody-conjugates

[0248] 1) Resuscitate PBMCs and incubate them at 37°C and 5% CO2 for 2 hours;

[0249] 2) Collect the PBMC cultured in step 1) of this embodiment into a 15mL centrifuge tube, centrifuge at 500g for 5min, discard the supernatant, and wash the PBMC precipitate once with 2mL of flow cytometry buffer.

[0250] 3) Resuspend the PBMCs using flow cytometry buffer, count them, and take 2.5 × 10⁻⁶ samples. 6 Cells were centrifuged, resuspended in 50 μL of flow cytometry buffer, and 0.3125 μL of the antibody conjugate obtained in Example 3 was added. After mixing, the mixture was incubated at room temperature for 15 min.

[0251] 4) Add 500 μL of flow cytometry buffer to terminate the reaction, transfer to MS Columns for cell sorting and enrichment. After the cell suspension has been completely dropped, wash the MS Columns twice with 500 μL of flow cytometry buffer and collect the cell waste for later use.

[0252] 5) Add 1 mL of flow cytometry buffer to MS Columns and use the stopcock to collect the sorted and enriched cells into a 1.5 mL centrifuge tube.

[0253] 4.2 Determination of the efficiency and purity of antibody-conjugates in the sorting and enrichment of CD4 T cells

[0254] 1) Take 3 × 10⁻⁶ cells each from the cell waste fluid obtained in step 4) of Example 4.1, the sorted and enriched cells obtained in step 5), and the PBMCs before sorting and enrichment. 5 Centrifuge each cell at 4°C and 500×g for 5 min, discard the supernatant, and wash once with flow cytometry buffer.

[0255] 2) Resuspend the cells in 100 μL of flow cytometry buffer, add 1 μg of APC-labeled goat anti-mouse IgG secondary antibody (purchased from BioLegend), and incubate on ice for 30 min to obtain labeled cells;

[0256] 3) After washing twice with flow cytometry buffer, the labeled cells were resuspended in 100 μL of flow cytometry buffer, and 1 μg of PE-anti-CD3 antibody (purchased from BioLegend) was added. The cells were incubated on ice for 30 min. After washing twice with flow cytometry buffer, the cells were resuspended in 300 μL of flow cytometry buffer. The proportion of CD4 T cells in the pre-enriched PBMCs, cell waste, and enriched cells was detected by flow cytometry.

[0257] Meanwhile, a control group was set up. The control group differed from the antibody conjugate of the present invention in that: in step 3) of Example 5.1, the antibody magnetic bead conjugate was not added, but anti-CD4-2 conjugate, anti-CD4-4 conjugate, anti-CD4-8 conjugate, anti-CD4-6 conjugate, anti-CD4-7 conjugate, anti-CD4-9 conjugate, anti-CD4-10 conjugate, and anti-CD4-13 conjugate were added, and the rest were the same.

[0258] Figure 6A This figure shows the proportion of CD4T cells in the cells before and after enrichment using antibody-drug conjugates in this embodiment.

[0259] Depend on Figure 6A It can be seen that the proportion of CD4 T cells in PBMC before sorting and enrichment was 31.9%. After sorting and enrichment, the proportion of CD4 T cells increased significantly, exceeding 90%, indicating that the antibody conjugate of the present invention can enrich high-purity CD4 T cells from PBMC.

[0260] Figure 6B This figure shows the proportion of CD4T cells in the cell waste fluid after sorting and enrichment using antibody-conjugates in this embodiment.

[0261] Depend on Figure 6B As can be seen, only a small number of CD4 T cells remain in the cell waste fluid. As can be seen from the proportion in the figure, more than 90% of CD4 T cells are enriched, indicating that the antibody conjugate of the present invention can efficiently enrich CD4 T cells from PBMCs.

[0262] Figure 7A This figure shows the proportion of CD4T cells in the cells before and after sorting and enrichment using anti-CD4-3 conjugates in this embodiment.

[0263] Depend on Figure 7A It can be seen that before sorting and enrichment using anti-CD4-3 conjugates, the proportion of CD4 T cells in PBMCs was 33.7%; after sorting and enrichment, the proportion of CD4 T cells decreased to 23%.

[0264] Figure 7B This figure shows the proportion of CD4T cells in the cell waste fluid after sorting and enrichment using anti-CD4-3 conjugates in this embodiment.

[0265] Depend on Figure 7B It can be seen that after sorting and enriching with anti-CD4-3 conjugates, the proportion of CD4 T cells in the cell waste fluid is relatively high, reaching 25.8%.

[0266] Figure 8A This figure shows the proportion of CD4T cells in the cells before and after enrichment using anti-CD4-2 conjugate, anti-CD4-4 conjugate, and anti-CD4-8 conjugate, respectively, in this embodiment.

[0267] Depend on Figure 8A It can be seen that before sorting and enrichment using anti-CD4-2 conjugate, anti-CD4-4 conjugate, and anti-CD4-8 conjugate, the proportion of CD4 T cells in PBMCs was 31.0%; after sorting and enrichment, the proportions of CD4 T cells were 38.1%, 19.8%, and 52.7%, respectively.

[0268] Figure 8B The figure shows the proportion of CD4T cells in the cell waste fluid after sorting and enrichment using anti-CD4-2 conjugate, anti-CD4-4 conjugate, and anti-CD4-8 conjugate, respectively, in this embodiment.

[0269] Depend on Figure 8B It can be seen that after sorting and enriching with anti-CD4-2 conjugate, anti-CD4-4 conjugate and anti-CD4-8 conjugate, the proportions of CD4 T cells in the cell waste fluid were 24.1%, 34.8% and 4.52%, respectively.

[0270] Figure 9A This figure shows the proportion of CD4 T cells in the cells before and after enrichment using anti-CD4-6 conjugate, anti-CD4-7 conjugate, anti-CD4-9 conjugate, anti-CD4-10 conjugate, and anti-CD4-13 conjugate, respectively, in this embodiment.

[0271] Depend on Figure 9A It can be seen that before sorting and enrichment using anti-CD4-6 conjugates, anti-CD4-7 conjugates, anti-CD4-9 conjugates, anti-CD4-10 conjugates, and anti-CD4-13 conjugates, the proportion of CD4 T cells in PBMCs was 29.0%; after sorting and enrichment, the proportions of CD4 T cells were 65.8%, 85.0%, 22.1%, 80.1%, and 80.8%, respectively.

[0272] Figure 9B The figure shows the proportion of CD4 T cells in the cell waste fluid after sorting and enrichment using anti-CD4-6 conjugate, anti-CD4-7 conjugate, anti-CD4-9 conjugate, anti-CD4-10 conjugate, and anti-CD4-13 conjugate, respectively, in this embodiment.

[0273] Depend on Figure 9BIt can be seen that after sorting and enriching with anti-CD4-6 conjugate, anti-CD4-7 conjugate, anti-CD4-9 conjugate, anti-CD4-10 conjugate and anti-CD4-13 conjugate, the proportions of CD4 T cells in the cell waste fluid were 25.2%, 55.35%, 27.2%, 12.3% and 9.68%, respectively.

[0274] In summary, the separation and enrichment of CD4 T cells using the separation antibodies of the present invention can obtain high-purity CD4 T cells. However, when PBMCs are sorted and enriched using anti-CD4-2, anti-CD4-4, anti-CD4-8, anti-CD4-6, anti-CD4-7, anti-CD4-9, anti-CD4-10 and anti-CD4-13 antibodies, a large number of other contaminating cells are present in the enriched cells, and a large number of unenriched CD4 T cells remain in the waste liquid.

[0275] Example 5 Lentiviral Packaging and Concentration

[0276] This embodiment involves packaging and concentrating lentiviruses, and then detecting the lentivirus titer. The steps include:

[0277] 5.1 Packaging of Lentivirals

[0278] 5.1.1 with 1.6 × 10 7 293T cells were seeded in 15cm diameter culture dishes and cultured overnight at 37°C with 5% CO2 to prepare for virus packaging. The culture medium was DMEM with 10% fetal bovine serum (FBS) added.

[0279] 5.1.2 Dissolve 30 μg of HD SIN03 CD22(V29)41BBz(ka) lentiviral vector, 12.5 μg of helper plasmid gag / pol, and 10 μg of envelope plasmid VSVg in 2000 μL of serum-free DMEM culture medium, mix well, and obtain DNA mixture;

[0280] 5.1.3 Dissolve 157.5 μg PEI (1 μg / μL) in 2000 μL of serum-free DMEM culture medium, vortex at 1000 rpm for 5 seconds, and incubate at 25℃ for 5 min to obtain PEI mixture;

[0281] 5.1.4 Add the PEI mixture to the DNA mixture obtained in step 5.1.2 of this embodiment. Vortex mix or gently mix immediately after addition. Incubate at 25°C for 20 min to obtain the transfection complex.

[0282] 5.1.5 Add 4 mL of the transfection complex obtained in step 5.1.4 of this embodiment to a 15 cm culture dish containing 25 mL of DMEM medium. After 4 hours, replace with fresh medium.

[0283] 5.1.6 After 48 hours, collect the viral supernatant to obtain lentivirus supernatant.

[0284] 5.2 Lentiviral Concentration

[0285] The viral supernatant prepared in 5.1 of this embodiment was filtered through a 0.45 μm filter membrane and collected into a 50 mL centrifuge tube. 1 / 4 of the PEG-NaCl viral concentrate was added, and the mixture was mixed by inverting. The mixture was then incubated overnight at 4 °C. The mixture was centrifuged at 3500 rpm for 30 min at 4 °C. The supernatant was removed, and RPMI 1640 medium (containing 10% FBS) was added to dissolve and resuspend the viral precipitate. The concentrated lentivirus suspension was aliquoted into 50 μL portions and stored in finished tubes at -80 °C.

[0286] 5.3 Lentiviral titer detection

[0287] 500 μL of Jurkat cells (1 × 10⁻⁶) 5 (10 cells) were seeded into 24-well culture plates to obtain Jurkat cell suspension;

[0288] The concentrated lentivirus suspension from step 5.2 of this example was added to Jurkat cell suspension at doses of 1 μL, 0.2 μL, and 0.04 μL, respectively, and polybrene was added to a final concentration of 5 μg / mL. After incubation overnight at 37°C with 5% CO2, the culture medium was replaced with fresh medium. 72 h after infection, the cells were centrifuged at 400 × g for 5 min, the supernatant was discarded, and the cells were resuspended in 100 μL PBS + 2% FBS. 1 μg of PE-anti-VHH antibody was added, and the cells were incubated on ice for 30 min. After washing twice with PBS + 2% FBS, the cells were resuspended in 300 μL PBS + 2% FBS, and the infection efficiency was detected by flow cytometry. Cell samples with a positivity rate of 15% were considered optimal, and the titer was calculated to be 4.5 × 10⁻⁶. 8 TU / mL.

[0289] Titer (TU / mL) = Cell number (10) 5 ) × Positive rate / Viral volume (mL).

[0290] Example 6 Lentiviral transduction of CD4 T cells

[0291] In this embodiment, the concentrated lentivirus suspension obtained in Example 5 was transduced into CD4T cells sorted and enriched in Example 4, and the lentivirus infection efficiency was detected. The steps included are as follows:

[0292] 6.1 Lentiviral transduction of CD4 T cells

[0293] Anti-human CD3 antibody and anti-human CD28 antibody were diluted with PBS to final concentrations of 1 μg / mL and 0.5 μg / mL, respectively, and coated with the solutions in well plates. The plates were then incubated overnight at 4°C. The antibody coating solution in the well plates was discarded, and the plates were washed twice with 1 mL of PBS. The CD4 T cells sorted and enriched in Example 4 were adjusted to a density of 1 × 10⁻⁶ cells using T cell culture medium (X-VIVO + 10% FBS + IL-2 (300 U / mL)). 6 / mL, and then seeded into CD3 and CD28 antibody-coated well plates for activation for 48h; the activated CD4 T cells were collected and the cell density was adjusted to 1×10⁶. 6 / mL, add the concentrated lentivirus suspension prepared in Example 5 according to the multiplicity of infection (MOI) = 10, add polybrene to the final concentration of 5 μg / mL; culture overnight at 37°C and 5% CO2, then replace with fresh medium, passage every 2 days to obtain CAR-T cells that specifically target CD22.

[0294] 6.2 Detection of Lentiviral Infection Efficiency

[0295] 6.2.1 In this example, after lentivirus transduction of CD4 T cells in Example 6.1, cell counts were performed at 5, 8, 11, and 14 days after infection. Figure 10 (The day of infection is recorded as day 0) and 3 × 10⁻⁶ samples are collected. 5 T cells were centrifuged at 400×g for 5 min at 4℃, the supernatant was discarded, and the cells were washed once with PBS + 2% FBS.

[0296] 6.2.2. Resuspend cells in 100 μL PBS + 2% FBS, add 1 μg of iF488-anti-VHH antibody (purchased from GenScript), and incubate on ice for 30 min. Wash twice with PBS + 2% FBS, then resuspend cells in 300 μL PBS + 2% FBS. Using uninfected T cells as a control, infection efficiency was detected by flow cytometry. The infection efficiencies on days 5, 8, 11, and 14 after viral infection are shown in the figures below. Figure 11A , 11B 11C and 11D.

[0297] Figure 10 This is a graph showing the total cell count of CD4 T cells on days 5, 8, 11, and 14 after viral infection in this embodiment.

[0298] from Figure 10 It can be seen that CD4 T cells proliferated more than 1,000 times on the 14th day after infection with the virus compared to the day of infection, indicating that sorted and enriched CD4 T cells can proliferate rapidly under normal T cell culture conditions.

[0299] Figure 11A , 11B 11C and 11D are the infection rate results of CD4 T cells infected with the virus on days 5, 8, 11, and 14, respectively, in this embodiment.

[0300] from Figure 11A , 11B According to 11C and 11D, the chimeric antigen receptor can be expressed relatively stably on days 5, 8, 11 and 14 after CD4 T cells are infected with the virus, with an infection rate of over 77.3%.

[0301] Example 7 In vitro toxicity and cytokine release experiments

[0302] In this embodiment, CAR-T cells specifically targeting CD22 prepared in Example 6 were used to conduct in vitro toxicity experiments and cytokine release experiments, including the following steps:

[0303] 7.1 In vitro toxicity test

[0304] 7.1.1 Target cell inoculation

[0305] Using tumor cells K562-luci(CD22) - K562-CD22-luci(CD22) + As target cells, the target cell concentration was adjusted to 2 × 10⁻⁶. 5 / mL, take 50μL and inoculate into a 96-well plate.

[0306] 7.1.2 Effector cell seeding

[0307] CAR-T cells specifically targeting CD22 prepared in Example 6 and control T cells (not infected with lentivirus) were used as effector cells; CAR-T cells and control T cells were added to 96-well plates after seeding target cells in step 7.1.1 of this example at effector-to-target ratios of 0.3:1, 1:1 and 3:1.

[0308] Each group had two replicates, and the average value of the two replicates was taken. The experimental group and the control group are as follows:

[0309] Experimental group (CD4-14 CD22-CAR): CAR-T cells + target cells;

[0310] Control group (Control CD4 T): Control T cells + target cells;

[0311] 7.1.3. After co-culturing effector cells and target cells for 18 hours, use... The luciferase assay kit is used for detection; please refer to the specific detection steps. The instructions for the luciferase assay kit are as follows: Figure 12Aand 12B As shown.

[0312] from Figure 12A and 12B It is known that the CAR-T cells constructed in this invention have high killing activity against CD22-positive tumor cells, but no killing effect on CD22-negative cells. This indicates that the CAR-T cells specifically targeting CD22 constructed in this invention have high specificity. The CAR-T cells specifically targeting CD22 can specifically kill CD22-positive cells, and the killing effect is positively correlated with the effector-to-target ratio.

[0313] 7.2 In vitro cytokine release assay

[0314] 7.2.1 Cell Culture Supernatant

[0315] The cell culture with an intermediate-to-target ratio of 1:1 as described in 7.1 of this embodiment was centrifuged at 400×g for 10 min to remove the precipitate. The cell culture supernatant was then stored at -80℃ for testing. The cell culture supernatant was designated as K562-CD22-luci.

[0316] Meanwhile, CAR-T cell cultures alone and cultures of K562-luci and CAR-T cells were used as negative control groups.

[0317] 7.2.2 Reagent Preparation

[0318] The detection was performed using the Linko Bio ELISA kit (catalog number: Human Gamma Interferon ELISA Kit: EK180-96). Before the detection, all reagents and samples were brought to 25°C. 1× washing buffer and 1× detection buffer were prepared according to the instructions for use, and the antibody was detected.

[0319] 7.2.3 Preparation of Standards and Samples

[0320] Standards: The stock solution of the standard was diluted twice using 5% 1640 culture medium, with a total of 8 dilution gradients, including zero concentration.

[0321] Samples: Dilute the samples using 5% 1640 medium.

[0322] 7.2.4 Detection Procedure

[0323] (1) Soaking the microplate: Add 300 μL of 1× washing solution and let it stand for 30 seconds. After discarding the washing solution, pat the microplate dry on absorbent paper.

[0324] (2) Add standard: Add 100 μL of 2-fold serially diluted standard to the standard wells and add 100 μL of 5% 1640 culture medium to the blank wells;

[0325] (3) Add sample: Add 100 μL of cell culture supernatant to the sample well;

[0326] (4) Add detection antibody: Add 50 μL of diluted detection antibody (1:100 dilution) to each well;

[0327] (5) Incubation: Seal the plate with sealing film, shake at 300 rpm, and incubate at 25°C for 2 hours;

[0328] (6) Washing: Discard the liquid, add 300 μL of washing solution to each well and wash the plate 6 times;

[0329] (7) Enzyme incubation: Add 100 μL of diluted horseradish peroxidase-labeled streptavidin (1:100 dilution) to each well;

[0330] (8) Incubation: Seal the plate with a new sealing film, shake at 300 rpm, and incubate at 25°C for 45 min;

[0331] (9) Washing: Repeat step (6);

[0332] (10) Adding substrate for color development: Add 100 μL of TMB substrate to each well, incubate in the dark at 25°C for 15 min;

[0333] (11) Add stop solution: Add 100 μL of stop solution to each well and mix thoroughly;

[0334] (12) Detection reading: The OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm was measured using an ELISA reader. The calibrated OD value is the measured value at 450nm minus the measured value at 630nm.

[0335] Figure 13 This is a diagram showing the IFN-γ factor secretion results in this embodiment.

[0336] from Figure 13 It was found that trace amounts of IFN-γ were detected in spontaneous and K562-luci co-cultures with CAR-T, while higher levels of IFN-γ were detected in K562-CD22-luci co-cultures with CAR-T. This indicates that the CD22-specific CAR-T cells constructed in this invention can also release cytokines to CD22-positive tumor cells to exert a killing function, and have high specificity, with no significant cytokine secretion on CD22-negative cells.

[0337] In summary, this invention provides a highly efficient isolated antibody targeting the CD4 antigen. The antibody conjugate obtained by coupling this isolated antibody with magnetic particles can efficiently sort and enrich high-purity CD4 T cells from PBMCs. Before sorting and enrichment, the proportion of CD4 T cells was 31.9%, and after sorting and enrichment, the proportion of CD4 T cells increased significantly, exceeding 90%. Meanwhile, the proportion of CD4 T cells in the waste liquid was only 2.38%, indicating that the isolated antibody of this invention can efficiently enrich high-purity CD4 T cells from PBMCs.

[0338] Furthermore, this invention introduces the CD22-targeting chimeric antigen receptor gene into sorted and enriched CD4 T cells to prepare chimeric antigen receptor cells that specifically target CD22. These chimeric antigen receptor cells that specifically target CD22 can grow rapidly and express stably, and can effectively and specifically target and kill CD22-positive tumor cells in vitro. They can also activate cytokines to exert their killing function and have high specificity.

[0339] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. Use of the isolated antibody in the preparation of a product enriched from CD4 T cells in PBMCs, said isolated antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of CDR-H1 of the heavy chain variable region is shown in SEQ ID No. 1, the amino acid sequence of CDR-H2 is shown in SEQ ID No. 2 and the amino acid sequence of CDR-H3 is shown in SEQ ID No. 3; The amino acid sequences of the light chain variable regions CDR-L1 are shown in SEQ ID No. 4, CDR-L2 are shown in VAS, and CDR-L3 are shown in SEQ ID No.

5.

2. The use as described in claim 1, characterized in that, The product comprises an antibody-drug conjugate, which contains isolated antibodies and markers or separation aids.

3. The use as described in claim 2, characterized in that, The marker is selected from one or more of biotin, fluorescein, and horseradish peroxidase.

4. The use as described in claim 2, characterized in that, The separation aids are selected from magnetic beads and agarose beads.

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