Anti-cd4 antibodies and uses thereof

Abstract

The application relates to the technical field of biological medicine, and discloses an anti-CD4 antibody and application thereof. The anti-CD4 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region has the following technical features: 1-1) CDR-H1 with an amino acid sequence as shown in SEQ ID No. 1; 1-2) CDR-H2 with an amino acid sequence as shown in SEQ ID No. 2; and 1-3) CDR-H3 with an amino acid sequence as shown in SEQ ID No. 3. The anti-CD4 antibody of the application can efficiently target CD4 antigens, can efficiently enrich and sort CD4 T cells from PBMC to obtain CD4 T cells with high purity, can transduce a CD22-targeted chimeric antigen receptor gene into the CD4 T cells to obtain CD22-CAR-T cells which can specifically target CD22, and can specifically recognize CD22-positive tumor cells and kill the tumor cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an anti-CD4 antibody and its uses. Background Technology

[0002] 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.

[0003] 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.

[0004] 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.

[0005] Antibodies are essential for cell sorting. Therefore, developing new anti-CD4 antibodies for sorting and enriching CD4 T cells is of great significance. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an anti-CD4 antibody and its uses.

[0007] One objective of this invention is to provide an anti-CD4 antibody, which includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the following technical features;

[0008] 1-1) The amino acid sequence is CDR-H1 as shown in SEQ ID No. 1;

[0009] 1-2) The amino acid sequence is CDR-H2 as shown in SEQ ID No. 2;

[0010] 1-3) The amino acid sequence is CDR-H3 as shown in SEQ ID No.3.

[0011] According to the technical solution of this application, the amino acid sequence of the heavy chain variable region includes:

[0012] A1) The amino acid sequence shown in SEQ ID No. 4;

[0013] A2) An amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID No. 4 and has the function of the amino acid sequence defined in A1).

[0014] According to the technical solution of this application, 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.

[0015] According to the technical solution of this application, the light chain variable region has the following technical features:

[0016] 2-1) The amino acid sequence is CDR-L1 as shown in SEQ ID No. 5;

[0017] 2-2) The amino acid sequence is CDR-L2 of YAS;

[0018] 2-3) The amino acid sequence is CDR-L3 as shown in SEQ ID No. 6.

[0019] According to the technical solution of this application, the amino acid sequence of the light chain variable region includes:

[0020] B1) The amino acid sequence as shown in SEQ ID No. 7;

[0021] B2) An amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID No. 7 and has the function of the amino acid sequence defined in B1).

[0022] According to the technical solution of this application, 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.

[0023] According to the technical solution of this application, the antibody further includes a light chain constant region and a heavy chain constant region.

[0024] Preferably, the nucleotide sequence of the light chain constant region includes, for example, SEQ ID No. 18.

[0025] Preferably, the sequence of the heavy chain constant region includes that shown in SEQ ID No. 19.

[0026] In this invention, the encoding gene of the anti-CD4 antibody is inserted into an expression vector to obtain a construct. The construct is then introduced into host cells and cultured, followed by separation and purification to obtain the anti-CD4 antibody.

[0027] A second objective of this invention is to provide an isolated polypeptide, wherein the polypeptide comprises a transmembrane domain, an extracellular domain, and an intracellular domain, and the extracellular domain comprises an antibody as described above.

[0028] According to the technical solution of this application, the polypeptide is a chimeric antigen receptor.

[0029] According to the technical solution of this application, the polypeptide includes, from the N-terminus to the C-terminus, the anti-CD4 antibody, the transmembrane domain, and the intracellular domain as described above.

[0030] A third objective of this invention is to provide biomaterials related to the anti-CD4 antibody or the polypeptide described above, wherein the biomaterials include one or more of the following:

[0031] a) Nucleotides encoding the heavy chain variable region and / or light chain variable region of the anti-CD4 antibody as described above, or nucleotides encoding the polypeptide as described above;

[0032] b) A construct containing the nucleotides described in a);

[0033] c) A host cell containing the nucleotides described in a), or a host cell containing the construct described in b).

[0034] According to the technical solution of this application, in a), the nucleotide sequence encoding the heavy chain variable region of the antibody as described above includes the sequence shown in SEQ ID No. 16.

[0035] According to the technical solution of this application, in a), the nucleotide sequence encoding the light chain variable region of the antibody as described above includes the sequence shown in SEQ ID No. 17.

[0036] According to the technical solution of this application, in b), the construct is a viral vector or a non-viral vector.

[0037] According to the technical solution of this application, in c), the host cell is one or both of T cells and NK cells.

[0038] The fourth objective of this invention is to provide an antibody conjugate comprising an anti-CD4 antibody as described above and a marker or separation aid, wherein the marker is selected from one or more of biotin, fluorescein, and horseradish peroxidase; preferably, the separation aid is selected from magnetic beads and agarose beads.

[0039] Preferably, the separation aid is a magnetic bead.

[0040] A fifth objective of this invention is to provide a pharmaceutical composition comprising, as described above, antibodies and / or peptides and / or biomaterials and / or antibody conjugates, and pharmaceutically acceptable carriers.

[0041] The sixth objective of this invention is to provide a product comprising the anti-CD4 antibody and / or the antibody conjugate described above.

[0042] According to the technical solution of this application, the product is selected from reagent kits, chips, and membrane strips.

[0043] The seventh objective of this invention is to provide the use of the antibody, peptide, biomaterial, antibody-drug conjugate, pharmaceutical composition, or product described above in the preparation of at least one of the following products:

[0044] Products for in vitro CD4 detection;

[0045] Products for in vitro enrichment of CD4 T cells;

[0046] In the preparation or screening of preventive and / or therapeutic drugs.

[0047] The eighth objective of this invention is to provide a method for in vitro enrichment of CD4 T cells, which involves adding the antibodies and / or antibody-drug conjugates described above to peripheral blood mononuclear cells, causing CD4 T cells to bind to the antibodies and / or antibody-drug conjugates, and then separating them to obtain the CD4 T cells.

[0048] As described above, the present invention has the following beneficial effects:

[0049] 1) The anti-CD4 antibody of the present invention can efficiently target the CD4 antigen and is easy to prepare;

[0050] 2) The anti-CD4 antibody magnetic beads constructed in this invention can enrich and sort high-purity CD4 T cells from PBMCs. Furthermore, lentiviruses containing chimeric antigen receptor genes targeting CD22 are transduced into CD4 T cells to obtain CD22-CAR-T cells that specifically target CD22. In in vitro toxicity tests, these cells have been shown to be specifically activated by the antigen.

[0051] 3) The CD22-CAR-T cells of the present invention can specifically recognize CD22-positive tumor cells and kill them efficiently. At the same time, they can also release a variety of cytokines, including IFN-γ factor, to exert cell killing effects. Attached Figure Description

[0052] Figure 1 This is a graph showing the affinity assay results of the anti-CD4-1 antibody in Example 2 of the present invention.

[0053] Figure 2A This is a diagram showing the results of the anti-CD4-1 antibody recognizing the CD4 antigen on the cell surface in Example 2 of the present invention.

[0054] Figure 2B This is a diagram showing the results of the anti-CD4-3 antibody recognizing the CD4 antigen on the cell surface in Example 2 of the present invention.

[0055] Figure 2C 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 2 of the present invention.

[0056] Figure 2D 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 antibody recognizing the CD4 antigen on the cell surface in Example 2 of the present invention.

[0057] Figure 3A This is a graph showing the proportion of CD4T cells in PBMC cells before and after enrichment using anti-CD4-1 magnetic beads in Example 5 of the present invention.

[0058] Figure 3B This is a diagram showing the proportion of CD4 T cells in the waste liquid after enrichment and sorting using anti-CD4-1 magnetic beads in Example 5 of the present invention.

[0059] Figure 4A This is a graph showing the proportion of CD4T cells in PBMC cells before and after enrichment using anti-CD4-3 magnetic beads in Example 5 of the present invention.

[0060] Figure 4B This is a graph showing the proportion of CD4T cells in the cell waste fluid after sorting and enrichment with anti-CD4-3 magnetic beads in Example 5 of the present invention.

[0061] Figure 5A The figure shows the proportion of CD4T cells in PBMC cells before and after sorting and enrichment using anti-CD4-2 magnetic beads, anti-CD4-4 magnetic beads, and anti-CD4-8 magnetic beads in Example 5 of the present invention.

[0062] Figure 5B The figure shows the proportion of CD4T cells in the cell waste fluid after sorting and enrichment using anti-CD4-2 magnetic beads, anti-CD4-4 magnetic beads, and anti-CD4-8 magnetic beads, respectively, in Example 5 of the present invention.

[0063] Figure 6A The figure shows the proportion of CD4T cells in PBMC cells before and after sorting and enrichment using anti-CD4-6 magnetic beads, anti-CD4-7 magnetic beads, anti-CD4-9 magnetic beads, anti-CD4-10 magnetic beads, and anti-CD4-13 magnetic beads in Example 5 of the present invention.

[0064] Figure 6B The figure shows the proportion of CD4T cells in the cell waste fluid after sorting and enrichment using anti-CD4-6 magnetic beads, anti-CD4-7 magnetic beads, anti-CD4-9 magnetic beads, anti-CD4-10 magnetic beads, and anti-CD4-13 magnetic beads, respectively, in Example 5 of the present invention.

[0065] Figure 7 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 10 of the present invention.

[0066] Figure 8A 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 shown in Example 10 of the present invention.

[0067] Figure 8B 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 10 of the present invention.

[0068] Figure 8CThis is a graph showing the expression rate of chimeric antigen receptor in CAR-T cells 11 days after CD4 T cells were infected with the virus, as described in Example 10 of the present invention.

[0069] Figure 8D 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 10 of the present invention.

[0070] Figure 9A The CAR-T cells (CD4+) in Example 11 of the present invention + Image showing the killing effect of CD22-CAR-T on K562-luci cells.

[0071] Figure 9B The CAR-T cells (CD4+) in Example 11 of the present invention + Image showing the killing effect of CD22-CAR-T on K562-CD22-luci cells.

[0072] Figure 10 The CAR-T cells (CD4+) in Example 12 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

[0073] Through in-depth research, the inventors have provided an anti-CD4 antibody that specifically binds to the CD4 antigen. This anti-CD4 T antibody is coupled to magnetic nanoparticles, and high-purity CD4 T cells are obtained by enriching and sorting them from human peripheral blood mononuclear cells. Subsequently, a CD22-targeting chimeric antigen receptor gene is 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.

[0074] The first aspect of this invention protects an anti-CD4 antibody, wherein the anti-CD4 antibody includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region has the following technical features;

[0075] 1-1) The amino acid sequence is CDR-H1 as shown in SEQ ID No. 1;

[0076] 1-2) The amino acid sequence is CDR-H2 as shown in SEQ ID No. 2;

[0077] 1-3) The amino acid sequence is CDR-H3 as shown in SEQ ID No.3.

[0078] GYTFTDYF(SEQ ID No.1)

[0079] IYPNNGGT(SEQ ID No.2)

[0080] ARRGYDEGFAY (SEQ ID No. 3)

[0081] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region includes:

[0082] A1) The amino acid sequence shown in SEQ ID No. 4;

[0083] A2) An amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID No. 4 and has the function of the amino acid sequence defined in A1).

[0084] EVQLQQSGPELVKPGDSVKMSCKASGYTFTDYFMDWVKQSHGKSLEWIGYIYPNNGGTNNNQKFKGKATLTVDKSSSTAYMELHSLTSEDSAVYYCARRGYDEGFAYWGQGTLVTVSS(SEQ ID No.4)

[0085] In some embodiments of the present invention, the light chain variable region has the following technical features:

[0086] 2-1) The amino acid sequence is CDR-L1 as shown in SEQ ID No. 5;

[0087] 2-2) The amino acid sequence is CDR-L2 as shown in YAS;

[0088] 2-3) The amino acid sequence is CDR-L3 as shown in SEQ ID No. 6.

[0089] QIIGTS (SEQ ID No. 5)

[0090] QQSNTWPFT (SEQ ID No. 6)

[0091] In some embodiments of the present invention, according to the technical solution of this application, the amino acid sequence of the light chain variable region includes:

[0092] B1) The amino acid sequence as shown in SEQ ID No. 7;

[0093] B2) An amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID No. 7 and has the function of the amino acid sequence defined in B1).

[0094] DILLTQSPAILSVSPGERVSFSCRASQIIGTSILWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNTWPFTFGGGTKLEMK(SEQ ID No.7)

[0095] In some embodiments of the present invention, the heavy chain variable region and / or light chain variable region further include a framework region (FR). In some specific embodiments of the present invention, 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 (specifically, 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids, and the framework region sequence has 80%, 85%, 90%, 93%, 95%, 97% or more homology with the framework region sequence of the human monoclonal antibody variable region.

[0096] Preferably, the heavy chain variable region further includes framework regions HCFR1-HCFR4: the amino acid sequence of framework region HCFR1 is as shown in SEQ ID No. 8, the amino acid sequence of framework region HCFR2 is as shown in SEQ ID No. 9, the amino acid sequence of framework region HCFR3 is as shown in SEQ ID No. 10, and the amino acid sequence of framework region HCFR4 is as shown in SEQ ID No. 11.

[0097] EVQLQQSGPELVKPGDSVKMSCKAS(SEQ ID No.8)

[0098] MDWVKQSHGKSLEWIGY(SEQ ID No.9)

[0099] NNNQKFKGKATLTVDKSSSTAYMELHSLTSEDSAVYYC(SEQ ID No.10)

[0100] WGQGTLVTVSS (SEQ ID No. 11)

[0101] Preferably, the light chain variable region further includes framework regions LCFR1-LCFR4: the amino acid sequence of framework region LCFR1 is as shown in SEQ ID No. 12, the amino acid sequence of framework region LCFR2 is as shown in SEQ ID No. 13, the amino acid sequence of framework region LCFR3 is as shown in SEQ ID No. 14, and the amino acid sequence of framework region LCFR4 is as shown in SEQ ID No. 15.

[0102] DILLTQSPAILSVSPGERVSFSCRAS(SEQ ID No.12)

[0103] ILWYQQRTNGSPRLLIK(SEQ ID No.13)

[0104] ESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYC(SEQ ID No.14)

[0105] FGGGTKLEMK (SEQ ID No. 15)

[0106] In some embodiments of the present invention, the anti-CD4 antibody further includes a light chain constant region and a heavy chain constant region.

[0107] Preferably, the nucleotide sequence of the light chain constant region includes, for example, SEQ ID No. 18.

[0108] The nucleotide sequence shown in SEQ ID No. 18 is as follows:

[0109] Agagcagatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctgaac agttggactgatcaggacagcaaagacagcacctacagcatgagcagcaccctcacgttgaccaaggacgagtatgaacgacataacagctatacctgtgaggccactcacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgt(SEQ ID No.18)

[0110] Preferably, the sequence of the heavy chain constant region includes that shown in SEQ ID No. 19.

[0111] The nucleotide sequence shown in SEQ ID No. 20 is as follows:

[0112]

[0113] This invention involves inserting the encoding gene of the anti-CD4 antibody into an expression vector to obtain a construct. The construct is then introduced into host cells and cultured, followed by separation and purification to obtain the anti-CD4 antibody. The host cells may be E. coli cells, simian COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulins. The separation and purification methods may include protein A agarose gel chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. In a preferred embodiment, CHO cells are used. The construct is introduced into the host cells via electroporation and purified using affinity chromatography.

[0114] A second aspect of the present invention provides an isolated polypeptide comprising a transmembrane domain, an intracellular domain, and an extracellular domain, wherein the extracellular domain comprises an antibody as described above.

[0115] In some embodiments of the present invention, the polypeptide is a chimeric antigen receptor.

[0116] In some embodiments of the present invention, the transmembrane domain may include transmembrane domains of protein molecules such as CD8α, CD28, and DAP10. For example, the sequence of CD8α can be found in NM_001145873, the sequence of CD28 can be found in NM_006139, and the sequence of DAP10 can be found in NM_014266.

[0117] In some embodiments of the present invention, the intracellular domain may include a co-stimulatory domain and / or a signaling domain. For example, the intracellular domain may include the signal transduction domains of protein molecules such as 4-1BB, CD28, OX40, ICOS, CD3 zeta, and DAP 10. For example, the sequence of 4-1BB can be found in NM_001561, the sequence of CD28 in NM_006139, the sequence of OX40 in NM_003327, the sequence of ICOS in NM_012092, the sequence of CD3 zeta in NM_198053, and the sequence of DAP 10 in NM_014266. In a specific embodiment of the present invention, the intracellular domain includes 4-1BB and CD3 zeta sequentially from the N-terminus to the C-terminus.

[0118] In some embodiments of the present invention, the extracellular domain further includes a hinge region selected from CD8.

[0119] In some embodiments of the present invention, the extracellular domain further includes a signal peptide (SP), which is selected from CD8 SP.

[0120] A third aspect of the present invention provides biomaterials related to the anti-CD4 antibody or the polypeptide described above, said biomaterials comprising one or more of the following:

[0121] a) Nucleotides encoding the heavy chain variable region and / or light chain variable region of the anti-CD4 antibody as described above, or nucleotides encoding the polypeptide as described above;

[0122] b) A construct containing the nucleotides described in a);

[0123] c) A host cell containing the nucleotides described in a), or a host cell containing the construct described in b).

[0124] In some embodiments of the present invention, in a), the nucleotide sequence encoding the heavy chain variable region of the anti-CD4 antibody as described above includes the sequence shown in SEQ ID No. 16.

[0125] Gaggtgcagctgcagcagagcgggccagagctggtgaagccaggagacagcgtgaagatgagctgtaaggccagcggctacacattcaccgactatttcatggactgggtgaagcagagccatggaaaaagcctggaatggatcggctacatttaccccaataacggcggaaccaacaa caaccagaagttcaaaggcaaggccacactgaccgtggacaagagcagcagcaccgcctacatggaactgcacagcctgaccagcgaggacagcgccgtgtactactgcgccagaagaggatacgacgagggatttgcctactggggccagggaacactggtgaccgtgagctcc(SEQ ID No.16)

[0126] In some embodiments of the present invention, in a), the nucleotide sequence encoding the light chain variable region of the anti-CD4 antibody as described above includes the sequence shown in SEQ ID No. 17.

[0127] Gacattctgctgacccagagccccgccatcctgagcgtgagccccggagagagagtgagctttagctgcagagccagccagatcatcggaaccagcatcctgtggtatcagcagagaaccaacggcagccccagactgctgatcaaatacgccagtgagagc atcagcggaatccccagcagattcagcggaagcggcagcggcaccgacttcaccctgagcatcaacagcgtggagagcgaggacatcgccgactactactgccagcagagcaacacctggccctttaccttcggaggcggcacaaagctggaaatgaag(SEQ ID No.17)

[0128] In some embodiments of the present invention, in b), the construct can be constructed by inserting the nucleotides into the multiple cloning site of the expression vector. The expression vector can be transformed, transduced, or transfected into host cells, allowing the genetic material elements it carries to be expressed within the host cells. The construct can be a viral vector or a non-viral vector. For example, non-viral vectors include: plasmids, phage particles, 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. In addition, the vector may also contain a replication initiation site. The carrier may also include components that facilitate its entry into the cell, including but not limited to viral particles, liposomes, or protein coats.

[0129] In some embodiments of the present invention, in c), the host cell is a construct or genome introduced above that integrates exogenous nucleotides described above. Any cell suitable for expression via an expression vector can serve as a host cell. For example, the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. The host cell 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. In a preferred embodiment of this application, the host cell is selected from one or both of T cells and NK cells.

[0130] A fourth aspect of the present invention provides an antibody conjugate comprising an anti-CD4 antibody as described above and a label or separation aid.

[0131] In some embodiments of the present invention, the marker is selected from one or more of biotin, fluorescein, and horseradish peroxidase.

[0132] In some embodiments of the present invention, the separation aid is selected from one or more of magnetic beads and agarose beads. In a preferred embodiment of the present invention, the marker is magnetic beads.

[0133] In certain embodiments of the present invention, the anti-CD4 antibody is conjugated with magnetic beads, and its preparation method includes the following steps:

[0134] (1) Carboxyl magnetic beads and the anti-CD4 antibody as described above were coupled in a buffer solution;

[0135] (2) Centrifuge, remove the supernatant, add BSA aqueous solution to seal the unreacted activated carboxyl groups on the surface of the magnetic beads;

[0136] (3) Centrifuge, collect the precipitate, wash, and resuspend in PBS preservation solution to obtain.

[0137] Preferably, the mass ratio of the carboxyl magnetic beads to the anti-CD4 antibody is (1-3):1.

[0138] Preferably, the concentration of the BSA aqueous solution is 10–30 mg / mL. More preferably, in a preferred embodiment, it is 20 mg / mL.

[0139] In some embodiments of the present invention, the method for preparing the cells includes transferring the construct to host cells. In some specific embodiments of the present invention, the transfer method may be electroporation or viral transfer.

[0140] A fifth aspect of the present invention provides a pharmaceutical composition comprising an antibody as described above and / or a biological material as described above and / or a cell as described above, and a pharmaceutically acceptable carrier.

[0141] In some embodiments of the present invention, 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, one or more of water, saline, phosphate buffer, levulose, glycerol, ethanol, and other similar substances, as well as combinations of the foregoing.

[0142] In some embodiments of the present invention, 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.

[0143] A sixth aspect of the present invention provides a product comprising the anti-CD4 antibody and / or the antibody conjugate described above.

[0144] In some embodiments of the present invention, the product is selected from reagent kits, chips, and membrane strips.

[0145] In some embodiments of the present invention, the product is typically targeted at the CD4 antigen, using the CD4 antigen as a biomarker for diagnosis. The product may also include a marker for anti-CD4 antibodies, which can typically be used to label anti-CD4 antibodies. The types of markers that can be used include, but are not limited to, one or more of biotin, fluorescein, horseradish peroxidase, magnetic beads, and agarose beads.

[0146] The seventh aspect of the present invention provides the use of the antibody described above, or the polypeptide described above, or the biological material described above, or the pharmaceutical composition described above, or the antibody conjugate described above, in the preparation of at least one of the following products:

[0147] Products for in vitro CD4 detection;

[0148] Products for in vitro enrichment of CD4 T cells;

[0149] In the preparation or screening of preventive and / or therapeutic drugs.

[0150] The eighth aspect of the present invention provides a method for in vitro enrichment of CD4 T cells, wherein the antibody and / or antibody-drug conjugate as described above are added to peripheral blood mononuclear cells, so that CD4 T cells bind to the antibody and / or antibody-drug conjugate, and are then separated to obtain the CD4 T cells.

[0151] In some embodiments of the present invention, the ratio of peripheral blood mononuclear cells to antibodies is 16 × 10⁻⁶. 6 Cells: (0.5~1.5)μg.

[0152] 1) The antibody conjugate and PBMC were incubated in flow cytometry buffer to obtain an incubation solution;

[0153] 2) Over-select the column, discard the cells that flow down, rinse the column with flow cytometry buffer, and collect the liquid that flows down to obtain the CD4 T cells.

[0154] In some embodiments of the present invention, the flow cytometry buffer is a 1×PBS solution containing 1.8%–2.2% FBS and 1.8 mM–2.2 mM EDTA. Preferably, the flow cytometry buffer is a 1×PBS solution containing 2% FBS and 2 mM EDTA.

[0155] Using the antibody of this invention to sort and enrich PBMCs, the proportion of CD4 T cells was 31.9% before sorting and enrichment. After sorting and enrichment, the proportion of CD4 T cells increased significantly, exceeding 90%, while the CD4 T cells in the waste liquid were only 0.64%, indicating that the anti-CD4 antibody of this invention can efficiently enrich high-purity CD4 T cells from PBMCs. Furthermore, using CD4 T cells enriched after lentiviral infection, CAR-T cells specifically targeting CD22 (CD4+) were obtained. + CD22-CAR-T cells, after infection, can be activated and / or stimulated under normal culture conditions to rapidly proliferate and stably express anti-CD22 antibodies. In other words, CD22-specific CAR-T cells can express CARs containing anti-CD22 antibodies on the surface of T lymphocytes, thereby guiding T lymphocytes to act on cells expressing CD22 antigens (such as tumor cells). The effect can be killing cells expressing CD22 antigens, etc.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] In the following embodiments of this application:

[0160] 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.

[0161] The flow cytometry buffer was a 1×PBS solution containing 2% FBS and 2mM EDTA.

[0162] Example 1: Construction of anti-CD4 antibody expression vector

[0163] In this embodiment, an anti-CD4 (CD4-1) antibody expression vector was constructed. This includes the following:

[0164] The amino acid sequence of the heavy chain variable region of the anti-CD4 antibody includes the sequence shown in SEQ ID No. 4.

[0165] EVQLQQSGPELVKPGDSVKMSCKAS MDWVKQSHGKSLEWIGY NNNQKFKGKATLTVDKSSSTAYMELHSLTSEDSAVYYC WGQGTLVTVSS (SEQ ID No. 4, the bold and underlined parts are the heavy chain variable regions, namely CDR-H1, CDR-H2 and CDR-H3 in sequence)

[0166] The amino acid sequence of the light chain variable region of the anti-CD4 antibody includes the sequence shown in SEQ ID No. 7.

[0167] DILLTQSPAILSVSPGERVSFSCRAS ILWYQQRTNGSPRLLIK ESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYC FGGGTKLEMK (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)

[0168] 1.1 Obtaining the target fragment of antibody CD4-1

[0169] The target fragment of the antibody CD4-1 was obtained through two rounds of PCR amplification. The expression vector for the anti-CD4 antibody was developed by Shanghai Baiying Biotechnology Co., Ltd.

[0170] The primers, reaction system, and reaction conditions for the first round of PCR are shown in Table 1. The first round of PCR products were obtained.

[0171] Table 1

[0172]

[0173]

[0174] The primers, reaction system, and reaction conditions for the second round of PCR are shown in Table 2, and the CD4-1 target fragment was obtained.

[0175] Table 2

[0176]

[0177] Whole-genome synthetic nucleotide sequence

[0178]

[0179] 1.2 Construction of recombinant expression vectors

[0180] The CD4-1 target fragment from step 1.1 of the gel recovery process (refer to the Axygen product manual) was subjected to homologous recombination. The reagents and reaction conditions used are shown in Table 3.

[0181] Table 3

[0182]

[0183] The CD4-1 target fragment and the linearized vector were ligated to obtain the recombinant expression vector.

[0184] 1.3 Colony Screening Experiment

[0185] 1.3.1 Pick a single colony from the overnight plate;

[0186] 1.3.2 The colonies obtained in step 1.3.1 were amplified by PCR using primers Bl-CMV-F and Bl-SEQ-R;

[0187] Bl-CMV-F: CGCAAATGGGCGGTAGGCGTG (SEQ ID No. 21)

[0188] Bl-SEQ-R: AGCGTAAAAGGAGCAACATAGT (SEQ ID No. 22)

[0189] 1.3.3 Electrophoresis identification of positive clones;

[0190] 1.3.4 Four positive bacteria were randomly selected, and the positive bacterial solutions were sequenced using Bl-CMV-F / Bl-SEQ-R primers.

[0191] 1.3.5 Select positive bacterial cultures with correctly sequenced clones, expand the culture, extract low endotoxin plasmids, and verify with sequencing.

[0192] Example 2: Expression and purification of anti-CD4 antibody

[0193] In this embodiment, prokaryotic expression and purification of anti-CD4-1 antibody were performed, and the affinity of the anti-CD4-1 antibody and its recognition of cell surface CD4 antigen were detected. The steps included:

[0194] 2.1 Prokaryotic expression of anti-CD4-1 antibody

[0195] 2.2.1 Culture of CHO cells

[0196] 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.

[0197] 2.2.2 Transfection and Expression

[0198] 1) Take 145M of the cells obtained in step 2.2.1 of this embodiment, centrifuge to remove the supernatant, and retain the precipitate.

[0199] 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 (concentration 500 ng / μL) obtained in Example 1 to obtain a cell plasmid suspension.

[0200] 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.

[0201] 4) After electroporation, aliquot the cells from the electroporation tube into a shaker containing 20 mL of culture medium and incubate for 40 min.

[0202] 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.

[0203] 2.2 Antibody Purification

[0204] Experimental method: Protein A affinity chromatography column purification

[0205] (1) Equilibration chromatography column: l×PBS, flow rate l mL / min, 20 mL;

[0206] (2) Sample loading: flow rate 1 mL / min;

[0207] (3) Washing: 1×PBS, flow rate 1 mL / min, 20 mL;

[0208] (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;

[0209] (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 anti-CD4-1 antibody.

[0210] 2.3 Basic Antibody Quality Control

[0211] 1) Concentration detection;

[0212] 2) Purity detection (SEC-HPLC);

[0213] Experimental materials: High performance liquid chromatograph, gel chromatography column, deionized water, mobile phase (Na2HPO4·12H2O, NaH2PO4·2H2O, NaCl);

[0214] Experimental methods:

[0215] SEC experiments were performed using a high-performance liquid chromatograph LC-20AT and a gel chromatography column. The experimental conditions are shown in Table 4.

[0216] Table 4

[0217] Flow rate 1mL / min Injection volume 20μL Column temperature 35℃ Detection wavelength 214nm, 280nm Collection time 15min

[0218] 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.

[0219] The results showed that the antibody purity was >90%.

[0220] 3. Affinity assay of anti-CD4-1 antibody

[0221] Method: 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.

[0222] Figure 1 This is a graph showing the affinity assay results of the CD4-1 antibody in this embodiment.

[0223] from Figure 1 It can be seen that the EC50 of the affinity of the anti-CD4-1 antibody to the CD4 antigen is 0.1008 μg / mL.

[0224] 4. Anti-CD4-1 antibodies recognize CD4 antigens on the cell surface.

[0225] Anti-CD4-1, anti-CD4-3, 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 were used to recognize the CD4 antigen on the cell surface.

[0226] 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, and 1 μg of CD4-3 antibody was added. 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. 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. A control group without anti-CD4-3 antibody was used.

[0227] Mix K562-CD4 cells and K562 cells, and take 3×10 5 A mixture of cells was 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 cells were resuspended in 100 μL of flow cytometry buffer, and 1 μg of anti-CD4-1 antibody was added. The cells were incubated on ice for 30 min, washed twice with flow cytometry buffer, and then resuspended again in 100 μL of flow cytometry buffer. 1 μg of APC-labeled goat anti-mouse IgG secondary antibody (purchased from BioLegend, corresponding to CD4-1) was added, and the cells were incubated on ice for 30 min. The cells were washed twice with flow cytometry buffer, and then resuspended in 300 μL of flow cytometry buffer. Flow cytometry analysis was performed. A control group without anti-CD4-1 antibody was used.

[0228] K562-CD4 cells and K562 cells (purchased from ATCC) were mixed, and 3 × 10⁻⁶ cells were taken. 5The mixed cells 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 cells were resuspended in 100 μL of flow cytometry buffer, and 1 μg of 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 were added respectively. The cells were incubated on ice for 30 min, washed twice with flow cytometry buffer, and resuspended in 100 μL of flow cytometry buffer. Then, 1 μg of APC-labeled goat anti-mouse IgG was added to the cells resuspended in the buffers containing anti-CD4-2 and anti-CD4-4 antibodies. Anti-CD4 antibodies (purchased from BioLegend, corresponding to CD4-2 and anti-CD4-4, respectively), anti-CD4-8, anti-CD4-6, anti-CD4-7, anti-CD4-9, anti-CD4-10, and anti-CD4-13 antibodies were added to 1 μg of APC-labeled goat anti-human IgG secondary antibody (purchased from BioLegend, corresponding to anti-CD4-8, anti-CD4-6, anti-CD4-7, anti-CD4-9, anti-CD4-10, and anti-CD4-13, respectively). The cells were incubated on ice for 30 min, washed twice with flow cytometry buffer, and then resuspended in 300 μL of flow cytometry buffer for analysis using flow cytometry. A blank control group was used without CD4 antibody.

[0229] CD4-3 was purchased from BioLegend, item number: 317402.

[0230] CD4-2 amino acid sequence:

[0231] Heavy chain:

[0232] EVKLQESGPELVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCAREKDNYATGAWFAYWGQGTTVTVSS(SEQ ID No. 23)

[0233] Light chain:

[0234] DIVMTQSPSSLAVSVGEKVTMICKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYRTFGGGTKLEIK(SEQ ID No.24)

[0235] CD4-4 amino acid sequence:

[0236] Heavy chain:

[0237] QVLLQQSGPELVKPGASVKMSCKASGYFFTDYVINWVKQRTGQGLEWIGEIYPGSGSSYYSEKFKGKATLTADKSSNTAYMQLSSLTSADSAVYFCARRGTGLGFAYWGQGTLVTVSS(SEQ ID No.25)

[0238] Light chain:

[0239] DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYVASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPPTFGGGTKLEIK(SEQ ID No.26)

[0240] CD4-6 amino acid sequence:

[0241] Heavy chain:

[0242] QVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSS(SEQ ID No.27)

[0243] Light chain:

[0244] DIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIK(SEQ ID No.28)

[0245] CD4-7 amino acid sequence:

[0246] Heavy chain:

[0247] QVQLQQSGAEVARPRASVKLSCKASGYTFTSNGINWVKYRTGQGLEWIGEIYPRSGNIFYNERFEGKATLTADKSSTTAYMELRSLTSEDSAVYFCARRVPYFDHWGQGTTLTVSS(SEQ ID No.29)

[0248] Light chain:

[0249] DVVMTQNLRFLPVSAGDRVAMTCKASQSVGNNVAWYQRKPGQSPKLLIYYASNRYTGVPDRFTGSGSGTDFTFTISSVQVEDLAVYFCQQHYSSPFTFGTGTKLEIK(SEQ ID No.30)

[0250] CD4-8 amino acid sequence:

[0251] Heavy chain:

[0252] QVQLQEAGPGLVKPSETLSLTCSVSGGSISGDYYWFWIRQSPGKGLEWIGYIYGSGGGTNYNPSLNNRVSISIDTSKNLFSLKLRSVTAADTAVYYCASNILKYLHWLLYWGQGVLVTVSS(SEQ ID No.31)

[0253] Light chain:

[0254] SYELSQPRSVSVSPGQTAGFTCGGDNVGRKSVQWYQQKPPQAPVLVIYADSERPSGIPARFSGSNSGNTATLTISGVEAGDEADYYCQVWDSTADHWVFGGGTRLTVL(SEQ ID No.32)

[0255] CD4-9 amino acid sequence:

[0256] Heavy chain:

[0257] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVINWFDPWGQGTLVTVSS(SEQ ID No.33)

[0258] Light chain:

[0259] DIQMTQSPSSVSASVGDRVTITCRASQDISSWLAWYQHKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPYTFGQGTKLEIK(SEQ ID No.34)

[0260] CD4-10 amino acid sequence:

[0261] Heavy chain:

[0262] EEQLVESGGGLVKPGGSLRLSCAASGFSFSDCRMYWVRQAPGKGLEWIGVISVKSENYGANYAESVRGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCSASYYRYDVGAWFAYWGQGTLVTVSS(SEQ ID No.35)

[0263] Light chain:

[0264] DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYIYWYQQKPGQPPKLLIYLASILESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRELPWTFGQGTKVEIK(SEQ ID No. 36)

[0265] CD4-13 amino acid sequence:

[0266] Heavy chain:

[0267] QVQLVQSGAEVKKPGASVKVSCKASGYTFTAYVISWVRQAPGQGLEWMGEIYPGSGSSYYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSGDGSRFVYWGQGTLVTVSS(SEQ ID No. 37)

[0268] Light chain:

[0269] DIVMTQSPDSLAVSLGERATINCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYVASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSLQDPPTFGGGTKVEIK(SEQ ID No.38)

[0270] Figure 2A This is a diagram showing the results of the anti-CD4-1 antibody recognizing the CD4 antigen on the cell surface in this embodiment.

[0271] Figure 2B This is a diagram showing the results of the anti-CD4-3 antibody recognizing the CD4 antigen on the cell surface in this embodiment.

[0272] Figure 2C 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.

[0273] Figure 2DThis 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.

[0274] from Figure 2A , 2B As can be seen from 2C and 2D, the anti-CD4-1 antibody of the present invention can specifically recognize the CD4 antigen on the cell surface.

[0275] Example 3 Antibody conjugation with magnetic beads

[0276] In this embodiment, the purified anti-CD4-1 antibody obtained in step 2.2 of Example 2 is conjugated to magnetic nanoparticles to obtain an antibody conjugate (labeled as anti-CD4-1 magnetic beads). The experimental steps are as follows:

[0277] 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.

[0278] 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.

[0279] 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.

[0280] 4) Block the reaction with an aqueous solution containing 20 mg / mL BSA for 1 h, shake the reaction in a shaker at 37 °C, then centrifuge at 20000 g for 30 min, remove the supernatant, and obtain the precipitate.

[0281] 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.

[0282] 6) Centrifuge at 20000g for 30min, remove the supernatant, resuspend in PBS preservation solution to 500μL, filter through a 0.22μm filter membrane to obtain the antibody conjugate (labeled as anti-CD4-1 magnetic beads).

[0283] Example 4: Sorting and enrichment of PBMCs with antibodies

[0284] In this embodiment, the anti-CD4-1 magnetic beads obtained in Example 3 are used to sort and enrich PBMCs. The experimental steps are as follows:

[0285] 1) Resuscitate PBMCs and incubate them at 37°C and 5% CO2 for 2 hours;

[0286] 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.

[0287] 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 anti-CD4-1 magnetic beads were added. After mixing, the cells were incubated at room temperature for 15 min.

[0288] 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.

[0289] 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.

[0290] Example 5

[0291] In this embodiment, the efficiency and purity of anti-CD4-1 magnetic beads in sorting and enriching CD4 T cells from PBMCs were tested.

[0292] 1) Take 3 × 10⁻⁶ cells each from the cell waste fluid obtained in step 4) of Example 4, 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.

[0293] 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;

[0294] 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.

[0295] Meanwhile, using anti-CD4-2 antibody, anti-CD4-4 antibody, anti-CD4-8 antibody, anti-CD4-6 antibody, anti-CD4-7 antibody, anti-CD4-9 antibody, anti-CD4-10 antibody, and anti-CD4-13 antibody as controls, anti-CD4-2 magnetic beads, anti-CD4-4 magnetic beads, anti-CD4-8 magnetic beads, anti-CD4-6 magnetic beads, anti-CD4-7 magnetic beads, anti-CD4-9 magnetic beads, anti-CD4-10 magnetic beads, and anti-CD4-13 magnetic beads were prepared using the same method as for anti-CD4-1 antibody. CD4 T cells were then sorted and enriched. The proportion of CD4 T cells in PBMCs before enrichment and sorting, cell waste liquid, and enriched cells was detected by flow cytometry.

[0296] Figure 3A This figure shows the proportion of CD4T cells in the cells before and after enrichment using anti-CD4-1 magnetic beads in this embodiment.

[0297] Depend on Figure 3A 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 anti-CD4-1 magnetic beads can enrich high-purity CD4 T cells from PBMC.

[0298] Figure 3B This figure shows the proportion of CD4T cells in the cell waste fluid after sorting and enrichment with anti-CD4-1 magnetic beads in this embodiment.

[0299] Depend on Figure 3B It can be seen that 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 anti-CD4-1 magnetic beads can efficiently enrich CD4 T cells from PBMCs.

[0300] Figure 4A This figure shows the proportion of CD4T cells in the cells before and after enrichment using anti-CD4-3 magnetic beads in this embodiment.

[0301] Depend on Figure 4A It can be seen that before sorting and enrichment with anti-CD4-3 magnetic beads, the proportion of CD4 T cells in PBMCs was 33.7%; after sorting and enrichment, the proportion of CD4 T cells decreased to 23%.

[0302] Figure 4B This figure shows the proportion of CD4T cells in the cell waste fluid after sorting and enrichment with anti-CD4-3 magnetic beads in this embodiment.

[0303] Depend on Figure 4B It can be seen that after sorting and enriching with anti-CD4-3 magnetic beads, the proportion of CD4 T cells in the cell waste liquid is relatively high, reaching 25.8%.

[0304] Figure 5A The figure shows the proportion of CD4T cells in the cells before and after sorting and enrichment using anti-CD4-2 magnetic beads, anti-CD4-4 magnetic beads, and anti-CD4-8 magnetic beads, respectively, in this embodiment.

[0305] Depend on Figure 5A It can be seen that before sorting and enrichment using anti-CD4-2 magnetic beads, anti-CD4-4 magnetic beads, and anti-CD4-8 magnetic beads, 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.

[0306] Figure 5B The figure shows the proportion of CD4T cells in the cell waste fluid after sorting and enrichment using anti-CD4-2 magnetic beads, anti-CD4-4 magnetic beads, and anti-CD4-8 magnetic beads, respectively, in this embodiment.

[0307] Depend on Figure 5B It can be seen that after sorting and enriching with anti-CD4-2 magnetic beads, anti-CD4-4 magnetic beads and anti-CD4-8 magnetic beads, the proportions of CD4 T cells in the cell waste liquid were 24.1%, 34.8% and 4.52%, respectively.

[0308] Figure 6A The figure shows the proportion of CD4T cells in the cells before and after sorting and enrichment using anti-CD4-6 magnetic beads, anti-CD4-7 magnetic beads, anti-CD4-9 magnetic beads, anti-CD4-10 magnetic beads, and anti-CD4-13 magnetic beads, respectively, in this embodiment.

[0309] Depend on Figure 6A It can be seen that before sorting and enrichment with anti-CD4-6 magnetic beads, anti-CD4-7 magnetic beads, anti-CD4-9 magnetic beads, anti-CD4-10 magnetic beads, and anti-CD4-13 magnetic beads, 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.

[0310] Figure 6B The figure shows the proportion of CD4T cells in the cell waste fluid after sorting and enrichment using anti-CD4-6 magnetic beads, anti-CD4-7 magnetic beads, anti-CD4-9 magnetic beads, anti-CD4-10 magnetic beads, and anti-CD4-13 magnetic beads, respectively, in this embodiment.

[0311] Depend on Figure 6B It can be seen that after sorting and enriching with anti-CD4-6 magnetic beads, anti-CD4-7 magnetic beads, anti-CD4-9 magnetic beads, anti-CD4-10 magnetic beads and anti-CD4-13 magnetic beads, the proportions of CD4 T cells in the cell waste liquid were 25.2%, 5.35%, 27.2%, 12.3% and 9.68%, respectively.

[0312] In summary, the sorting and enrichment of PBMCs using the anti-CD4-1 antibody of the present invention can obtain high-purity CD4 T cells. However, when PBMCs are sorted and enriched using anti-CD4-2 antibody, anti-CD4-4 antibody, anti-CD4-8 antibody, anti-CD4-6 antibody, anti-CD4-7 antibody, anti-CD4-9 antibody, anti-CD4-10 antibody, and anti-CD4-13 antibody, 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.

[0313] Example 6 Lentiviral Packaging

[0314] This embodiment describes the packaging of lentiviruses. It includes the following steps:

[0315] 6.1 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.

[0316] 6.2 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;

[0317] 6.3 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;

[0318] 6.4 Add the PEI mixture to the DNA mixture obtained in step 6.2 of this embodiment. Vortex mix or gently mix immediately after addition. Incubate at 25°C for 20 min to obtain the transfection complex.

[0319] 6.5 Add 4 mL of the transfection complex obtained in step 6.4 of this embodiment to a 15 cm culture dish containing 25 mL of DMEM medium. After 4 hours, replace with fresh medium.

[0320] 6.6 After 48 hours, the viral supernatant was collected to obtain lentivirus.

[0321] Example 7 Lentiviral Concentration

[0322] This embodiment involves lentivirus concentration. The steps include:

[0323] The viral supernatant prepared in Example 6 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 tube. The tube was then incubated overnight at 4 °C. The tube 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.

[0324] Example 8 Lentiviral titer detection

[0325] This embodiment performs lentivirus titer detection. The steps include:

[0326] 500 μL of Jurkat cells (1 × 10⁻⁶) 5 (10 cells) were seeded into 24-well culture plates to obtain Jurkat cell suspension;

[0327] The concentrated lentivirus suspension from Example 7 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.

[0328] Titer (TU / mL) = Cell number (10) 5 ) × Positive rate / Viral volume (mL).

[0329] Example 9 Lentiviral transduction of CD4 T cells

[0330] In this embodiment, the concentrated lentivirus suspension obtained in Example 7 is used to transduce the CD4 T cells sorted and enriched in Example 4. The steps include:

[0331] 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 enriched and sorted 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 7 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.

[0332] Example 10: Detection of Lentiviral Infection Efficiency

[0333] This embodiment describes the detection of lentiviral infection efficiency in the CD4 T cell cultures transduced with lentivirus from Example 9. The steps include:

[0334] 10.1. In Example 9, after lentivirus transduction of CD4 T cells, cell counts were performed at 5, 8, 11, and 14 days after infection. Figure 7 (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.

[0335] 10.2. 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 8A , 8B 8C and 8D.

[0336] Figure 7 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.

[0337] from Figure 7 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.

[0338] Figure 8A , 8B Figures 8C and 8D show the infection rates of CD4 T cells infected with the virus on days 5, 8, 11, and 14 in this embodiment.

[0339] from Figure 8A , 8B As shown in 8C and 8D, the chimeric antigen receptor can be expressed relatively stably on days 5, 8, 11 and 14 after CD4 T cell infection with the virus, with an infection rate of over 77.3%.

[0340] Example 11 In vitro toxicity test

[0341] In this embodiment, in vitro toxicity experiments were conducted using CAR-T cells specifically targeting CD22 prepared in Example 9, including the following steps:

[0342] 11.1 Target cell inoculation

[0343] With 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 it into a 96-well plate;

[0344] 11.2 Effector cell seeding

[0345] CAR-T cells specifically targeting CD22 prepared in Example 9 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 11.1 of this example at effector-to-target ratios of 0.3:1, 1:1 and 3:1.

[0346] 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:

[0347] Experimental group (CD4-1 CD22-CAR): CAR-T cells + target cells;

[0348] Control group (Control CD4 T): Control T cells + target cells;

[0349] 11.3 After co-culturing effector cells and target cells for 18 hours, Steady-Glo was used. The detection was performed using a luciferase assay kit. For specific detection steps, please refer to Steady-Glo. The instructions for the luciferase assay kit are as follows: Figure 9A and 9B As shown.

[0350] from Figure 9A and 9BIt is known that the CD22-specific 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 CD22-specific CAR-T cells constructed in this invention have high specificity. The CD22-specific CAR-T cells can specifically kill CD22-positive cells, and the killing effect is positively correlated with the effector-to-target ratio.

[0351] Example 12 In vitro cytokine release experiment

[0352] In this embodiment, an in vitro cytokine release experiment was conducted using CAR-T cells specifically targeting CD22 prepared in Example 9, including the following steps:

[0353] 12.1 Cell Culture Supernatant

[0354] The cell culture with an effect-to-target ratio of 1:1 in Example 11 was centrifuged at 400×g for 10 min to remove the precipitate. The cell culture supernatant was stored at -80℃ for testing. The cell culture supernatant was labeled K562-CD22-luci.

[0355] Meanwhile, CAR-T cell cultures alone and cultures of K562-luci and CAR-T cells were used as negative control groups.

[0356] 12.2 Reagent Preparation

[0357] 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.

[0358] 12.3 Preparation of Standards and Samples

[0359] 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.

[0360] Samples: Dilute the samples using 5% 1640 medium.

[0361] 12.4 Testing Procedures

[0362] (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.

[0363] (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;

[0364] (3) Add sample: Add 100 μL of cell culture supernatant to the sample well;

[0365] (4) Add detection antibody: Add 50 μL of diluted detection antibody (1:100 dilution) to each well;

[0366] (5) Incubation: Seal the plate with sealing film, shake at 300 rpm, and incubate at 25°C for 2 hours;

[0367] (6) Washing: Discard the liquid, add 300 μL of washing solution to each well and wash the plate 6 times;

[0368] (7) Enzyme incubation: Add 100 μL of diluted horseradish peroxidase-labeled streptavidin (1:100 dilution) to each well;

[0369] (8) Incubation: Seal the plate with a new sealing film, shake at 300 rpm, and incubate at 25°C for 45 min;

[0370] (9) Washing: Repeat step (6);

[0371] (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;

[0372] (11) Add stop solution: Add 100 μL of stop solution to each well and mix thoroughly;

[0373] (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.

[0374] IFN-γ factor secretion results as follows Figure 10 As shown.

[0375] from Figure 10 It was found that trace amounts of IFN-γ were detected in spontaneously generated cells, as well as in K562-luci and CAR-T cultures, while higher levels of IFN-γ were detected in K562-CD22-luci and CAR-T cultures. 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.

[0376] In summary, this invention prepares a highly efficient anti-CD4 antibody targeting the CD4 antigen. The antibody-conjugate (anti-CD4-1 magnetic beads) obtained by conjugating this anti-CD4 antibody with magnetic particles can efficiently enrich and sort 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 CD4 T cells in the waste liquid were only 0.64%, indicating that the CD4 of this invention can efficiently enrich high-purity CD4 T cells from PBMCs.

[0377] 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.

[0378] 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 an anti-CD4 antibody in the preparation of a product enriched from PBMCs for CD4 T cells in vitro, the anti-CD4 antibody comprising a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region being shown in SEQ ID No. 4 and the amino acid sequence of the light chain variable region being shown in SEQ ID No.

7.

2. The use as described in claim 1, characterized in that, The nucleotide sequence of the heavy chain variable region of the anti-CD4 antibody is shown in SEQ ID No. 16, and the nucleotide sequence of the light chain variable region is shown in SEQ ID No.

17.

3. The use as described in claim 1, characterized in that, The product comprises an antibody-drug conjugate, which comprises the anti-CD4 antibody and a marker or separation aid; And / or, the products are selected from kits, chips, and membrane strips.

4. The use as described in claim 3, characterized in that, The marker is selected from one or more of biotin, fluorescein, and horseradish peroxidase; And / or, the separation aid is selected from magnetic beads and agarose beads.

5. A method for in vitro enrichment of CD4 T cells, characterized in that, An antibody-drug conjugate containing anti-CD4 antibody is added to peripheral blood mononuclear cells, causing CD4 T cells to bind to the antibody-drug conjugate. The cells are then separated to obtain the CD4 T cells. The anti-CD4 antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 4 and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

7.

6. The method as described in claim 5, characterized in that, The nucleotide sequence of the heavy chain variable region of the anti-CD4 antibody is shown in SEQ ID No. 16, and the nucleotide sequence of the light chain variable region is shown in SEQ ID No. 17.

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