Anti-cd4 antibodies or antigen-binding fragments thereof and uses thereof

Abstract

This invention provides an anti-CD4 antibody or its antigen-binding fragment and its applications. The anti-CD4 antibody or its antigen-binding fragment includes a CDR selected from at least one of the following: a heavy chain variable region CDR: a CDR of the heavy chain variable region of the amino acid sequence shown in any of SEQ ID NO: 31, 33, 35, 37, 39, determined by any numbering scheme; and a light chain variable region CDR: a CDR of the light chain variable region of the amino acid sequence shown in any of SEQ ID NO: 32, 34, 36, 38, 40, determined by any numbering scheme. The anti-CD4 antibody or its antigen-binding fragment of this invention can specifically bind to CD4 molecules or CD4+. + The cells produce high signal intensity, which helps to accurately detect CD4 molecules or CD4+. + Cellular, highly accurate, for CD4 + It has important application value in the diagnosis, prevention, or treatment of lymphocyte-related diseases.

Description

Technical Field

[0001] This invention relates to the field of biology. Specifically, this invention relates to anti-CD4 antibodies or their antigen-binding fragments and their applications. Background Technology

[0002] CD4 is a membrane molecule and a receptor on the surface of T lymphocytes, also known as the T-helper cell co-receptor. The CD4 molecule consists of four exons and encodes a membrane glycoprotein of approximately 55 kDa, which plays an important role in lymphocyte development, recognition, and immune responses.

[0003] CD4 molecules are primarily expressed on the surface of T lymphocytes and play a crucial role in the T lymphocyte immune response. The main function of CD4 molecules is to bind to MHC-II molecules, promoting the interaction between T lymphocytes and antigen-presenting cells, thereby initiating the immune response. CD4 molecules can also enhance the immune response of B lymphocytes and participate in regulating the immune response of T cells.

[0004] CD4 expression levels also play an important role in many diseases, such as HIV, rheumatoid arthritis, and psoriasis, among other autoimmune diseases. HIV can infect and destroy CD4 cells. + T lymphocytes contribute to damage and dysfunction of the immune system. The occurrence and development of diseases such as rheumatoid arthritis and psoriasis are also related to CD4+. + The abnormal proliferation and activation state of T lymphocytes are related.

[0005] CD4 antibody is a substance used to detect CD4. + Monoclonal antibodies against T lymphocytes are commonly used reagents in immunological research and clinical diagnosis. CD4 + T lymphocytes play an important role in the diagnosis of immune-related diseases, such as HIV and autoimmune diseases. CD4 antibodies can be used to detect CD4. + The number and proportion of T lymphocytes help in the diagnosis and monitoring of disease progression and treatment effectiveness. CD4 + T lymphocytes play a crucial role in the immune response, therefore CD4 antibodies can be used to study the mechanisms and regulation of immune responses. Furthermore, the development of novel CD4 antibodies can provide more options and possibilities for treating immune-related diseases. For example, some bispecific antibodies can utilize the binding of CD4 to other molecules to enhance therapeutic effects. Therefore, the development of CD4 antibodies is of great significance for immunological research and clinical diagnosis, and can support the development of novel therapeutic strategies.

[0006] The development process of CD4 antibodies mainly includes the following steps: Preparation of the primary immunogen: The peripheral region of CD4 is prepared as an immunogen using biotechnology, generally through recombinant proteins, peptides, or cells expressing CD4; Immunization of animals: Suitable animals are selected for immunization, typically mice or rabbits. The immunogen is injected into the immunized animals to stimulate the production of CD4-specific antibodies; Screening and identification: CD4-specific antibodies with specificity and affinity are screened using methods such as ELISA and cell-mediated immunization. Further identification and evaluation of candidate antibodies are performed, including affinity assays and flow cytometry analysis; Cloning, antibody optimization, and improvement.

[0007] Although several CD4 antibodies have been developed, some shortcomings remain, mainly including the following: 1) Specificity issues: Some traditional CD4 antibodies may exhibit cross-reactivity, meaning they can bind to other cell surface molecules, producing false positive signals. This can lead to inaccurate CD4 antibody responses. + 1) Inaccurate results for T lymphocyte count and proportion. 2) Affinity issues: Some CD4 antibodies have low affinity, which may lead to inaccurate detection of CD4. + 3) Insufficient T lymphocyte count or large error: Some CD4 antibodies may only be applicable to specific sample types or CD4 counts of certain species. + T lymphocyte detection is limited in its applicability to various sample types and species. 4) Cost issues: Some CD4 antibodies are expensive, which may limit their application in clinical and research settings.

[0008] Therefore, CD4 antibodies still require further research. Summary of the Invention

[0009] The present invention aims to at least partially address one of the technical problems existing in the prior art. To this end, the present invention proposes a CD4 anti-CD4 antibody or its antigen-binding fragment, which can specifically bind to CD4 molecules or CD4... + The cells produce high signal intensity, which helps to accurately detect CD4 molecules or CD4+. + Cellular, highly accurate, for CD4 + It has important application value in the diagnosis, prevention, or treatment of lymphocyte-related diseases.

[0010] In one aspect of the invention, an anti-CD4 antibody or its antigen-binding fragment is provided. According to embodiments of the invention, the anti-CD4 antibody or its antigen-binding fragment comprises a CDR selected from at least one of the following: a heavy chain variable region CDR: a CDR of the heavy chain variable region of the amino acid sequence shown in any of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, and SEQ ID NO:39, determined by any numbering scheme; a light chain variable region CDR: a CDR of the light chain variable region of the amino acid sequence shown in any of SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, and SEQ ID NO:40, determined by any numbering scheme; wherein the numbering scheme is selected from Kabat, Chothia, Contact, and IMGT. The anti-CD4 antibody or its antigen-binding fragment of this application can specifically bind to CD4 molecules or CD4 + The cells produce high signal intensity, which helps to accurately detect CD4 molecules or CD4+. + Cellular, highly accurate, for CD4 + It has important application value in the diagnosis, prevention, or treatment of lymphocyte-related diseases.

[0011] In another aspect of the invention, a recombinant protein is proposed. According to an embodiment of the invention, the recombinant cell comprises: the aforementioned anti-CD4 antibody or its antigen-binding fragment.

[0012] In another aspect of the invention, a nucleic acid molecule is provided. According to embodiments of the invention, the nucleic acid molecule encodes the aforementioned anti-CD4 antibody or its antigen-binding fragment, or the aforementioned recombinant protein.

[0013] In another aspect, the present invention provides an expression vector. According to an embodiment of the invention, the expression vector carries the aforementioned nucleic acid molecule.

[0014] In another aspect of the invention, a recombinant cell is provided. According to embodiments of the invention, the recombinant cell carries at least one of the following: the aforementioned anti-CD4 antibody or its antigen-binding fragment, the expressed protein, the nucleic acid molecule, and the expression vector.

[0015] In another aspect of the invention, a pharmaceutical composition is provided. According to embodiments of the invention, the pharmaceutical composition comprises at least one of the following: the aforementioned anti-CD4 antibody or its antigen-binding fragment, the recombinant protein, the nucleic acid molecule, the expression vector, and the recombinant cell.

[0016] In another aspect of the invention, a conjugate is provided. According to an embodiment of the invention, the conjugate comprises: the aforementioned anti-CD4 antibody or its antigen-binding fragment; and a conjugation portion linked to the anti-CD4 antibody or its antigen-binding fragment or recombinant protein.

[0017] In another aspect of the invention, a kit is provided. According to embodiments of the invention, the kit comprises at least one of the following: the aforementioned anti-CD4 antibody or its antigen-binding fragment, the recombinant protein, the nucleic acid molecule, the expression vector, and the recombinant cells.

[0018] In another aspect of the invention, a method for detecting CD4 molecules or detecting cells expressing CD4 molecules is provided. According to an embodiment of the invention, the method involves contacting the aforementioned anti-CD4 antibody or its antigen-binding fragment, the recombinant protein, the conjugate, or the kit with the sample to be tested to form an immune complex.

[0019] In another aspect of the invention, the invention provides for the use of the aforementioned anti-CD4 antibody or its antigen-binding fragment, the recombinant protein, the pharmaceutical composition, or the conjugate in the preparation of a medicament for the prevention and / or treatment of CD4. + Lymphocyte-related diseases.

[0020] In another aspect of the invention, the invention provides for the use of the anti-CD4 antibody or its antigen-binding fragment, the recombinant protein, the conjugate, or the kit in the preparation of a product for detecting CD4 molecules or cells expressing CD4 molecules.

[0021] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0022] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0023] Figure 1 A graph showing the purification results of a recombinant polypeptide according to an embodiment of the present invention is displayed;

[0024] Figure 2 The figure shows an analysis diagram of hCD4 immune serum titer detection according to an embodiment of the present invention;

[0025] Figure 3 The diagram shows the purification results of a positive antibody according to an embodiment of the present invention;

[0026] Figure 4 The diagram shows a positive clone affinity ELISA assay according to an embodiment of the present invention;

[0027] Figure 5 An antibody flow cytometry assay analysis diagram is shown according to an embodiment of the present invention. Detailed Implementation

[0028] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0029] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0030] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0031] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0032] In this paper, the term "fragment" refers to a target protein or polypeptide, and a target protein or polypeptide that has been truncated at an N-terminus (N-terminus) or C-terminus (C-terminus) and / or internally deleted.

[0033] In this paper, the term "knob into hole structure" refers to the formation of a button (hole) mutation in the CH3 region of the constant region of the antibody heavy chain, which facilitates heavy chain interlocking and the formation of a heterodimer. For example, this can be achieved by mutating the amino acids in the CH3 domain of the constant region of the human IgG1 heavy chain (T366S, L368A, Y407V, Y349C mutation in one chain, i.e., "hole"; and T366W, S354C mutation in the other chain, i.e., "knob").

[0034] In this paper, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including: Needleman et al. (1970) J. Mol. Biol. 48: 443, a homology alignment algorithm; Smith et al. (1981) Adv. Appl. Math. 2: 482, a local homology algorithm; Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444, a similarity search method; and the Smith-Waterman algorithm (Meth. Mol. Biol). .70:173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J.Mol.Biol. 215:403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0035] In this paper, the term "at least 80% homology" refers to a homology of at least 80% with each reference sequence, which may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0036] In this document, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule to host cells and / or between host cells. The expression vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The expression vector also includes vectors having multiple of the aforementioned functions. The expression vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the expression vector, the expression vector can produce the desired expression product.

[0037] In this document, the term "recombinant cell" generally refers to a cell in which the genetic material of a host cell is modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of this invention and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this invention include immortalized hybridoma cells, NS / O myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0038] In this document, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely chopped solid carrier, or both.

[0039] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients, except those that are incompatible with the compounds of the present invention, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0040] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The anti-CD4 antibody or its antigen-binding fragment, recombinant protein, multispecific antibody, or pharmaceutical composition of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, administration is performed via intravenous or subcutaneous injection.

[0041] In this document, the term "treatment" refers to the administration of a drug or compound to an individual to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in individuals susceptible to disease but not yet diagnosed with the disease; (b) suppression of disease, such as inhibiting disease progression; or (c) alleviating disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, improve, reduce, or suppress the individual's disease, including but not limited to administering an individual in need an anti-CD4 antibody or an altered antigen-binding fragment thereof as described herein.

[0042] This invention proposes methods and uses for anti-CD4 antibodies or their antigen-binding fragments, recombinant proteins, nucleic acid molecules, expression vectors, recombinant cells, pharmaceutical compositions, conjugates, kits, and detection of CD4 molecules or cells expressing CD4 molecules, which will be described in detail below.

[0043] Anti-CD4 antibody or its antigen-binding fragment

[0044] In one aspect of the invention, an anti-CD4 antibody or an antigen-binding fragment thereof is provided. According to embodiments of the invention, the anti-CD4 antibody or its antigen-binding fragment comprises a CDR selected from at least one of the following: a heavy chain variable region CDR: a CDR of the heavy chain variable region of the amino acid sequence shown in any one of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, and SEQ ID NO:39, determined by any numbering scheme; a light chain variable region CDR: a CDR of the light chain variable region of the amino acid sequence shown in any one of SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, and SEQ ID NO:40, determined by any numbering scheme; wherein the numbering scheme is selected from Kabat, Chothia, Contact, and IMGT.

[0045] This invention proposes a CD4 antibody or antigen-binding fragment that can specifically bind to CD4 molecules or CD4 antibodies. + The cells produce high signal intensity, which helps to accurately detect CD4 molecules or CD4+. + Cellular, highly accurate, for CD4 + It has important application value in the diagnosis, prevention, or treatment of lymphocyte-related diseases.

[0046] For ease of understanding, the method for obtaining the antibody of this invention is described in detail below:

[0047] This invention selects the extracellular amino acid sequence of CD4, expresses and purifies the CD4 extracellular protein, tests its immunogenicity, and then immunizes New Zealand white rabbits. After four immunizations, the antiserum titer is detected by enzyme-linked immunosorbent assay (ELISA). After the serum titer reaches a certain dilution, a shock immunization is performed, and the spleen is collected and ground. Total RNA is extracted from the spleen and reverse transcribed to synthesize the first-strand cDNA. Using a set of primers matching the variable regions FR1 and FR4 of the rabbit antibody light and heavy chains, the variable region fragments (VH / Vκ) of the immunized rabbits are amplified. A second amplification is performed using another set of primers, adding a linker sequence with complementary overlapping regions to the antibody light and heavy chain variable fragments. The VH and Vκ fragments are assembled into a single-chain antibody fragment (scFv) by overlap extension PCR (SOE PCR). After digesting the scFv fragment and the phage display vector with the same restriction endonuclease, the fragments are ligated by gel recovery. After cleaning the ligation product, the fragments are electroporated into E. coli TG1 to obtain the scFv phage display bacterial library. A bacterial library was cultured, and overinfection was performed using helper phage M13K07 to aid in the packaging and rescue of the phage library. The scFv fragment was displayed on the phage surface. The CD4 protein was biotinylated and then captured using streptavidin-conjugated magnetic beads. Phages displaying specific scFv fragments were captured due to their binding to the CD4 protein. Unbound phages were removed by rinsing. The phages were eluted from the magnetic beads and used to infect logarithmically growing TG1 hosts. After culturing, helper phages could be used again for overinfection and packaging rescue. After several rounds of selection, the TG1 bacterial cultures infected with the selected phages were serially diluted, plated, and single clones were picked and cultured in 96-well plates. Helper phages were added to package the single clones, and the binding activity of phages to CD4 in the single clone supernatant was detected by ELISA, yielding positive single clones that specifically bind to CD4.

[0048] Candidate positive clones were sequenced and constructed into the eukaryotic expression vector pcDNA3.1(+) containing the constant regions of the rabbit antibody heavy and light chains, obtaining a complete light and heavy chain expression vector. The light and heavy chain vector plasmids were co-transfected into HEK293F cells for expression, and the positive clone antibody proteins were purified from the culture supernatant using Protein A. The specificity and affinity of the positive clone antibody proteins for binding to CD4 were verified by ELISA and FACS, ultimately yielding five antibodies specifically binding to CD4, named C6, C34, C59, C68, and C71.

[0049] In this document, the term "antibody" is used in the broadest sense and can include full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, with no specific structural limitations, as long as they exhibit the desired biological activity. Antibody molecules typically consist of a lighter light chain and a heavier heavy chain linked by disulfide bonds. The amino-terminal (N-terminus) amino acid sequence of the peptide chain varies considerably and is called the variable region (V-terminus); the carboxyl-terminus (C-terminus) is relatively stable and varies little, and is called the constant region (C-terminus). The V-termini of the L-chain and H-chain are referred to as VL and VH, respectively. As used herein, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of an immunoglobulin, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of the antibody or its functional fragments to the antigens or epitopes it recognizes. In specific embodiments of this disclosure, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0050] In this document, the term "antigen-binding fragment" refers to a fragment containing part or all of an antibody that lacks at least some of the amino acids present in the full-length chain but still possesses the performance activity of specifically binding to an antigen. For example, the fragment may contain part or all of the antibody's CDR. Such fragments are biologically active because they bind to the antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from Fab, Fv, scFv, or single-domain antibodies. Such fragments can be generated using recombinant nucleic acid technology or through enzymatic or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0051] According to embodiments of this application, the anti-CD4 antibody or its antigen-binding fragment includes a CDR selected from at least one of the following: heavy chain variable region CDR: SEQ ID NO: 1-3, 7-9, 13-15, 19-21, 25-27 or the amino acid sequence of its conserved modified form; light chain variable region CDR: SEQ ID NO: 4-6, 10-12, 16-18, 22-24, 28-30 or the amino acid sequence of its conserved modified form.

[0052] In this document, "conservatively modified amino acid sequences" refers to amino acid modifications that do not significantly affect or alter the binding properties of antibodies containing that amino acid sequence. These modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of this invention using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions involve replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine). Therefore, one or more amino acid residues in the CDR region of the antibody of the present invention can be replaced by other amino acid residues from the same side chain family, and the retained function of the modified antibody can be tested using the functional assay methods described herein. Preferably, the number of conservative modifications does not exceed one or two.

[0053] According to embodiments of the present invention, the anti-CD4 antibody or its antigen-binding fragment comprises: heavy chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO:1, 2, and 3, respectively; or heavy chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO:7, 8, and 9, respectively; or heavy chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO:13, 14, and 15, respectively; or heavy chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO:19, 20, and 21, respectively; or heavy chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO:25, 26, and 27, respectively.

[0054] According to embodiments of the present invention, the anti-CD4 antibody or its antigen-binding fragment comprises: light chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO:4, 5, and 6, respectively; or light chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO:10, 11, and 12, respectively; or light chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO:16, 17, and 18, respectively; or light chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO:22, 23, and 24, respectively; or light chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO:28, 29, and 30, respectively.

[0055] According to embodiments of the present invention, the anti-CD4 antibody or its antigen-binding fragment comprises: heavy chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 4, 5, and 6, respectively; or heavy chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 7, 8, and 9, respectively, and light chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 10, 11, and 12, respectively; or heavy chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 13, 14, and 15, respectively, and light chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 16, 17, and 18, respectively; or as shown in the amino acid sequences of SEQ ID NO: 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 16, 17, and 18, respectively; or as shown in the amino acid sequences of SEQ ID NO: 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 1, 2, and 3, respectively. The heavy chain variable regions CDR1, CDR2, and CDR3 sequences shown in amino acid sequences NO:19, 20, and 21, and the light chain variable regions CDR1, CDR2, and CDR3 sequences shown in amino acid sequences SEQ ID NO:22, 23, and 24, respectively; or the heavy chain variable regions CDR1, CDR2, and CDR3 sequences shown in amino acid sequences SEQ ID NO:25, 26, and 27, and the light chain variable regions CDR1, CDR2, and CDR3 sequences shown in amino acid sequences SEQ ID NO:28, 29, and 30, respectively.

[0056] According to an embodiment of the present invention, the anti-CD4 antibody or its antigen-binding fragment includes a heavy chain framework region and / or a light chain framework region.

[0057] According to embodiments of the present invention, at least a portion of the heavy chain framework region and / or light chain framework region is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof, preferably at least one of mouse antibodies, human antibodies, and primate antibodies.

[0058] According to embodiments of the present invention, the anti-CD4 antibody or its antigen-binding fragment comprises: a heavy chain variable region of an amino acid sequence as shown in any one of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39 or an amino acid sequence having at least 90% homology therewith; and / or a light chain variable region of an amino acid sequence as shown in any one of SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40 or an amino acid sequence having at least 90% homology therewith.

[0059] According to embodiments of the present invention, the anti-CD4 antibody or its antigen-binding fragment comprises: a heavy chain variable region of the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 90% homology therewith, and a light chain variable region of the amino acid sequence shown in any one of SEQ ID NO:32 or an amino acid sequence having at least 90% homology therewith; or a heavy chain variable region of the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 90% homology therewith, and a light chain variable region of the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 90% homology therewith; or a heavy chain variable region of the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having at least 90% homology therewith, and a light chain variable region of the amino acid sequence shown in SEQ ID NO:36 or an amino acid sequence having at least 90% homology therewith; or a heavy chain variable region of the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having at least 90% homology therewith, and a light chain variable region of the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having at least 90% homology therewith, and a light chain variable region of the amino acid sequence shown in SEQ ID NO:31 ... The amino acid sequence shown in NO:38 or the light chain variable region of an amino acid sequence having at least 90% homology with it; or the amino acid sequence shown in SEQ ID NO:39 or the heavy chain variable region of an amino acid sequence having at least 90% homology with it, and the amino acid sequence shown in SEQ ID NO:40 or the light chain variable region of an amino acid sequence having at least 90% homology with it.

[0060] According to an embodiment of the present invention, the anti-CD4 antibody or its antigen-binding fragment further includes a constant region; wherein the constant region includes at least one of a heavy chain constant region and a light chain constant region.

[0061] According to embodiments of the present invention, at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof.

[0062] According to embodiments of the present invention, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or the light chain constant region includes a light chain constant region selected from κ-type or λ-type.

[0063] According to an embodiment of the present invention, both the light chain constant region and the heavy chain constant region are derived from mouse antibodies or their mutants, or human antibodies or their mutants.

[0064] According to an embodiment of the present invention, the N end of the heavy chain constant region is connected to the C end of the heavy chain variable region; and / or the N end of the light chain constant region is connected to the C end of the light chain variable region.

[0065] According to an embodiment of the present invention, the heavy chain constant region includes: a heavy chain constant region as shown in SEQ ID NO:51 or an amino acid sequence having at least 80% identity with it; the light chain constant region includes or is a light chain constant region as shown in SEQ ID NO:52 or an amino acid sequence having at least 80% identity with it.

[0066] According to embodiments of the present invention, the anti-CD4 antibody or its antigen-binding fragment comprises: a heavy chain of an amino acid sequence as shown in any one of SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49 or an amino acid sequence having at least 90% homology therewith; and / or a light chain of an amino acid sequence as shown in any one of SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 or an amino acid sequence having at least 90% homology therewith.

[0067] According to embodiments of the present invention, the anti-CD4 antibody or its antigen-binding fragment comprises: a heavy chain of an amino acid sequence as shown in SEQ ID NO:41 or a conserved modified form of an amino acid sequence thereof, and a light chain of an amino acid sequence as shown in SEQ ID NO:42 or a conserved modified form of an amino acid sequence thereof; or a heavy chain of an amino acid sequence as shown in SEQ ID NO:43 or a conserved modified form of an amino acid sequence thereof, and a light chain of an amino acid sequence as shown in SEQ ID NO:44 or a conserved modified form of an amino acid sequence thereof; or a heavy chain of an amino acid sequence as shown in SEQ ID NO:45 or a conserved modified form of an amino acid sequence thereof, and a light chain of an amino acid sequence as shown in SEQ ID NO:46 or a conserved modified form of an amino acid sequence thereof; or a heavy chain of an amino acid sequence as shown in SEQ ID NO:47 or a conserved modified form of an amino acid sequence thereof, and a light chain of an amino acid sequence as shown in SEQ ID NO:48 or a conserved modified form of an amino acid sequence thereof; or a heavy chain of an amino acid sequence as shown in SEQ ID NO:49 or a conserved modified form of an amino acid sequence thereof, and a light chain of an amino acid sequence as shown in SEQ ID NO:50 or a conserved modified form of an amino acid sequence thereof.

[0068] According to embodiments of the present invention, the antibody comprises at least one selected from polyclonal antibodies, full-length monoclonal antibodies, Fab antibodies, Fab' antibodies, F(ab')2 antibodies, Fv antibodies, single-chain antibodies, single-domain antibodies, and minimal recognition units; or the antigen-binding fragment comprises at least one selected from F(ab')2 fragments, Fab' fragments, Fab fragments, F(ab)2 fragments, Fv fragments, scFv fragments, scFv-Fc fusion proteins, scFv-Fv fusion proteins, and minimal recognition units.

[0069] In this article, the terms “full-length antibody”, “full-length monoclonal antibody” or “full-length monoclonal antibody” refer to antibodies composed of at least two identical light chains and at least two identical heavy chains linked by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE).

[0070] In this document, the terms "polyclonal antibody" and "multispecific antibody" are synonymous, both referring to antibodies that can recognize multiple antigenic epitopes. For example, antibodies that recognize two antigenic epitopes (bispecific antibodies, or simply biantibodies), three antigenic epitopes, or four antigenic epitopes are used in a broad sense, and their specific structures are not limited, as long as they can recognize multiple antigenic epitopes. In this invention, at least one of the multiple antigenic epitopes is derived from NKp46.

[0071] In this paper, the terms “single-domain antibody,” “nanobody,” and “VHH antibody” are used interchangeably. The antibody was originally described as an antigen-binding immunoglobulin (variable) domain of a “heavy chain antibody” (i.e., “antibody lacking light chains”) containing a heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically binds to antigen proteins (e.g., NKp46) through the heavy chain variable region.

[0072] In this article, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment containing only Fab molecules, which consists of the VH and CH1 of the heavy chain and the complete light chain, linked by a disulfide bond.

[0073] In this paper, the term “F(ab')2 antibody” or “F(ab')2 fragment” has two antigen-binding F(ab') parts linked together by disulfide bonds.

[0074] In this article, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment consisting only of a light chain variable region (VL) and a heavy chain variable region (VH) linked by non-covalent bonds. It is the smallest functional fragment of an antibody that retains the complete antigen-binding site.

[0075] In this paper, the terms "single-chain antibody" and "scFv fragment" refer to antibodies or fragments formed by linking the variable regions of the antibody heavy chain and light chain through short peptides.

[0076] In this article, the terms "minimum recognition unit" and "MRU" both refer to antibodies or fragments consisting of only one CDR, with a very small molecular weight, accounting for only about 1% of a complete antibody.

[0077] Recombinant protein

[0078] In another aspect, the present invention provides a recombinant protein. According to embodiments of the present invention, the recombinant cell comprises: the aforementioned anti-CD4 antibody or its antigen-binding fragment. The recombinant protein of the present invention can specifically bind to CD4 molecules or CD4 + The cells produce high signal intensity, which helps to accurately detect CD4 molecules or CD4+. + Cellular, highly accurate, for CD4 +It has important application value in the diagnosis, prevention, or treatment of lymphocyte-related diseases.

[0079] According to embodiments of the present invention, the recombinant protein further comprises at least one selected from bioactive proteins or fragments thereof, bioactive polypeptides or fragments thereof.

[0080] According to embodiments of the present invention, the bioactive protein or fragment thereof includes at least one selected from protein tags, protein toxins or fragment thereof, tumor necrosis factor or fragment thereof, interferon or fragment thereof, biological response regulators or fragment thereof, and Fc fragments.

[0081] In this article, "protein tag" generally refers to a polypeptide or protein fused together with a target protein (anti-CD4 antibody or its antigen-binding fragment) for expression, detection, retrieval, or purification of the target protein. Examples include, but are not limited to, His tags (also known as His-Tag, sequence HHHHHH), Flag tags (also known as Flag-Tag, sequence DYKDDDDK), GST tags (also known as GST-Tag, glutathione thiotransferase tag), MBP tags (also known as MBP-Tag, maltose-binding protein tag), SUMO tags, and C-Myc tags.

[0082] In this document, "toxin" generally refers to substances toxic to the host, including protein toxins and non-protein toxins. Protein toxins include, but are not limited to, abrin, ricin A, Pseudomonas exotoxin, and diphtheria toxin. In this invention, the protein toxin is preferably an enzymatic protein toxin.

[0083] In this article, "tumor necrosis factor" generally refers to substances that can cause hemorrhagic necrosis in various tumors, including but not limited to TNF-α and TNF-β.

[0084] In this article, "interferon" generally refers to a glycoprotein that has the ability to directly kill or inhibit viruses. This includes, but is not limited to, IFN-α, IFN-β, and IFN-γ.

[0085] In this article, "biological response modulators" generally refers to a class of protein substances that enhance the body's anti-tumor effects directly or indirectly through the immune system. These include, but are not limited to, lymphokines, IL-2, IL-6, IL-10, and GM-CSF.

[0086] In this document, "Fc fragment" generally refers to the Fc region of IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM, including CH2, CH3 regions and optionally hinge regions. Preferably, the IgG, IgA1, IgA2, IgD, IgE, or IgM are derived from mouse, human, primate, or alpaca sources.

[0087] Nucleic acid molecules, expression vectors and recombinant cells

[0088] In another aspect of the invention, a nucleic acid molecule is provided. According to embodiments of the invention, the nucleic acid molecule encodes the aforementioned anti-CD4 antibody or its antigen-binding fragment, or the aforementioned recombinant protein.

[0089] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0090] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is also disclosed. Furthermore, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0091] In another aspect of the invention, an expression vector is provided. According to an embodiment of the invention, the expression vector carries the aforementioned nucleic acid molecule. When the nucleic acid molecule is ligated to the vector, it can be directly or indirectly linked to control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. These control elements can be directly derived from the vector itself or are exogenous, i.e., not derived from the vector itself. Of course, the nucleic acid molecule and control elements need to be operably linked. In this context, "operably linked" means ligating a foreign gene to the vector so that control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform their intended function of regulating the transcription and translation of the foreign gene. Commonly used vectors include plasmids, bacteriophages, etc. After the expression vector of some specific embodiments of the invention is introduced into suitable recipient cells, under the mediation of a regulatory system, the expression of the aforementioned anti-CD4 antibody or its antigen-binding fragment or the aforementioned recombinant protein can be effectively achieved, thereby enabling the large-scale in vitro production of the anti-CD4 antibody or its antigen-binding fragment or recombinant protein.

[0092] According to embodiments of the present invention, the expression vector includes those selected from eukaryotic expression vectors or prokaryotic expression vectors.

[0093] According to an embodiment of the present invention, the expression vector is a plasmid expression vector.

[0094] In another aspect, the present invention provides a recombinant cell. According to embodiments of the invention, the recombinant cell carries at least one of the following: the aforementioned anti-CD4 antibody or its antigen-binding fragment, the expressed protein, the nucleic acid molecule, and the expression vector. Using this recombinant cell, under suitable conditions, the aforementioned anti-CD4 antibody or its antigen-binding fragment, or the recombinant protein, can be effectively expressed intracellularly.

[0095] It should be noted that the "suitable conditions" mentioned in this application specification refer to conditions suitable for the expression of the anti-CD4 antibody or its antigen-binding fragment or recombinant protein described in this invention. Those skilled in the art will readily understand that suitable conditions for the expression of the anti-CD4 antibody or its antigen-binding fragment or recombinant protein include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the anti-CD4 antibody or its antigen-binding fragment or recombinant protein according to the specific environment of their laboratory.

[0096] According to an embodiment of the present invention, the recombinant cells are obtained by introducing the aforementioned expression vector into host cells.

[0097] According to an embodiment of the present invention, the recombinant cell is a eukaryotic cell, preferably a mammalian cell.

[0098] Pharmaceutical Composition

[0099] In another aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises at least one of the following: the aforementioned anti-CD4 antibody or its antigen-binding fragment, the recombinant protein, the nucleic acid molecule, the expression vector, and the recombinant cell. The present invention proposes a drug that can specifically bind to CD4 molecules or CD4 + The cells produce high signal intensity, which helps to accurately detect CD4 molecules or CD4+. + Cellular, highly accurate, for CD4 + It has important application value in the diagnosis, prevention, or treatment of lymphocyte-related diseases.

[0100] According to embodiments of the present invention, pharmaceutically acceptable excipients are further included.

[0101] The pharmaceutical compositions of the present invention can be administered by any acceptable method of administration. The pharmaceutical compositions of the present invention can be formulated into solid, semi-solid, liquid, or gaseous forms, such as injections or lyophilized powders, and current methods for preparing these dosage forms are known or obvious to those skilled in the art. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, oral, rectal, vaginal, and intranasal routes. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. The pharmaceutical compositions of the present invention are formulated to allow the bioactive components contained therein to be bioavailable after administration to a patient.

[0102] In another aspect, the present invention provides a conjugate. According to an embodiment of the invention, the conjugate comprises: the aforementioned anti-CD4 antibody or its antigen-binding fragment; and a conjugation portion linked to the anti-CD4 antibody or its antigen-binding fragment or recombinant protein. The present invention proposes a conjugate capable of specifically binding to CD4 molecules or CD4... + The cells produce high signal intensity, which helps to accurately detect CD4 molecules or CD4+. + Cellular, highly accurate, for CD4 + It has important application value in the diagnosis, prevention, or treatment of lymphocyte-related diseases.

[0103] According to embodiments of the present invention, the coupling portion includes at least one selected from carriers, drugs, toxins, cytokines, protein tags, modifiers, and chemotherapeutic agents.

[0104] In this document, the carrier can be a substance capable of suspension or dispersion in a liquid phase (e.g., solid-phase carriers such as particles and magnetic beads), or a solid phase capable of containing or carrying a liquid phase (e.g., supports such as plates, membranes, and test tubes, as well as containers such as well plates, microfluidic pathways, glass capillaries, nanopillars, and monolithic columns); it can also be a labeling carrier for labeling anti-CD4 antibodies or their antigen-binding fragments or recombinant proteins, such as enzymes (e.g., peroxidase, alkaline phosphatase, luciferin, β-galactose). Glycosidases), affinity substances (e.g., one of streptavidin and biotin, one of complementary sense and antisense nucleic acids), fluorescent substances (e.g., luciferin, luciferin isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., insect luciferin, jellyfish luminescent protein (Aequorin), acridinium ester, tris(2,2'-bipyridine)ruthenium, luminol), radioactive isotopes (e.g., 3H, 14C, 32P, 35S, 125I), and gold colloids, etc.

[0105] According to embodiments of the present invention, the drug is a small molecule drug that can bind to an anti-CD4 antibody or its antigen-binding fragment or recombinant protein.

[0106] According to embodiments of the present invention, the protein tag includes, but is not limited to, His tag, Flag tag, GST tag, MBP tag, SUMO tag, and C-Myc tag.

[0107] According to embodiments of the present invention, the term "modifier" should be interpreted broadly and may refer to substances used to modify proteins. Exemplarily, it may be polyethylene glycol or a derivative thereof.

[0108] According to embodiments of the present invention, the chemotherapeutic agents include, but are not limited to, albumin-bound paclitaxel, cyclophosphamide, ifosfamide, phenylalanine nitrogen mustard, methotrexate, fluorouracil, actinomycin D, and vincristine.

[0109] It should be noted that the binding method between the conjugated portion and the anti-CD4 antibody or its antigen-binding fragment or recombinant protein can use methods known in the art. For example, methods such as physical adsorption, covalent binding, using affinity substances (e.g., biotin, streptavidin) and ion binding can be used.

[0110] In another aspect, the present invention provides a kit. According to embodiments of the present invention, the kit comprises at least one of the following: the aforementioned anti-CD4 antibody or its antigen-binding fragment, the recombinant protein, the nucleic acid molecule, the expression vector, and the recombinant cells. The kit of the present invention can specifically detect CD4 molecules or CD4+. + Cellular, highly accurate, helps CD4 + Diagnosis of lymphocyte-related diseases.

[0111] According to embodiments of the present invention, the kit further comprises at least one of the following reagents: a reagent for ELISA detection; a reagent for Western Bolt detection; a reagent for FACS detection; and a reagent for flow cytometry detection. The present invention does not strictly limit the reagents used for ELISA detection, Western Bolt detection, FACS detection, and flow cytometry detection; these can be commonly used reagents in the art and can be flexibly selected as needed.

[0112] method

[0113] In another aspect, the present invention provides a method for detecting CD4 molecules or detecting cells expressing CD4 molecules. According to an embodiment of the present invention, the method involves contacting the aforementioned anti-CD4 antibody or its antigen-binding fragment, the recombinant protein, the conjugate, or the kit with the sample to be tested to form an immune complex. The anti-CD4 antibody or its antigen-binding fragment, the recombinant protein, or the conjugate of the present invention can specifically detect CD4 molecules or CD4+. + Cellular detection offers high accuracy. Specifically, the detection method of this invention can be performed both in vitro and in vivo, allowing for flexible selection based on actual needs.

[0114] According to an embodiment of the present invention, based on the signal of the immune complex, it is determined whether the sample to be tested contains CD4 or whether it contains cells expressing CD4 molecules, or the content of CD4 or cells expressing CD4 molecules in the sample to be tested is determined.

[0115] According to embodiments of the present invention, the signal of the immune complex is detected by at least one of flow cytometry, ELISA, WB, and FACS.

[0116] According to an embodiment of the present invention, the signal includes a fluorescence signal.

[0117] In another aspect of the invention, a treatment or prevention method for CD4 is proposed. + Methods for treating lymphocyte-related diseases. According to embodiments of the present invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the aforementioned anti-CD4 antibody or its antigen-binding fragment, the aforementioned recombinant protein, the aforementioned pharmaceutical composition, or the aforementioned conjugate. The methods of the present invention can effectively treat or prevent CD4. + Lymphocyte-related diseases. The anti-CD4 antibody or its antigen-binding fragment, recombinant protein, pharmaceutical composition, or conjugate of the present invention can specifically bind to CD4, activate the immune response of T lymphocytes, enhance the immune response of B lymphocytes, and participate in the regulation of T cell immune responses, thereby effectively preventing or treating CD4-related diseases. + Lymphocyte-related diseases.

[0118] The effective amount of the anti-CD4 antibody or its antigen-binding fragment, recombinant protein, pharmaceutical composition, or conjugate described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0119] The anti-CD4 antibody or its antigen-binding fragment, recombinant protein, pharmaceutical composition, or conjugate of the present invention can be incorporated into medicaments suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These medicaments can be prepared in various forms, such as liquid, semi-solid, and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions) or lyophilized powders. Typical medicaments are in the form of injection solutions or infusion solutions. The aforementioned anti-CD4 antibody or its antigen-binding fragment, recombinant protein, pharmaceutical composition, or conjugate can be administered by intravenous infusion or injection, or by intramuscular or subcutaneous injection.

[0120] use

[0121] In another aspect of the invention, the invention provides for the use of the aforementioned anti-CD4 antibody or its antigen-binding fragment, the recombinant protein, the pharmaceutical composition, or the conjugate in the preparation of a medicament for the prevention and / or treatment of CD4. + Lymphocyte-related diseases.

[0122] According to an embodiment of the present invention, the CD4 + Lymphocyte-related diseases include those caused by tumors or cancer, AIDS, or autoimmune diseases.

[0123] According to embodiments of the present invention, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0124] In another aspect of the invention, the invention provides for the use of the anti-CD4 antibody or its antigen-binding fragment, the recombinant protein, the conjugate, or the kit in the preparation of a product for detecting CD4 molecules or cells expressing CD4 molecules.

[0125] According to an embodiment of the present invention, the product is used for detection by at least one of the following methods: flow cytometry, ELISA, Western blotting, and FACS.

[0126] The amino acid sequence boundaries of a CDR can be determined by one of those skilled in the art using any of the many known numbering schemes, including those described below: Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition (1991), Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al., Biochem. J.Mol.Biol., 1997, 273:927-948 (“Chothia” numbering scheme); MacCallum et al., Molecular Biology, 1996, 262:732-745 (“Contact” numbering scheme); Lefranc et al., Development of Competitive Immunology, 2003, 27:55-77 (“IMGT” numbering scheme).

[0127] Table 1 provides sequence information for heavy chain CDRs and light chain CDRs identified using the Kabat numbering scheme, while Table 2 provides sequence information for heavy chain CDRs and light chain CDRs identified using the Chothia, Contact, and IMGT numbering schemes.

[0128] Table 1. Sequence information such as heavy chain CDR and light chain CDR identified by the Kabat numbering scheme.

[0129]

[0130]

[0131]

[0132]

[0133]

[0134] Note: The underscore in the sequence indicates the CDR sequence of the antibody.

[0135] Table 2. Heavy chain CDR and light chain CDR sequence information identified by the Contact, Chothia, and IMGT numbering schemes.

[0136]

[0137]

[0138] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0139] Unless otherwise specified, the practice of this disclosure will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994) and "Current Protocols in Immunology" (JEColigan et al., ed., 2011), each of which is explicitly incorporated herein by reference.

[0140] In this embodiment of the invention, the nucleotide sequence used to prepare the expression vector can be obtained from its amino acid sequence using conventional methods or conventional software (such as the online program Vectorbuilder (website: https: / / www.vectorbuilder.cn / tool / codon-optimization.html), GeneOptimizer online program, etc.).

[0141] Example 1: Eukaryotic expression and purification of recombinant hCD4 extracellular protein

[0142] Recombinant protein expression: The extracellular region sequence of hCD4 was synthesized by Guangzhou Liuhe and the codon sequence was optimized before being constructed into the pCDNA3.1(+) vector. After obtaining the vector, the plasmid was extracted, and then the light and heavy chain vectors were mixed at a molar ratio of 1:1. 300 μg of plasmid was taken, added to 12.5 mL of OPM CD05 medium and 800 μL of 1 mg / mL PEI (POLYSCIENCE) solution, mixed well, and incubated at room temperature for half an hour. The mixture was then added to 250 mL of HEK293F cells that were being cultured. The cells were placed in a shaker and cultured at 37°C, 8% CO2, and 110 rpm. OPM 293 cells were added every other day, and the cells were cultured for 7 days. The cell supernatant was collected by centrifugation.

[0143] Recombinant protein purification: 2 mL of HisTrap FF packing material was added to each manual column (purchased from Sangon Biotech, model F506607-0001#, empty column) and the packing material was equilibrated by washing with 30 mL of binding buffer. Then, approximately 30 mL of filtered cell supernatant was added. The column was washed 10 times (10 mL / wash) with wash buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 10 mM imidazole), followed by elution with 500 μL of elution buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 300 mM imidazole) 4-5 times. The eluted protein was collected. The final purified sample was dialyzed or ultrafiltered into PBS, flash-frozen in liquid nitrogen, and stored at -80°C. The gel electrophoresis results of the purified protein are shown in the attached figure. Figure 1 The band size was consistent with expectations, and the purity was greater than 95%. This antigen was then used for immunization.

[0144] Example 2: Animal Immunity and Serum Titer Test

[0145] In this example, the immunization was performed by a CRO company, with each peptide immunizing two rabbits.

[0146] 1. Collect 1 mL of blood from the ears of two rabbits and use the collected serum as a control. Immunize each rabbit with 1 mg of antigen, emulsify with complete adjuvant, dilute 2 mg of antigen in 1.5 mL of PBS, mix 1.5 mL of antigen with 1.5 mL of complete adjuvant, and shake on a shaker for at least 15 minutes until it reaches a water-in-oil state; immunize each rabbit with approximately 1.5 mL of the antigen on its back.

[0147] 2. Two weeks later, administer a second immunization. Each rabbit is immunized with 0.5 mg of antigen, emulsified with incomplete adjuvant. Dilute 1 mg of antigen in 1 mL of PBS and mix 1 mL of antigen with 1 mL of incomplete adjuvant. Shake on a shaker for at least 15 minutes until it reaches a water-in-oil state. Immunize approximately 1 mL of the antigen on the back of each rabbit.

[0148] 3. One week later, each rabbit was immunized with 0.5 mg of antigen, emulsified with incomplete adjuvant, and 1 mg of antigen was diluted in 1 mL of PBS. The mixture was 1 mL of antigen + 1 mL of incomplete adjuvant and shaken on a shaker for more than 15 minutes until it reached a water-in-oil state. Each rabbit was immunized with about 1 mL of the antigen on its back.

[0149] 4. One week later, each rabbit was immunized with 0.5 mg of antigen, emulsified with incomplete adjuvant, and 1 mg of antigen was diluted in 1 mL of PBS. The mixture was 1 mL of antigen + 1 mL of incomplete adjuvant and shaken on a shaker for more than 15 minutes until it reached a water-in-oil state. Each rabbit was immunized with about 1 mL of the antigen on its back.

[0150] 5. One week later, take 1mL of blood from the ear and test the titer with ELISA. If the titer reaches 1:100,000 or higher, then perform a shock immunization.

[0151] 6. For shock immunization, immunize each rabbit with 1 mg of antigen, emulsify with complete adjuvant, dilute 2 mg of antigen in 1.5 mL of PBS, mix 1.5 mL of antigen + 1.5 mL of complete adjuvant, shake on a shaker for more than 15 minutes until it reaches a water-in-oil state; immunize each rabbit with about 1.5 mL on its back.

[0152] 7. Three days later, blood was collected from the ears. 5 mL of blood was collected from each rabbit to test the titer. The titer met the expectations. Whole blood was collected using anticoagulant tubes and serum was separated.

[0153] Example 3: ELISA detection of antiserum titer

[0154] 1. Dilute the recombinant hCD4 protein to 0.1 μg / mL with PBS, add 100 μL to each well of the immunoassay plate, and incubate overnight at 4°C.

[0155] 2. Wash the microplate with PBST 3 times, 300 μL per well, and pat off any remaining liquid on a paper towel on the last wash.

[0156] 3. Add 300 μL of 2% BSA blocking solution to each well and incubate at 37°C for 1 hour.

[0157] 4. Wash the microplate with PBST 3 times, 300 μL per well, and pat off any remaining liquid on a paper towel on the last wash.

[0158] 5. Dilute the antiserum with PBS and add 100 μL to each well of the immunoassay plate. Incubate at 37°C for 1 hour.

[0159] 6. Wash the microplate with PBST 6 times, 300 μL per well, and blot off any remaining liquid on a paper towel on the last wash.

[0160] 7. Add 100 μL of Anti-rabbit Antibody (HRP) diluted in PBS to each well and incubate at room temperature for 1 hour.

[0161] 8. Wash the microplate with PBST 6 times, 300 μL per well, and blot off any remaining liquid on a paper towel on the last wash.

[0162] 9. Remove solutions A and B from the TMB Substrate Solution Set from 4°C, allow them to return to room temperature, mix them in equal proportions to prepare an appropriate amount of colorimetric solution, add 100 μL to each well of the microplate, and incubate in the dark for 10 min.

[0163] 10. Add 50 μL of 1M HCl to each well to stop the colorimetric reaction, and read the OD450 value using a microplate reader.

[0164] The potency test results are attached. Figure 2 valence greater than 10 -5 Immunization was successful.

[0165] Example 4: Screening of scFv display libraries of immunized rabbit antibodies

[0166] Rabbit spleen cells were ground and RNA extracted to construct a cDNA library. Specifically, total RNA was extracted from the spleen and reverse transcribed to synthesize first-strand cDNA. Using a set of primers matching the variable regions FR1 and FR4 of the rabbit antibody light and heavy chains, the variable region fragments (VH / Vκ) of the immunized rabbit were amplified. A second amplification was performed using another set of primers, adding a linker sequence with complementary overlapping regions to the antibody light and heavy chain variable fragments. The VH and Vκ fragments were then assembled into a single-stranded antibody fragment (scFv) using overlap extension PCR (SOE PCR). After digesting the scFv fragments and the phage display vector with the same restriction endonuclease, ligation was performed via gel recovery. After cleaning the ligation product, the fragments were electroporated into *E. coli* TG1 to obtain the scFv phage display bacterial library. The bacterial library was cultured, and superinfection was performed using helper phage M13K07 to aid in packaging and rescue the phage library, displaying the scFv fragments on the phage surface. The CD4 protein was biotinylated and then captured using streptavidin-conjugated magnetic beads. A phage library was added, demonstrating that phages with specific scFv fragments were captured due to their binding to the CD4 protein. Unbound phages were removed by rinsing. The phages were eluted from the magnetic beads and used to infect TG1 hosts in the logarithmic growth phase. After culturing, helper phages could be used again for superinfection and packaging rescue. After several rounds of screening, the TG1 bacterial cultures infected with the selected phages were serially diluted, plated, and single clones were picked and cultured in 96-well plates. Helper phages were added to package the single clones, and the phage-CD4 binding activity in the single clone supernatant was detected by ELISA. 192 single clones were picked, and 73 positive clones were obtained. The five preferred clones were C6, C34, C59, C68, and C71.

[0167] Example 5: Expression and purification of positive clone antibodies

[0168] Recombinant antibody expression: The C6, C34, C59, C68, and C71 light and heavy chain sequences were synthesized by Guangzhou Liuhe and their codon sequences were optimized before being constructed into the pCDNA3.1(+) vector. After obtaining the vector, the plasmid was extracted, and then the light and heavy chain vectors were mixed at a 1:1 molar ratio. 300 μg of plasmid was added to 12.5 mL of LOPM CD05 medium and 800 μL of 1 mg / mL PEI (POLYSCIENCE) solution, mixed well, and incubated at room temperature for half an hour. This mixture was then added to 250 mL of HEK293F cells and cultured on a shaker at 37°C, 8% CO2, and 110 rpm. OPM 293 cell culture feed was added every other day, and the cells were cultured for 7 days. The cell supernatant was collected by centrifugation.

[0169] Recombinant antibody purification: 1 ml of Protein A packing material (Sino Biotech) was added to the received supernatant and incubated at room temperature for half an hour. The packing material was then removed and loaded into an empty purification column. 20 ml of PBS solution was added for washing, followed by elution with 10 ml of 100 mM glycine solution (pH 3.0). The eluent was neutralized to pH 7.0 with 1 M Tris solution. The neutralized eluent was concentrated to 1 ml, protein concentration was determined, and representative antibodies were subjected to SDS-PAGE for purity assessment. Figure 3 As can be seen, the antibody meets the electrophoretic purity standard, and no other obvious bands are observed besides the main band.

[0170] Example 6: Positive clone affinity ELISA assay

[0171] 1. Conjugate BSA to the phosphorylated antigen peptide and the non-phosphorylated peptide of the same sequence, respectively. Then, coat the enzyme strip with 100 μL of the conjugated antigen at a concentration of 1 μg / ml BSA-peptide overnight. Add 200 μL of PBST and wash three times with shaking for 10 min each time, followed by three washes with PBS.

[0172] 2. Add 200 μL of 1% BSA-PBS and block at 37°C for 2 h. Add 200 μL of PBST and wash with shaking for 3 x 10 min, then add 200 μL of PBS and wash with shaking for 3 x 10 min.

[0173] 3. Dilute the antibody to different concentrations, add 100 μL, and incubate at 37°C for 2 hours. Add 200 μL of PBST and wash with shaking for 3 x 10 min, then add 200 μL of PBS and wash with shaking for 3 x 10 min.

[0174] 4. Add 100 μL of diluted commercial horseradish peroxidase-conjugated goat anti-rabbit antibody and incubate at 37°C for 1 h. Add 200 μL of PBST and wash with vortexing for 3 x 10 min, then add 200 μL of PBS and wash with vortexing for 3 x 10 min.

[0175] 5. Use tetramethylbenzidine microporous peroxidase substrate (TMB) as substrate for color development for 10 min;

[0176] 6. After terminating the reaction by adding 50 μL of 0.1 mol / L sulfuric acid, measure its absorbance at 450 nm.

[0177] The affinity test results are attached. Figure 4 Based on the ELISA results, the calculated affinity of the three antibodies is 8 × 10⁻⁶. -9 about.

[0178] Example 7: Antibody Flow Cytometry Test

[0179] 1. Collect TIL cells or HEK293 cells, count them, divide them into 1x106 cells / tube, centrifuge at 300g for 5min, and resuspend them in 3% BSA / PBS.

[0180] 2. Add 100 μL of primary antibody diluted with 3% BSA / PBS (final concentration 10 μg / mL) and incubate at 4°C for 1 h. Wash 3 times with 500 μL of cold PBS.

[0181] 3. Add FITC-labeled goat anti-rabbit secondary antibody at a ratio of 1:200 and incubate at 4°C in the dark for 30 min. Wash three times with 500 μL of cold PBS.

[0182] 4. Resuspend in 200 μL PBS, and add 1 μL of PE (1 mg / mL) dye at a ratio of 1:200 before instrumentation.

[0183] The results are attached. Figure 5 FACS testing on positive clones clearly showed that the antibody bound to CD4-expressing TIL cells, but not to HEK293 cells, indicating that the candidate CD4 antibody had good specificity and a better signal than the control antibody (Abcam EPR6855).

[0184] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An anti-CD4 antibody or its antigen-binding fragment, characterized in that, Includes CDRs selected from at least one of the following: Heavy chain variable region CDR: The CDR of the heavy chain variable region of any of the amino acid sequences shown in SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, determined by any numbering scheme; Light chain variable region CDR: The light chain variable region of any of the amino acid sequences shown in SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, determined by any numbering scheme; The numbering scheme is selected from Kabat, Chothia, Contact, and IMGT; Optionally, the anti-CD4 antibody or its antigen-binding fragment includes a CDR selected from at least one of the following: Heavy chain variable region CDR: SEQ ID NO: 1~3, 7~9, 13~15, 19~21, 25~27 or their conserved modified amino acid sequences; Light chain variable region CDR: SEQ ID NO: 4~6, 10~12, 16~18, 22~24, 28~30 or their conserved modified amino acid sequences.

2. The anti-CD4 antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-CD4 antibody or its antigen-binding fragment includes: The heavy chain variable region CDR1, CDR2, and CDR3 sequences are shown in the amino acid sequences of SEQ ID NO:1, 2, and 3, respectively; or The heavy chain variable region CDR1, CDR2, and CDR3 sequences are shown in the amino acid sequences of SEQ ID NO:7, 8, and 9, respectively; or The heavy chain variable region CDR1, CDR2, and CDR3 sequences are shown in the amino acid sequences of SEQ ID NO:13, 14, and 15, respectively; or The heavy chain variable region CDR1, CDR2, and CDR3 sequences are shown in the amino acid sequences of SEQ ID NO:19, 20, and 21, respectively; or The heavy chain variable regions CDR1, CDR2, and CDR3 sequences are shown in the amino acid sequences of SEQ ID NO:25, 26, and 27, respectively; Optionally, the anti-CD4 antibody or its antigen-binding fragment comprises: The light chain variable region CDR1, CDR2, and CDR3 sequences are shown in the amino acid sequences of SEQ ID NO:4, 5, and 6, respectively; or The light chain variable region CDR1, CDR2, and CDR3 sequences are shown in the amino acid sequences of SEQ ID NO:10, 11, and 12, respectively; or The light chain variable region CDR1, CDR2, and CDR3 sequences are shown in the amino acid sequences of SEQ ID NO:16, 17, and 18, respectively; or The light chain variable region CDR1, CDR2, and CDR3 sequences are shown in the amino acid sequences of SEQ ID NO:22, 23, and 24, respectively; or The light chain variable region CDR1, CDR2, and CDR3 sequences are shown in the amino acid sequences of SEQ ID NO:28, 29, and 30, respectively; Optionally, the anti-CD4 antibody or its antigen-binding fragment comprises: The heavy chain variable regions CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NO:1, 2, and 3, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NO:4, 5, and 6, respectively; or The heavy chain variable regions CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NO:7, 8, and 9, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NO:10, 11, and 12, respectively; or The heavy chain variable regions CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NO:13, 14, and 15, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NO:16, 17, and 18, respectively; or The heavy chain variable regions CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NO:19, 20, and 21, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NO:22, 23, and 24, respectively; or The heavy chain variable regions CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NO:25, 26, and 27, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NO:28, 29, and 30, respectively.

3. The anti-CD4 antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The anti-CD4 antibody or its antigen-binding fragment includes a heavy chain framework region and / or a light chain framework region; Optionally, at least a portion of the heavy chain framework region and / or light chain framework region is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof, preferably at least one of mouse antibodies, human antibodies, and primate antibodies; Optionally, the anti-CD4 antibody or its antigen-binding fragment comprises: The heavy chain variable region of any of the amino acid sequences shown in SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39, or an amino acid sequence having at least 90% homology with them; and / or The light chain variable region of any of the amino acid sequences shown in SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, or SEQ ID NO:40, or an amino acid sequence having at least 90% homology with them; Optionally, the anti-CD4 antibody or its antigen-binding fragment comprises: The heavy chain variable region of an amino acid sequence as shown in SEQ ID NO:31 or an amino acid sequence having at least 90% homology therewith, and the light chain variable region of an amino acid sequence as shown in any one of SEQ ID NO:32 or an amino acid sequence having at least 90% homology therewith; or The heavy chain variable region of the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 90% homology therewith, and the light chain variable region of the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 90% homology therewith; or The heavy chain variable region of the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having at least 90% homology therewith, and the light chain variable region of the amino acid sequence shown in SEQ ID NO:36 or an amino acid sequence having at least 90% homology therewith; or The heavy chain variable region of the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having at least 90% homology therewith, and the light chain variable region of the amino acid sequence shown in SEQ ID NO:38 or an amino acid sequence having at least 90% homology therewith; or The heavy chain variable region of the amino acid sequence shown in SEQ ID NO:39 or an amino acid sequence having at least 90% homology with it, and the light chain variable region of the amino acid sequence shown in SEQ ID NO:40 or an amino acid sequence having at least 90% homology with it. Optionally, the anti-CD4 antibody or its antigen-binding fragment further includes a constant region; The constant region includes at least one of the heavy chain constant region and the light chain constant region; Optionally, at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof; Optionally, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD; or The light chain constant region includes a light chain constant region selected from κ-type or λ-type; Optionally, both the light chain constant region and the heavy chain constant region are derived from mouse antibodies or their mutants, or human antibodies or their mutants. Optionally, the N-end of the heavy chain constant region is connected to the C-end of the heavy chain variable region; and / or The N-end of the constant region of the light chain is connected to the C-end of the variable region of the light chain; Optionally, the heavy chain constant region includes: As shown in SEQ ID NO:51 or an amino acid sequence having at least 80% identity with it, the heavy chain constant region; The light chain constant region includes or is a light chain constant region as shown in SEQ ID NO:52 or an amino acid sequence having at least 80% identity with it; Optionally, the anti-CD4 antibody or its antigen-binding fragment comprises: Such as the amino acid sequence shown in any one of SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, or a heavy chain having at least 90% homology with the amino acid sequence shown therein; and / or Such as the amino acid sequence shown in any one of SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, or a light chain having at least 90% homology with the amino acid sequence shown therein; Optionally, the anti-CD4 antibody or its antigen-binding fragment comprises: Heavy chains of amino acid sequences as shown in SEQ ID NO:41 or their conserved modified forms, and light chains of amino acid sequences as shown in SEQ ID NO:42 or their conserved modified forms; or Heavy chains of amino acid sequences as shown in SEQ ID NO:43 or their conserved modified forms, and light chains of amino acid sequences as shown in SEQ ID NO:44 or their conserved modified forms; or Heavy chains of amino acid sequences as shown in SEQ ID NO:45 or their conserved modified forms, and light chains of amino acid sequences as shown in SEQ ID NO:46 or their conserved modified forms; or Heavy chains of amino acid sequences as shown in SEQ ID NO:47 or their conserved modified forms, and light chains of amino acid sequences as shown in SEQ ID NO:48 or their conserved modified forms; or Heavy chains of amino acid sequences as shown in SEQ ID NO:49 or their conserved modified forms, and light chains of amino acid sequences as shown in SEQ ID NO:50 or their conserved modified forms. Optionally, the antibody comprises at least one selected from polyclonal antibodies, full-length monoclonal antibodies, Fab antibodies, Fab' antibodies, F(ab')2 antibodies, Fv antibodies, single-chain antibodies, single-domain antibodies, and the smallest recognition unit; or The antigen-binding fragment includes at least one selected from the following: F(ab')2 fragment, Fab' fragment, Fab fragment, F(ab)2 fragment, Fv fragment, scFv fragment, scFv-Fc fusion protein, scFv-Fv fusion protein, and minimum recognition unit.

4. A recombinant protein, characterized in that, include: The anti-CD4 antibody or its antigen-binding fragment as described in any one of claims 1 to 3; Optionally, it further includes at least one selected from bioactive proteins or fragments thereof, bioactive peptides or fragments thereof; Optionally, the bioactive protein or fragment thereof includes at least one selected from protein tags, protein toxins or fragments thereof, tumor necrosis factor or fragments thereof, interferon or fragments thereof, biological response regulators or fragments thereof, and Fc fragments.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-CD4 antibody or its antigen-binding fragment as described in any one of claims 1 to 3, or the recombinant protein as described in claim 4; Optionally, the nucleic acid molecule is DNA.

6. An expression carrier, characterized in that, The expression vector carries the nucleic acid molecule as described in claim 5; Optionally, the expression vector includes a selection from eukaryotic expression vectors or prokaryotic expression vectors; Preferably, the expression vector is a plasmid expression vector.

7. A recombinant cell, characterized in that, The recombinant cells carry at least one of the following: the anti-CD4 antibody or its antigen-binding fragment as described in any one of claims 1 to 3, the expression protein as described in claim 4, the nucleic acid molecule as described in claim 5, and the expression vector as described in claim 6; Optionally, the recombinant cells are obtained by introducing the expression vector of claim 6 into a host cell; Optionally, the recombinant cells are eukaryotic cells, and preferably, the recombinant cells are mammalian cells.

8. A pharmaceutical composition, characterized in that, It includes at least one of the following: the anti-CD4 antibody or its antigen-binding fragment as described in any one of claims 1 to 3, the recombinant protein as described in claim 4, the nucleic acid molecule as described in claim 5, the expression vector as described in claim 6, and the recombinant cell as described in claim 7; Optionally, pharmaceutically acceptable excipients may be further included.

9. A coupling, characterized in that, Include: The anti-CD4 antibody or its antigen-binding fragment according to any one of claims 1 to 3; and The coupling portion is linked to the anti-CD4 antibody or its antigen-binding fragment or recombinant protein; Optionally, the coupling portion includes at least one selected from carriers, drugs, toxins, cytokines, protein tags, modifiers, and chemotherapeutic agents.

10. A reagent kit, characterized in that, Includes at least one of the following: The anti-CD4 antibody or its antigen-binding fragment according to any one of claims 1 to 3, the recombinant protein according to claim 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6, and the recombinant cell according to claim 7; Optionally, the kit further comprises at least one of the following reagents: Reagents used for ELISA detection; Reagents used for Western Bolt detection; Reagents used for FACS detection; Reagents used for flow cytometry analysis.

11. A method for detecting CD4 molecules or detecting cells expressing CD4 molecules, characterized in that, include: The anti-CD4 antibody or its antigen-binding fragment according to any one of claims 1 to 3, the recombinant protein according to claim 4, the conjugate according to claim 9, or the kit according to claim 10 are brought into contact with the sample to be tested to form an immune complex; Optionally, based on the signal of the immune complex, it is determined whether the sample to be tested contains CD4 or whether it contains cells expressing CD4 molecules, or the content of CD4 or cells expressing CD4 molecules in the sample to be tested is determined; Optionally, the signal of the immune complex is detected by at least one of flow cytometry, ELISA, WB, and FACS. Optionally, the signal includes a fluorescence signal.

12. Use in the preparation of a medicament of the anti-CD4 antibody or its antigen-binding fragment according to any one of claims 1 to 3, the recombinant protein according to claim 4, the pharmaceutical composition according to claim 8, or the conjugate according to claim 9, wherein the medicament is used for the prevention and / or treatment of CD4. + Lymphocyte-related diseases; Optionally, the CD4 + Lymphocyte-related diseases include those caused by tumors or cancer, AIDS, or autoimmune diseases; Optionally, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

13. Use of the anti-CD4 antibody or its antigen-binding fragment according to any one of claims 1 to 3, the recombinant protein according to claim 4, the conjugate according to claim 9, or the kit according to claim 10 in the preparation of a product, wherein the product is used to detect CD4 molecules or cells expressing CD4 molecules; Optionally, the product is used for detection by at least one of the following methods: flow cytometry, ELISA, Western blotting, and FACS.

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