Anti-CD8 antibody

Abstract

Provided are an anti-CD8 antibody, and a multispecific antibody, fusion protein, conjugate and lipid nanoparticle containing the antibody or an antigen-binding fragment thereof. Further provided are a detection reagent and pharmaceutical composition containing the antibody or the antigen-binding fragment thereof, which can be used for CD8+ cell detection, localization, and / or imaging, and for treating or preventing a disease.

Description

Anti-CD8 antibody Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an anti-CD8 antibody. Background Technology

[0002] CD8 (differentiation antigen cluster 8) is a transmembrane glycoprotein mainly expressed on the surface of cytotoxic T lymphocytes (CTLs) and some natural killer T cells (NKTs). As a co-receptor of the T cell receptor (TCR), CD8 participates in antigen recognition and the transmission of T cell activation signals by specifically binding to major histocompatibility complex class I molecules (MHC-I), thus playing a central role in the activation, differentiation, and target cell killing of cytotoxic T cells (CTLs). Summary of the Invention

[0003] This disclosure provides an anti-CD8 antibody or its antigen-binding fragment thereof, wherein the anti-CD8 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO:1, and the heavy chain variable region further comprising a heavy chain framework region, the light chain variable region comprising LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO:5, and the light chain variable region further comprising a light chain framework region, wherein:

[0004] (1) The heavy chain frame region includes:

[0005] HFR1 in the heavy chain variable region shown in SEQ ID NO:4;

[0006] HFR2 in the heavy chain variable region shown in SEQ ID NO:4; or

[0007] HFR3 in the heavy chain variable region shown in SEQ ID NO:1 or 4;

[0008] Or, (2) the light chain framework region includes:

[0009] LFR1 in the variable region of the light chain shown in SEQ ID NO:7; or

[0010] LFR2 in the variable region of the light chain shown in SEQ ID NO:7.

[0011] In some implementations, the heavy chain framework region includes:

[0012] HFR3 in the heavy chain variable region shown in SEQ ID NO:1;

[0013] HFR3 in the heavy chain variable region shown in SEQ ID NO:4;

[0014] HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:4; or

[0015] HFR1, HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:4.

[0016] In some implementations, the light chain framework region includes:

[0017] LFR1 in the variable region of the light chain shown in SEQ ID NO:7;

[0018] LFR2 in the variable region of the light chain shown in SEQ ID NO:7; or

[0019] LFR1 and LFR2 in the variable region of the light chain shown in SEQ ID NO:7.

[0020] In some implementations, the heavy chain frame region and the light chain frame region are a group of the following:

[0021] (1) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:1, and the light chain frame region includes LFR1 in the light chain variable region shown in SEQ ID NO:5;

[0022] (2) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:1, and the light chain frame region includes LFR2 in the light chain variable region shown in SEQ ID NO:6.

[0023] (3) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:1, and the light chain frame region includes LFR1 and LFR2 in the light chain variable region shown in SEQ ID NO:7;

[0024] (4) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:1, and the light chain frame region includes LFR1, LFR2, LFR3 and LFR4 in the light chain variable region shown in SEQ ID NO:28;

[0025] (5) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:2, and the light chain frame region includes LFR1 in the light chain variable region shown in SEQ ID NO:5;

[0026] (6) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:2, and the light chain frame region includes LFR2 in the light chain variable region shown in SEQ ID NO:6;

[0027] (7) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:2, and the light chain frame region includes LFR1 and LFR2 in the light chain variable region shown in SEQ ID NO:7;

[0028] (8) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:2, and the light chain frame region includes LFR1, LFR2, LFR3 and LFR4 in the light chain variable region shown in SEQ ID NO:28;

[0029] (9) The heavy chain frame region includes HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:3, and the light chain frame region includes LFR1 in the light chain variable region shown in SEQ ID NO:5;

[0030] (10) The heavy chain frame region includes HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:3, and the light chain frame region includes LFR2 in the light chain variable region shown in SEQ ID NO:6;

[0031] (11) The heavy chain frame region includes HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:3, and the light chain frame region includes LFR1 and LFR2 in the light chain variable region shown in SEQ ID NO:7;

[0032] (12) The heavy chain frame region includes HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:3, and the light chain frame region includes LFR1, LFR2, LFR3 and LFR4 in the light chain variable region shown in SEQ ID NO:28;

[0033] (13) The heavy chain frame region includes HFR1, HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:4, and the light chain frame region includes LFR1 in the light chain variable region shown in SEQ ID NO:5;

[0034] (14) The heavy chain frame region includes HFR1, HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:4, and the light chain frame region includes LFR2 in the light chain variable region shown in SEQ ID NO:6;

[0035] (15) The heavy chain frame region includes HFR1, HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:4, and the light chain frame region includes LFR1 and LFR2 in the light chain variable region shown in SEQ ID NO:7.

[0036] (16) The heavy chain frame region includes HFR1, HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:4, and the light chain frame region includes LFR1, LFR2, LFR3 and LFR4 in the light chain variable region shown in SEQ ID NO:28.

[0037] (17) The heavy chain frame region includes HFR1, HFR2, HFR3 and HFR4 in the heavy chain variable region shown in SEQ ID NO:27, and the light chain frame region includes LFR1 in the light chain variable region shown in SEQ ID NO:5;

[0038] (18) The heavy chain frame region includes HFR1, HFR2, HFR3 and HFR4 in the heavy chain variable region shown in SEQ ID NO:27, and the light chain frame region includes LFR2 in the light chain variable region shown in SEQ ID NO:6; and

[0039] (19) The heavy chain frame region includes HFR1, HFR2, HFR3 and HFR4 in the heavy chain variable region shown in SEQ ID NO:27, and the light chain frame region includes LFR1 and LFR2 in the light chain variable region shown in SEQ ID NO:7.

[0040] In some embodiments, the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively.

[0041] In some implementations, the heavy chain framework region includes:

[0042] HFR1 as shown in SEQ ID NO:15;

[0043] HFR2 as shown in SEQ ID NO:17;

[0044] HFR3 as shown in SEQ ID NO:18 or 19;

[0045] Alternatively, the light chain framework region may include:

[0046] LFR1 as shown in SEQ ID NO:21; or

[0047] LFR2 as shown in SEQ ID NO:24.

[0048] In some implementations, the heavy chain framework region includes:

[0049] HFR3 as shown in SEQ ID NO:18;

[0050] HFR3 as shown in SEQ ID NO:19;

[0051] HFR2 shown in SEQ ID NO:17, HFR3 shown in SEQ ID NO:19; or

[0052] HFR1 shown in SEQ ID NO:15, HFR2 shown in SEQ ID NO:17, and HFR3 shown in SEQ ID NO:19.

[0053] In some implementations, the light chain framework region includes:

[0054] LFR1 as shown in SEQ ID NO:21;

[0055] LFR2 as shown in SEQ ID NO:24; or

[0056] LFR1 shown in SEQ ID NO:21 and LFR2 shown in SEQ ID NO:24.

[0057] In some implementations, the heavy chain frame region and the light chain frame region are a group of the following:

[0058] (1) The heavy chain frame region includes HFR3 as shown in SEQ ID NO:18, and the light chain frame region includes LFR1 as shown in SEQ ID NO:21;

[0059] (2) The heavy chain frame region includes HFR3 as shown in SEQ ID NO:18, and the light chain frame region includes LFR2 as shown in SEQ ID NO:24;

[0060] (3) The heavy chain frame region includes HFR3 shown in SEQ ID NO:18, and the light chain frame region includes LFR1 shown in SEQ ID NO:21 and LFR2 shown in SEQ ID NO:24;

[0061] (4) The heavy chain frame region includes HFR3 shown in SEQ ID NO:18, and the light chain frame region includes LFR1 shown in SEQ ID NO:22, LFR2 shown in SEQ ID NO:23, LFR3 shown in SEQ ID NO:25 and LFR4 shown in SEQ ID NO:26;

[0062] (5) The heavy chain frame region includes HFR3 as shown in SEQ ID NO:19, and the light chain frame region includes LFR1 as shown in SEQ ID NO:21;

[0063] (6) The heavy chain frame region includes HFR3 as shown in SEQ ID NO:19, and the light chain frame region includes LFR2 as shown in SEQ ID NO:24;

[0064] (7) The heavy chain frame region includes HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:21 and LFR2 shown in SEQ ID NO:24;

[0065] (8) The heavy chain frame region includes HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:22, LFR2 shown in SEQ ID NO:23, LFR3 shown in SEQ ID NO:25 and LFR4 shown in SEQ ID NO:26;

[0066] (9) The heavy chain frame region includes HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:21;

[0067] (10) The heavy chain frame region includes HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR2 shown in SEQ ID NO:24;

[0068] (11) The heavy chain frame region includes HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:21 and LFR2 shown in SEQ ID NO:24;

[0069] (12) The heavy chain frame region includes HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:22, LFR2 shown in SEQ ID NO:23, LFR3 shown in SEQ ID NO:25 and LFR4 shown in SEQ ID NO:26;

[0070] (13) The heavy chain frame region includes HFR1 shown in SEQ ID NO:15, HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:21;

[0071] (14) The heavy chain frame region includes HFR1 shown in SEQ ID NO:15, HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR2 shown in SEQ ID NO:24;

[0072] (15) The heavy chain frame region includes HFR1 shown in SEQ ID NO:15, HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:21 and LFR2 shown in SEQ ID NO:24.

[0073] (16) The heavy chain frame region includes HFR1 shown in SEQ ID NO:15, HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:22, LFR2 shown in SEQ ID NO:23 and LFR3 shown in SEQ ID NO:25 and LFR4 shown in SEQ ID NO:26;

[0074] (17) The heavy chain frame region includes HFR1 shown in SEQ ID NO:14, HFR2 shown in SEQ ID NO:16, HFR3 shown in SEQ ID NO:29 and HFR4 shown in SEQ ID NO:20, and the light chain frame region includes LFR1 shown in SEQ ID NO:21;

[0075] (18) The heavy chain frame region comprises HFR1 shown in SEQ ID NO:14, HFR2 shown in SEQ ID NO:16, HFR3 shown in SEQ ID NO:29, and HFR4 shown in SEQ ID NO:20; and the light chain frame region comprises LFR2 shown in SEQ ID NO:24; and

[0076] (19) The heavy chain frame region includes HFR1 shown in SEQ ID NO:14, HFR2 shown in SEQ ID NO:16, HFR3 shown in SEQ ID NO:29 and HFR4 shown in SEQ ID NO:20, and the light chain frame region includes LFR1 shown in SEQ ID NO:21 and LFR2 shown in SEQ ID NO:24.

[0077] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 1-4 and 27; and / or

[0078] The light chain variable region contains an amino acid sequence as shown in any of SEQ ID NO:5-7 and 28.

[0079] In some embodiments, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:6 or 7.

[0080] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:1, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:5;

[0081] The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:1, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:6;

[0082] The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:1, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:7;

[0083] The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:1, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:28.

[0084] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:2, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:5;

[0085] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:2, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:6;

[0086] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:2, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7;

[0087] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:2, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:28;

[0088] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:3, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:5;

[0089] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:3, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:6;

[0090] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:3, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7;

[0091] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:3, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:28;

[0092] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:4, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:5;

[0093] The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:4, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:6;

[0094] The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:4, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:7;

[0095] The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:4, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:28.

[0096] The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:27, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:5;

[0097] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:6; or

[0098] The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:27, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:7.

[0099] In some implementations, the anti-CD8 antibody or its antigen-binding fragment is scFv, Fab, scFab, F(ab'), F(ab')2, scFv-Fc, or a microantibody.

[0100] In some embodiments, the anti-CD8 antibody or its antigen-binding fragment further comprises human Ig Fc.

[0101] In some implementations, the human Ig is human IgG1, human IgG2, human IgG3, or human IgG4.

[0102] In some implementations, the human Ig Fc is human IgG4 Fc;

[0103] In some implementations, the human IgG4 Fc has S228P, F234A, and L235A mutations.

[0104] This disclosure also provides a multispecific antibody comprising an anti-CD8 antibody or an antigen-binding fragment thereof from any of the above embodiments.

[0105] This disclosure also provides a fusion protein comprising an anti-CD8 antibody or its antigen-binding fragment and an active molecule according to any of the above embodiments.

[0106] This disclosure also provides a nucleic acid encoding an anti-CD8 antibody or its antigen-binding fragment of any of the above embodiments, a multispecific antibody of any of the above embodiments, or a fusion protein of any of the above embodiments.

[0107] In some implementations, the nucleic acid is mRNA.

[0108] This disclosure also provides a DNA that encodes an anti-CD8 antibody or an antigen-binding fragment thereof of any of the above embodiments, a multispecific antibody of any of the above embodiments, or a fusion protein of any of the above embodiments.

[0109] This disclosure also provides a genetic engineering vector comprising DNA from any of the above embodiments.

[0110] This disclosure also provides a host cell comprising nucleic acid of any of the above embodiments, DNA of any of the above embodiments, or a genetic engineering vector of any of the above embodiments.

[0111] This disclosure also provides a conjugate comprising an anti-CD8 antibody or its antigen-binding fragment and an active molecule conjugated thereto, or a multispecific antibody and an active molecule conjugated thereto, according to any of the above embodiments.

[0112] This disclosure also provides a lipid nanoparticle coupled with a targeting domain, the targeting domain comprising an anti-CD8 antibody or its antigen-binding fragment according to any of the above embodiments, or a multispecific antibody according to any of the above embodiments.

[0113] In some embodiments, the lipid nanoparticles comprise ionizable lipids.

[0114] In some embodiments, the lipid nanoparticles also contain accessory lipids, structural lipids, and polymer-lipids.

[0115] In some implementations, polymer-lipids include functionalized polymer-lipids and unfunctionalized polymer-lipids.

[0116] In some embodiments, the auxiliary lipid is a phospholipid, the structural lipid is cholesterol, the unfunctionalized polymer-lipid is a PEG-lipid, and the functionalized polymer-lipid is a maleimide-modified PEG-lipid.

[0117] In some embodiments, the lipid nanoparticles are encapsulated with active molecules.

[0118] This disclosure also provides a pharmaceutical composition comprising an anti-CD8 antibody or its antigen-binding fragment according to any of the above embodiments, a multispecific antibody according to any of the above embodiments, a fusion protein according to any of the above embodiments, a nucleic acid according to any of the above embodiments, DNA according to any of the above embodiments, a genetically engineered vector according to any of the above embodiments, a host cell according to any of the above embodiments, a conjugate according to any of the above embodiments, or a lipid nanoparticle according to any of the above embodiments.

[0119] The anti-CD8 antibody or its antigen-binding fragment, the multispecific antibody, the fusion protein, the nucleic acid, the DNA, the gene-engineered vector, the host cell, the conjugate, the lipid nanoparticle, or the pharmaceutical composition of any of the above embodiments can be used in the preparation of a drug targeting CD8. + Applications of cell-based drugs.

[0120] This disclosure also provides a detection reagent comprising an anti-CD8 antibody or its antigen-binding fragment according to any of the above embodiments, a multispecific antibody according to any of the above embodiments, a fusion protein according to any of the above embodiments, or a conjugate according to any of the above embodiments.

[0121] The application of anti-CD8 antibody or antigen-binding fragment thereof of any of the above embodiments, multispecific antibody of any of the above embodiments, fusion protein of any of the above embodiments, nucleic acid of any of the above embodiments, DNA of any of the above embodiments, genetic engineering vector of any of the above embodiments, host cell of any of the above embodiments, and conjugate of any of the above embodiments in the preparation of detection reagents.

[0122] This disclosure also provides a method for delivering an active molecule to CD8-positive cells, the method comprising administering to a subject in need a fusion protein of any of the above embodiments, a nucleic acid encoding a fusion protein of any of the above embodiments, a conjugate of any of the above embodiments, or a lipid nanoparticle of any of the above embodiments.

[0123] This disclosure also provides a method for treating or preventing a disease, the method comprising administering to a subject an anti-CD8 antibody or antigen-binding fragment thereof comprising or conjugated with an active molecule of any of the above embodiments, a multispecific antibody of any of the above embodiments, a fusion protein of any of the above embodiments, a nucleic acid of any of the above embodiments, DNA of any of the above embodiments, a genetically engineered vector of any of the above embodiments, a conjugate of any of the above embodiments, or a lipid nanoparticle of any of the above embodiments encapsulating an active molecule. Attached Figure Description

[0124] Figure 1 shows the FACS detection of anti-CD8 antibody and human CD3. + Results of T-cell binding.

[0125] Figures 2A and 2B show the binding ability of anti-CD8 antibodies to CD8+ T cells as detected by FACS.

[0126] Figures 3A-3B show the in vitro delivery capability evaluation results of lipid nanoparticles conjugated with anti-CD8 antibodies. In Figures 3A-3B, "hIgG4isotype" represents the lipid nanoparticle group conjugated with antibody hIgG4 isotype, "H2H4" represents the lipid nanoparticle group conjugated with antibody H2H4, "H4H4" represents the lipid nanoparticle group conjugated with antibody H4H4, and "PBS" represents the PBS group.

[0127] Invention Details

[0128] I. Definition

[0129] All patents, patent applications, scientific publications, manufacturers' specifications and guidelines, etc., cited herein, are incorporated herein in their entirety, whether mentioned above or below. Nothing herein should be construed as an admission that this disclosure is not entitled to precede such disclosure.

[0130] Unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, and microbiology used herein are all widely used terms in their respective fields (see, for example, *Molecular Cloning: A Laboratory Manual, 2nd Edition*, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). To better understand this application, definitions and explanations of relevant terms are provided below.

[0131] As used herein, the expressions “comprising,” “including,” “containing,” and “having” are open-ended, meaning they include the listed elements, steps, or components but do not exclude other unlisted elements, steps, or components. The expression “composed of” excludes any unspecified elements, steps, or components. The expression “substantially composed of” means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the essential and novel nature of the claimed subject matter. It should be understood that the expressions “substantially composed of” and “composed of” are encompassed within the meaning of the expression “including.”

[0132] As used herein, unless the context otherwise indicates, the singular forms of “a,” “an,” and “the,” and similar references used in the context of describing this application (particularly in the context of the claims) should be interpreted as encompassing both the singular and plural. The terms “one or more” or “at least one” cover 1, 2, 3, 4, 5, 6, 7, 8, 9, or more.

[0133] The numerical ranges described herein should be understood to encompass any and all subranges contained therein. For example, the range “1 to 10” should be understood to include not only the explicitly stated values ​​of 1 and 10, but also any single value within the range of 1 to 10 (e.g., 2, 3, 4, 5, 6, 7, 8, and 9) and subranges (e.g., 1 to 2, 1.5 to 2.5, 1 to 3, 1.5 to 3.5, 2.5 to 4, 3 to 4.5, etc.). This principle also applies to ranges that use only one numerical value as their minimum or maximum value.

[0134] As used herein, the terms “and / or,” “any combination thereof,” and their grammatical equivalents are used interchangeably. These terms can express any combination specifically. For example, the phrases “A, B, and / or C” or “A, B, C, or any combination thereof” can refer to “A alone; B alone; C alone; A and B; B and C; A and C; and A, B, and C.”

[0135] Unless otherwise stated, all methods described herein may be performed in any suitable order.

[0136] As used herein, the term "% identity" or "% similarity" refers to the percentage of identical nucleotides or amino acids in the best alignment between sequences to be compared. Differences between the two sequences can be distributed across local regions (segments) or the entire length of the sequences being compared. Identity between two sequences is typically determined after the best alignment of a segment or "comparison window." The best alignment can be performed manually or using algorithms known in the art. Algorithms known in the art include, but are not limited to, the local homology algorithms described in Smith and Waterman, 1981, Ads App. Math. 2, 482 and Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443; the similarity search methods described in Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444; or the use of computer programs such as GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. For example, the percentage similarity between two sequences can be determined using the BLASTN or BLASTP algorithms publicly available on the National Center for Biotechnology Information (NCBI) website.

[0137] "% identity" or "% similarity" can be obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100. In some embodiments, a degree of similarity is given for at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the region. In some embodiments, a degree of similarity is given for the entire length of the reference sequence. Alignment for determining sequence similarity can be performed using tools known in the art, preferably using optimal sequence alignment, such as Align, using standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, or Gap Extend 0.5.

[0138] As used herein, "nucleotide" includes deoxyribonucleotides, deoxyribonucleotides, deoxyribonucleotide derivatives, and ribonucleotide derivatives. As used herein, "ribonucleotide" is a constituent of ribonucleic acid (RNA), consisting of one base, one pentose sugar, and one phosphate molecule; it refers to a nucleotide with a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. "Deoxyribonucleotide" is a constituent of deoxyribonucleic acid (DNA), also consisting of one base, one pentose sugar, and one phosphate molecule; it refers to a nucleotide where the hydroxyl group at the 2' position of the β-D-ribofuranosyl group is replaced by hydrogen, and is a major chemical component of chromosomes.

[0139] Nucleotides are usually identified by a single letter representing the bases in them. "A" or "A nucleotide" refers to adenine deoxyribonucleotide or adenine ribonucleotide containing adenine; "C" or "C nucleotide" refers to cytosine deoxyribonucleotide or cytosine ribonucleotide containing cytosine; "G" or "G nucleotide" refers to guanine deoxyribonucleotide or guanine ribonucleotide containing guanine; "U" or "U nucleotide" refers to uracil ribonucleotide containing uracil; and "T" or "T nucleotide" refers to thymine deoxyribonucleotide containing thymine.

[0140] As used herein, the term "nucleic acid" generally refers to any compound comprising a polymer of deoxyribonucleotides (deoxyribonucleic acid, or DNA) or a polymer of ribonucleotides (ribonucleic acid, or RNA), or a combination thereof. Additionally, "nucleic acid" as used herein also includes derivatives of nucleic acids. The term "derivatives of nucleic acids" includes chemical derivatization of nucleic acids at the bases, sugars, or phosphates of nucleotides, as well as nucleic acids containing non-natural nucleotides and nucleotide analogs. Furthermore, in this document, nucleic acids can be in the form of single-stranded or double-stranded linear or covalently closed circular molecules.

[0141] The terms "polynucleotide sequence," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably to refer to the sequence of nucleotides in a polynucleotide. Those skilled in the art should understand that the DNA coding strand (sense strand) and its encoded RNA can be considered to have the same nucleotide sequence, and the deoxythymidine nucleotide in the DNA coding strand sequence corresponds to the uridine nucleotide in its encoded RNA sequence. The RNA corresponding to DNA refers to a polynucleotide in DNA where all T atoms are replaced with U atoms.

[0142] A polynucleotide may contain one or more segments (nucleic acid fragments) (e.g., segments 1, 2, 3, 4, 5, 6, 7, and 8). For example, a polynucleotide may contain a segment encoding a polypeptide of interest. In a particular embodiment, a polynucleotide may contain a segment encoding a polypeptide of interest as well as a regulatory segment (including, but not limited to, segments for transcriptional and translational regulation). In one embodiment, the regulatory segment comprises a polynucleotide corresponding to one or more of the following regulatory elements: a promoter, a 5' untranslated region (5'-UTR), a 3' untranslated region (3'-UTR), and a poly(A) tail.

[0143] As used herein, the term "promoter" refers to a polynucleotide located upstream of the 5' end of the coding region of a gene. It contains a conserved sequence required for the specific binding of RNA polymerase and transcription initiation, activates RNA polymerase, and enables precise binding of RNA polymerase to the template DNA, thus achieving transcription initiation specificity. Promoters can originate from viruses, bacteria, fungi, plants, insects, and animals. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operon-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter, or SV40 late promoter and CMV IE promoter.

[0144] As used herein, the term "5' untranslated region" or "5'-UTR" can refer to an RNA sequence in mRNA that is upstream of the coding sequence and is not translated into protein. A 5'-UTR in a gene typically begins at the transcription start site and ends with a nucleotide upstream of the translation start codon in the coding sequence. The 5'-UTR can contain elements that control gene expression, such as ribosome binding sites, 5'-terminal oligopyrimidine bundles, and translation initiation signals such as the Kozak sequence. mRNA can undergo post-transcriptional modification by adding a 5' cap. Therefore, the 5'-UTR in mature mRNA can also refer to the RNA sequence between the 5' cap and the start codon.

[0145] As used herein, the term "3' untranslated region" or "3'-UTR" can refer to an RNA sequence in mRNA that is downstream of the coding sequence and is not translated into a protein. The 3'-UTR in mRNA is located between a stop codon and a poly(A) sequence of the coding sequence, for example, starting from a nucleotide downstream of the stop codon and ending at a nucleotide upstream of the poly(A) sequence.

[0146] As used herein, the terms “poly(A) nucleotide,” “poly(A) sequence,” and “poly(A) tail” are used interchangeably. Naturally occurring poly(A) sequences typically consist of adenine ribonucleotides. According to this application, the term “modified poly(A) sequence” refers to a poly(A) sequence containing nucleotides or nucleotide segments other than adenine ribonucleotides. Poly(A) sequences are typically located at the 3' end of mRNA, such as the 3' end (downstream) of the 3'-UTR.

[0147] As used herein, the term "5'-cap structure" refers to a structure that is typically located at the 5' end of mature mRNA. In some embodiments, the 5'-cap structure is linked to the 5' end of the mRNA via a 5'-5'-triphosphate bond. 5'-cap structures are typically formed from modified (e.g., methylated) ribonucleotides, particularly guanine nucleotide derivatives. For example, m7GpppN (cap0, or "cap0") is a cap structure formed by the interaction of the 5' phosphate group of hnRNA with the 5' phosphate group of m7GTP via guanylate transferase to form a 5',5'-phosphodiester bond, where N is the terminal 5' nucleotide of the nucleic acid carrying the 5'-cap structure. In some embodiments, the 5'-cap structure includes, but is not limited to, cap 0, cap 1 (a cap structure formed by further methylation of the 2'-OH of the first nucleotide glycosyl group of hnRNA on the basis of cap 0, or "cap1"), cap 2 (a cap structure formed by further methylation of the 2'-OH of the second nucleotide glycosyl group of hnRNA on the basis of cap 1, or "cap2"), cap 4, cap 0 analogue, cap 1 analogue, cap 2 analogue, or cap 4 analogue.

[0148] As used herein, the term “expression” includes the transcription and / or translation of a nucleotide sequence. Therefore, expression can involve the production of transcripts and / or peptides. The term “transcription” refers to the process of transcribing the genetic code in a DNA sequence into RNA (transcription). The term “in vitro transcription” refers to the in vitro synthesis of RNA, particularly mRNA, in a cell-free system (e.g., in a suitable cell extract) (see, e.g., Pardi N., Muramatsu H., Weissman D., Karikó K. (2013). In: Rabinovich P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology (Methods and Protocols), vol 969. Humana Press, Totowa, NJ.). Vectors that can be used to produce transcripts are also called “transcription vectors,” which contain the regulatory sequences required for transcription. The term “transcription” encompasses “in vitro transcription.”

[0149] The term “antibody” is used in the broadest sense herein and encompasses a wide range of antibody structures, including but not limited to genetically engineered or otherwise modified forms of immunoglobulins, such as intracellular antibodies, chimeric antibodies, fully human antibodies, humanized antibodies, antibody fragments, and conjugated antibodies (e.g., bispecific antibodies, bivalent antibodies, trivalent antibodies, tetravalent antibodies, etc.).

[0150] The term "multispecific antibody" refers to an antibody that has binding specificity to at least two different sites (i.e., different epitopes on different antigens or different epitopes on the same antigen).

[0151] The term "coupling" refers to the association between atoms or molecules. This association can be direct (e.g., through covalent bonds) or indirect (e.g., through non-covalent bonds). Non-covalent bonds include, but are not limited to, electrostatic interactions (e.g., ionic bonds, hydrogen bonds, halogen bonds), van der Waals forces, ring stacking (π effect), and hydrophobic interactions.

[0152] As used herein, the term "host cell" refers to a cell used to receive, maintain, replicate, and express polynucleotides or vectors. The term "host cell" includes prokaryotic cells (e.g., *Escherichia coli*) or eukaryotic cells (e.g., yeast cells and insect cells). Examples include cells derived from humans, mice, hamsters, pigs, goats, and primates. Cells can be derived from a variety of tissue types and include primary cells and cell lines. Some specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells, and embryonic stem cells. In other embodiments, the host cell is an antigen-presenting cell, particularly dendritic cells, monocytes, or macrophages. Nucleic acids may be present in the host cell in single or multiple copies. In some embodiments, the host cell may be a cell in which the polypeptide of this application is expressed.

[0153] In the context of this application, the term "plasmid" generally refers to a circular DNA molecule, but the term can also encompass linearized DNA molecules. Specifically, the term "plasmid" also encompasses molecules obtained by linearizing a circular plasmid, for example, by digesting the circular plasmid with a restriction enzyme, thereby converting the circular plasmid molecule into a linear molecule. Plasmids can replicate, i.e., amplify genetic information in a cell independently of chromosomal DNA, and can be used for cloning, i.e., for amplifying genetic information in bacterial cells. In one optional specific example, the DNA plasmid is a medium-copy or high-copy plasmid. In another optional specific example, the DNA plasmid is a high-copy plasmid. Examples of such high-copy plasmids include, for example, pUC and pTZ plasmids or any other plasmid (e.g., pMB1, pCoIE1) containing a replication origin that supports high copy numbers.

[0154] The term "vaccine" is typically understood as a preventive or therapeutic material that provides at least one antigen or has antigenic function, which can stimulate the body's adaptive immune system to provide an adaptive immune response.

[0155] The term "treatment" and similar terms are used herein to generally mean achieving a desired pharmacological and / or physiological effect. Therefore, the treatment of this application may involve the treatment of a disease state, but may also involve preventative treatment with regard to the complete or partial prevention of the disease or its symptoms. In some embodiments, the term "treatment" should be understood as being therapeutic in terms of partially or completely curing the disease and / or the adverse effects and / or symptoms attributable to the disease. Treatment can also be prophylactic or preventive, i.e., measures taken to prevent disease, such as to prevent infection and / or the onset of disease.

[0156] As used herein, the terms "subject" and "patient" are used interchangeably. In some embodiments, the subject is a mammal, such as a human, a non-human primate (e.g., apes, chimpanzees, monkeys, and orangutans), a domesticated animal (including dogs and cats, and livestock (e.g., horses, cattle, pigs, sheep, and goats)), or other mammals. Other mammals include, but are not limited to, mice, rats, guinea pigs, rabbits, hamsters, etc. In a particular embodiment, the subject is a human. In one embodiment, the subject is a mammal (e.g., a human) suffering from an infectious disease or a neoplastic disease. In another embodiment, the subject is a mammal (e.g., a human) at risk of developing an infectious disease or a neoplastic disease.

[0157] As used herein, the term "administration" means the provision or administration of a drug to a subject by any effective route. Exemplary routes of administration include, but are not limited to, one or more of the following: injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intravenous, intraventricular, or intravenous), oral, intraluminal bile duct, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation. When used to treat a disease, condition, symptom, or symptom, the substance is usually administered after the onset of the disease, condition, symptom, or symptom. When used to prevent a disease, condition, symptom, or symptom, the substance is usually administered before the onset of the disease, condition, symptom, or symptom.

[0158] This document describes some elements of the present application. These elements are listed together with specific embodiments; however, it should be understood that they can be combined in any manner and in any number to produce other embodiments. The examples and preferred embodiments described differently should not be construed as limiting the present application to the explicitly described embodiments. This specification should be understood to support and include embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, unless the context otherwise indicates, any permutation and combination of all descriptive elements in this application should be considered as disclosed in the specification of this application.

[0159] II. Anti-CD8 antibodies or their antigen-binding fragments

[0160] This disclosure provides an anti-CD8 antibody or its antigen-binding fragment thereof. The anti-CD8 antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes heavy chain complementarity-determining region 1 (HCDR1), heavy chain complementarity-determining region 2 (HCDR2), and heavy chain complementarity-determining region 3 (HCDR3) as shown in SEQ ID NO:1. The heavy chain variable region also includes a heavy chain framework region. The light chain variable region includes light chain complementarity-determining region 1 (LCDR1), light chain complementarity-determining region 2 (LCDR2), and light chain complementarity-determining region 3 (LCDR3) as shown in SEQ ID NO:5. The light chain variable region also includes a light chain framework region, wherein:

[0161] (1) The heavy-chain frame region contains:

[0162] Heavy chain frame region 1 (HFR1) in the heavy chain variable region shown in SEQ ID NO:4;

[0163] Heavy chain frame region 2 (HFR2) in the heavy chain variable region shown in SEQ ID NO:4; or

[0164] Heavy chain frame region 3 (HFR3) in the heavy chain variable region shown in SEQ ID NO:1 or 4;

[0165] Or, (2) the light chain framework region includes:

[0166] Light chain frame region 1 (LFR1) in the light chain variable region shown in SEQ ID NO:7; or

[0167] Light chain frame region 2 (LFR2) in the light chain variable region shown in SEQ ID NO:7.

[0168] In some implementations, the heavy chain sequentially includes heavy chain frame region 1 (HFR1), heavy chain complement determination region 1 (HCDR1), heavy chain frame region 2 (HFR2), heavy chain complement determination region 2 (HCDR2), heavy chain frame region 3 (HFR3), heavy chain complement determination region 3 (HCDR3), and heavy chain frame region 4 (HFR4), while the light chain sequentially includes light chain frame region 1 (LFR1), light chain complement determination region 1 (LCDR1), light chain frame region 2 (LFR2), light chain complement determination region 2 (LCDR2), light chain frame region 3 (LFR3), light chain complement determination region 3 (LCDR3), and light chain frame region 4 (LFR4).

[0169] In some implementations, the heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:1.

[0170] In some implementations, the heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:4.

[0171] In some implementations, the heavy chain frame region includes HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:4.

[0172] In some implementations, the heavy chain frame region includes HFR1, HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:4.

[0173] In some embodiments, the heavy chain frame region includes HFR1, HFR2, HFR3, and HFR4 from any one of the heavy chain variable regions shown in SEQ ID NO:1 to 4. In some embodiments, the light chain frame region includes LFR1 from the light chain variable region shown in SEQ ID NO:7.

[0174] In some implementations, the light chain frame region includes LFR2 in the light chain variable region shown in SEQ ID NO:7.

[0175] In some implementations, the light chain framework region includes LFR1 and LFR2 in the light chain variable region shown in SEQ ID NO:7.

[0176] In some implementations, the light chain framework region includes LFR1, LFR2, LFR3 and LFR4 of any one of the light chain variable regions shown in SEQ ID NO:5 to 7.

[0177] In some implementations, the heavy chain frame region includes HFR1, HFR2, or HFR3 of the heavy chain variable region shown in SEQ ID NO:4, or the heavy chain variable region shown in SEQ ID NO:1 or 4; the light chain frame region includes LFR1 or LFR2 of the light chain variable region shown in SEQ ID NO:7.

[0178] In some embodiments, the heavy chain frame region includes HFR1, HFR2, HFR3, and HFR4 of the heavy chain variable region shown in SEQ ID NO:1 or 4; the light chain frame region includes LFR1, LFR2, LFR3, and LFR4 of the light chain variable region shown in SEQ ID NO:1 or 4; and LFR4 of the light chain variable region shown in SEQ ID NO:7.

[0179] In some implementations, the heavy chain framework region and the light chain framework region are one of the following:

[0180] (1) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:1, and the light chain frame region includes LFR1 in the light chain variable region shown in SEQ ID NO:5.

[0181] (2) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:1, and the light chain frame region includes LFR2 in the light chain variable region shown in SEQ ID NO:6.

[0182] (3) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:1, and the light chain frame region includes LFR1 and LFR2 in the light chain variable region shown in SEQ ID NO:7.

[0183] (4) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:1, and the light chain frame region includes LFR1, LFR2, LFR3 and LFR4 in the light chain variable region shown in SEQ ID NO:28.

[0184] (5) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:2, and the light chain frame region includes LFR1 in the light chain variable region shown in SEQ ID NO:5.

[0185] (6) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:2, and the light chain frame region includes LFR2 in the light chain variable region shown in SEQ ID NO:6.

[0186] (7) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:2, and the light chain frame region includes LFR1 and LFR2 in the light chain variable region shown in SEQ ID NO:7.

[0187] (8) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:2, and the light chain frame region includes LFR1, LFR2, LFR3 and LFR4 in the light chain variable region shown in SEQ ID NO:28.

[0188] (9) The heavy chain frame region includes HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:3, and the light chain frame region includes LFR1 in the light chain variable region shown in SEQ ID NO:5.

[0189] (10) The heavy chain frame region includes HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:3, and the light chain frame region includes LFR2 in the light chain variable region shown in SEQ ID NO:6.

[0190] (11) The heavy chain frame region includes HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:3, and the light chain frame region includes LFR1 and LFR2 in the light chain variable region shown in SEQ ID NO:7.

[0191] (12) The heavy chain frame region includes HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:3, and the light chain frame region includes LFR1, LFR2, LFR3 and LFR4 in the light chain variable region shown in SEQ ID NO:28.

[0192] (13) The heavy chain frame region includes HFR1, HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:4, and the light chain frame region includes LFR1 in the light chain variable region shown in SEQ ID NO:5;

[0193] (14) The heavy chain frame region includes HFR1, HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:4, and the light chain frame region includes LFR2 in the light chain variable region shown in SEQ ID NO:6.

[0194] (15) The heavy chain frame region includes HFR1, HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:4, and the light chain frame region includes LFR1 and LFR2 in the light chain variable region shown in SEQ ID NO:7.

[0195] (16) The heavy chain frame region includes HFR1, HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:4, and the light chain frame region includes LFR1, LFR2, LFR3 and LFR4 in the light chain variable region shown in SEQ ID NO:28.

[0196] (17) The heavy chain frame region includes HFR1, HFR2, HFR3 and HFR4 in the heavy chain variable region shown in SEQ ID NO:27, and the light chain frame region includes LFR1 in the light chain variable region shown in SEQ ID NO:5;

[0197] (18) The heavy chain frame region includes HFR1, HFR2, HFR3, and HFR4 in the heavy chain variable region shown in SEQ ID NO:27, and the light chain frame region includes LFR2 in the light chain variable region shown in SEQ ID NO:6; and

[0198] (19) The heavy chain frame region includes HFR1, HFR2, HFR3 and HFR4 in the heavy chain variable region shown in SEQ ID NO:27, and the light chain frame region includes LFR1 and LFR2 in the light chain variable region shown in SEQ ID NO:7.

[0199] In some embodiments, the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively.

[0200] In some implementations, the heavy chain frame region includes HFR1 as shown in SEQ ID NO:14 or 15.

[0201] In some implementations, the heavy chain frame region includes HFR2 as shown in SEQ ID NO:16 or 17.

[0202] In some implementations, the heavy chain frame region includes HFR3 as shown in SEQ ID NO:18, 19 or 29.

[0203] In some implementations, the heavy chain frame region includes HFR3 as shown in SEQ ID NO:18 or 19.

[0204] In some implementations, the heavy chain frame region includes HFR1 as shown in SEQ ID NO:14.

[0205] In some implementations, the heavy chain frame region includes HFR1 as shown in SEQ ID NO:15.

[0206] In some implementations, the heavy chain frame region includes HFR2 as shown in SEQ ID NO:16.

[0207] In some implementations, the heavy chain frame region includes HFR2 as shown in SEQ ID NO:17.

[0208] In some implementations, the heavy chain frame region includes HFR3 as shown in SEQ ID NO:18.

[0209] In some implementations, the heavy chain frame region includes HFR3 as shown in SEQ ID NO:19.

[0210] In some implementations, the heavy chain frame region includes HFR4 as shown in SEQ ID NO:20.

[0211] In some implementations, the heavy chain frame region includes HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19.

[0212] In some implementations, the heavy chain frame region includes HFR1 shown in SEQ ID NO:15, HFR2 shown in SEQ ID NO:17, and HFR3 shown in SEQ ID NO:19.

[0213] In some implementations, the light chain frame region includes LFR1 as shown in SEQ ID NO:21 or 22.

[0214] In some implementations, the light chain frame region includes LFR2 as shown in SEQ ID NO:23 or 24.

[0215] In some implementations, the light chain framework region includes LFR1 as shown in SEQ ID NO:21.

[0216] In some implementations, the light chain framework region includes LFR1 as shown in SEQ ID NO:22.

[0217] In some implementations, the light chain framework region includes LFR2 as shown in SEQ ID NO:23.

[0218] In some implementations, the light chain framework region includes LFR2 as shown in SEQ ID NO:24.

[0219] In some implementations, the light chain framework region includes LFR3 as shown in SEQ ID NO:25.

[0220] In some implementations, the light chain framework region includes LFR4 as shown in SEQ ID NO:26.

[0221] In some implementations, the light chain frame region includes LFR1 shown in SEQ ID NO:21 and LFR2 shown in SEQ ID NO:24.

[0222] In some implementations, the heavy chain framework region and the light chain framework region are one of the following:

[0223] (1) The heavy chain frame region contains HFR3 as shown in SEQ ID NO:18, and the light chain frame region contains LFR1 as shown in SEQ ID NO:21;

[0224] (2) The heavy chain frame region contains HFR3 as shown in SEQ ID NO:18, and the light chain frame region contains LFR2 as shown in SEQ ID NO:24;

[0225] (3) The heavy chain frame region includes HFR3 as shown in SEQ ID NO:18, and the light chain frame region includes LFR1 as shown in SEQ ID NO:21 and LFR2 as shown in SEQ ID NO:24;

[0226] (4) The heavy chain frame region includes HFR3 shown in SEQ ID NO:18, and the light chain frame region includes LFR1 shown in SEQ ID NO:22, LFR2 shown in SEQ ID NO:23, LFR3 shown in SEQ ID NO:25 and LFR4 shown in SEQ ID NO:26;

[0227] (5) The heavy chain frame region contains HFR3 as shown in SEQ ID NO:19, and the light chain frame region contains LFR1 as shown in SEQ ID NO:21;

[0228] (6) The heavy chain frame region contains HFR3 as shown in SEQ ID NO:19, and the light chain frame region contains LFR2 as shown in SEQ ID NO:24;

[0229] (7) The heavy chain frame region includes HFR3 as shown in SEQ ID NO:19, and the light chain frame region includes LFR1 as shown in SEQ ID NO:21 and LFR2 as shown in SEQ ID NO:24;

[0230] (8) The heavy chain frame region includes HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:22, LFR2 shown in SEQ ID NO:23, LFR3 shown in SEQ ID NO:25 and LFR4 shown in SEQ ID NO:26;

[0231] (9) The heavy chain frame region includes HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:21;

[0232] (10) The heavy chain frame region includes HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR2 shown in SEQ ID NO:24;

[0233] (11) The heavy chain frame region includes HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:21 and LFR2 shown in SEQ ID NO:24;

[0234] (12) The heavy chain frame region includes HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:22, LFR2 shown in SEQ ID NO:23, LFR3 shown in SEQ ID NO:25 and LFR4 shown in SEQ ID NO:26;

[0235] (13) The heavy chain frame region includes HFR1 shown in SEQ ID NO:15, HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:21;

[0236] (14) The heavy chain frame region includes HFR1 shown in SEQ ID NO:15, HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR2 shown in SEQ ID NO:24;

[0237] (15) The heavy chain frame region includes HFR1 shown in SEQ ID NO:15, HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:21 and LFR2 shown in SEQ ID NO:24.

[0238] (16) The heavy chain frame region includes HFR1 shown in SEQ ID NO:15, HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:22, LFR2 shown in SEQ ID NO:23, LFR3 shown in SEQ ID NO:25 and LFR4 shown in SEQ ID NO:26;

[0239] (17) The heavy chain frame region includes HFR1 shown in SEQ ID NO:14, HFR2 shown in SEQ ID NO:16, HFR3 shown in SEQ ID NO:29 and HFR4 shown in SEQ ID NO:20, and the light chain frame region includes LFR1 shown in SEQ ID NO:21;

[0240] (18) The heavy chain frame region includes HFR1 shown in SEQ ID NO:14, HFR2 shown in SEQ ID NO:16, HFR3 shown in SEQ ID NO:29, and HFR4 shown in SEQ ID NO:20; the light chain frame region includes LFR2 shown in SEQ ID NO:24; and

[0241] (19) The heavy chain frame region includes HFR1 shown in SEQ ID NO:14, HFR2 shown in SEQ ID NO:16, HFR3 shown in SEQ ID NO:29 and HFR4 shown in SEQ ID NO:20, and the light chain frame region includes LFR1 shown in SEQ ID NO:21 and LFR2 shown in SEQ ID NO:24.

[0242] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO:1-4 and 27; and / or, the light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO:5-7 and 28.

[0243] In some embodiments, the heavy chain variable region contains an amino acid sequence as shown in any of SEQ ID NO:1-4 and 27.

[0244] In some implementations, the heavy chain variable region contains an amino acid sequence as shown in any one of SEQ ID NO:1 to 4.

[0245] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NO:1 to 4.

[0246] In some embodiments, the light chain variable region contains an amino acid sequence as shown in any of SEQ ID NO:5-7 and 28.

[0247] In some implementations, the light chain variable region contains an amino acid sequence as shown in any of SEQ ID NO:5 to 7.

[0248] In some implementations, the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO:5 to 7.

[0249] In some implementations, the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:6.

[0250] In some implementations, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:6.

[0251] In some implementations, the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:7.

[0252] In some implementations, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:7.

[0253] In some embodiments, the heavy chain variable region contains an amino acid sequence as shown in any one of SEQ ID NO:1-4 and 27, and the light chain variable region contains an amino acid sequence as shown in any one of SEQ ID NO:5-7 and 28.

[0254] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NO:1-4 and 27, and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO:5-7 and 28.

[0255] In some embodiments, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:1, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:5.

[0256] In some embodiments, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:1, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:6.

[0257] In some embodiments, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:1, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:7.

[0258] In some embodiments, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:1, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:28.

[0259] In some embodiments, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:2, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:5.

[0260] In some embodiments, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:2, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:6.

[0261] In some embodiments, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:2, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:7.

[0262] In some embodiments, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:2, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:28.

[0263] In some embodiments, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:3, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:5.

[0264] In some embodiments, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:3, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:6.

[0265] In some embodiments, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:3, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:7.

[0266] In some embodiments, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:3, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:28.

[0267] In some embodiments, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:4, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:5.

[0268] In some embodiments, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:4, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:6.

[0269] In some embodiments, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:4, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:7.

[0270] In some embodiments, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:4, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:28.

[0271] In some embodiments, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:27, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:5.

[0272] In some embodiments, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:27, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:6.

[0273] In some embodiments, the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:27, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:7.

[0274] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:5.

[0275] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:6.

[0276] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:7.

[0277] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:28.

[0278] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:5.

[0279] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:6.

[0280] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:7.

[0281] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:28.

[0282] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:5.

[0283] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:6.

[0284] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:7.

[0285] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:28.

[0286] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:5.

[0287] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:6.

[0288] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:7.

[0289] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:28.

[0290] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:27, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:5.

[0291] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:27, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:6.

[0292] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:27, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:7.

[0293] In some embodiments, the anti-CD8 antibody encompasses an amino acid sequence variant of the anti-CD8 antibody of any of the above embodiments. For example, it may be desirable to improve the binding affinity and / or other biological properties of the aforementioned anti-CD8 antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of residues within the amino acid sequence of the anti-CD8 antibody (such as in one or more CDR and / or framework sequences, or in the VH and / or VL domains). Amino acid sequence variants of the anti-CD8 antibody can be prepared by introducing suitable modifications into the nucleotide sequence encoding the protein or through peptide synthesis.

[0294] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the amino acid sequence shown in any one of SEQ ID NO: 1-4 and 27; and / or, the light chain variable region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and less than 100% identity with the amino acid sequence shown in any one of SEQ ID NO: 5-7 and 28.

[0295] In some implementations, the anti-CD8 antibody or its antigen-binding fragment is scFv, Fab, scFab, F(ab'), F(ab')2, scFv-Fc, or a minibodies.

[0296] In some implementations, the anti-CD8 antibody or its antigen-binding fragment also contains human Ig Fc.

[0297] In some implementations, human Ig is human IgG1, human IgG2, human IgG3, or human IgG4.

[0298] In some implementations, human Ig Fc has mutations that enhance its stability.

[0299] In some implementations, human Ig Fc has mutations that weaken its effects.

[0300] In some implementations, the anti-CD8 antibody or its antigen-binding fragment also contains human IgG4 Fc.

[0301] In some implementations, human IgG4 Fc has one or more of the following mutations: S228P, F234A, and L235A.

[0302] III. Multispecific antibodies

[0303] This disclosure also provides a multispecific antibody comprising an anti-CD8 antibody or an antigen-binding fragment thereof from any of the above embodiments.

[0304] In some implementations, the multispecific antibody is a bispecific antibody.

[0305] In some implementations, the multispecific antibody is a trispecific antibody or more antibodies that bind to specific antibodies.

[0306] In some implementations, multispecific antibodies also include an active molecule or a binding site that binds to the active molecule.

[0307] IV. Fusion Proteins and Conjugates

[0308] This disclosure also provides a fusion protein comprising an anti-CD8 antibody or its antigen-binding fragment and an active molecule from any of the above embodiments.

[0309] In some implementations, the active molecule is a detectable marker. Detectable markers include proteins or peptides that facilitate the diagnosis, detection, or visualization of the location and / or quantity of target molecules, cells, tissues, organs, etc.

[0310] In some implementations, the active molecule is a protein or polypeptide used to prevent or treat a disease.

[0311] In some embodiments, the active molecule is a protein or peptide that is beneficial for treating cancer, inflammation, other disease disorders, or other immune-suppressing responses. For example, a protein or peptide used for immunosuppression in organ transplantation. In some embodiments, the active molecule includes, but is not limited to, one or more of the following: therapeutic antibodies or antibody fragments (e.g., cetuximab, bevacizumab) and enzymes (e.g., enzymes that cleave a prodrug into a cytotoxic agent at the binding site of an anti-CD8 antibody or its antigen-binding fragment).

[0312] In some implementations, the active molecule is a protein or peptide used to treat tumors or cancer.

[0313] In some implementations, the active molecule is a cytotoxic or cell-inhibiting protein or peptide.

[0314] In some implementations, the active molecule is a therapeutic antibody or antibody fragment.

[0315] In some implementations, the active molecule is directly linked to the anti-CD8 antibody or its antigen-binding fragment via non-covalent bonds (e.g., electrostatic interactions, hydrogen bonds, hydrophobic interactions, van der Waals forces, etc.).

[0316] In some implementations, the active molecule is indirectly linked to the anti-CD8 antibody or its antigen-binding fragment via a coupling connector in a non-covalent manner.

[0317] In some implementations, the active molecule is directly covalently linked to the anti-CD8 antibody or its antigen-binding fragment.

[0318] In some implementations, the active molecule is indirectly linked to the anti-CD8 antibody or its antigen-binding fragment via a linker covalent bond.

[0319] In some implementations, the active molecule is one or more.

[0320] This disclosure also provides a conjugate comprising an anti-CD8 antibody or its antigen-binding fragment from any of the above embodiments and an active molecule conjugated thereto.

[0321] In some embodiments, the active molecule is a detectable marker. Detectable markers include atoms, molecules, or compounds that facilitate the diagnosis, detection, or visualization of the location and / or quantity of target molecules, cells, tissues, organs, etc. Detectable markers that can be used according to the embodiments described herein include, but are not limited to, radioactive substances (e.g., radioisotopes, radionuclides, radiolabeled substances, or radiotracers), dyes (e.g., IndoCyanine Green (ICG)), contrast agents, fluorescent compounds or molecules, bioluminescent compounds or molecules, enzymes, and enhancers (e.g., paramagnetic ions).

[0322] In some implementations, the active molecule is used to prevent or treat diseases.

[0323] In some embodiments, the active molecule is beneficial for treating cancer, inflammation, other disease disorders, or other immune response suppression. For example, it may be an atom, molecule, or compound used for immunosuppression in organ transplantation. In some embodiments, the active molecule includes, but is not limited to, one or more of the following: drugs, chemotherapeutic agents, therapeutic antibodies or antibody fragments, toxins, radioisotopes, enzymes (e.g., enzymes that cleave prodrugs into cytotoxic agents at the binding site of anti-CD8 antibodies or their antigen-binding fragments), ribozymes, hormones, immunomodulators, antisense oligonucleotides, chelating agents, and boron compounds.

[0324] In some implementations, the active molecule is used to treat tumors or cancer.

[0325] In some implementations, the active molecule is cytotoxic or cell-inhibiting.

[0326] In some implementations, the active molecule is a protein or polypeptide.

[0327] In some implementations, the active molecule is a therapeutic antibody or its antigen-binding fragment.

[0328] In some implementations, the active molecule is a nucleic acid.

[0329] In some embodiments, the active molecule is a nucleic acid based on RNA interference technology or gene therapy. In some embodiments, the active molecule is one or more of the following: small interfering RNA (siRNA), microRNA (miRNA), small ribonucleic acid (shRNA), sgRNA, or mRNA encoding a gene editing-related protein (e.g., Cas9).

[0330] In some implementations, the active molecule is mRNA.

[0331] In some implementations, the active molecule is a small molecule compound. Examples include carboplatin, epirubicin, paclitaxel, and gefitinib.

[0332] In some implementations, the active molecule is one or more.

[0333] It is understood that, in some implementation schemes, the active molecules used to prevent or treat diseases are not limited to proteins or peptides, nucleic acids and small molecule compounds, but may also include other substances that can be used to prevent or treat diseases, such as viruses, oncolytic bacteria, etc.

[0334] This disclosure also provides another conjugate comprising a multispecific antibody of any of the above embodiments and an active molecule conjugated thereto.

[0335] In some implementations, the active molecule is as described above.

[0336] This disclosure also provides another conjugate comprising the fusion protein of any of the above embodiments and the active molecule conjugated thereto.

[0337] In some implementations, the active molecule is as described above.

[0338] In some embodiments, the anti-CD8 antibody or its antigen-binding fragment, multispecific antibody or fusion protein of the conjugate is directly conjugated to the active molecule via covalent bonds (e.g., peptide bonds, ester bonds, thioether bonds, imine bonds, disulfide bonds, etc.).

[0339] In some embodiments, the conjugate's anti-CD8 antibody or its antigen-binding fragment, multispecific antibody or fusion protein is indirectly conjugated to the active molecule via covalent bonds (e.g., using a linker for indirect covalent conjugation).

[0340] In some embodiments, the conjugate's anti-CD8 antibody or its antigen-binding fragment, multispecific antibody or fusion protein is conjugated to the active molecule via non-covalent bonds (e.g., electrostatic interactions, hydrogen bonds, hydrophobic interactions, van der Waals forces, etc.).

[0341] In some embodiments, the conjugate comprises the fusion protein of any of the above embodiments and another active molecule conjugated thereto, which is different from the active molecule in the fusion protein.

[0342] V. DNA, nucleic acids and their preparation methods, gene engineering vectors, and host cells

[0343] This disclosure also provides a nucleic acid that encodes an anti-CD8 antibody or its antigen-binding fragment of any of the above embodiments, a multispecific antibody of any of the above embodiments, or a fusion protein of any of the above embodiments.

[0344] In some implementations, the nucleic acid is mRNA.

[0345] In some implementations, the nucleic acid is DNA.

[0346] This disclosure also provides an mRNA encoding an anti-CD8 antibody or its antigen-binding fragment according to any of the above embodiments, a multispecific antibody according to any of the above embodiments, or a fusion protein according to any of the above embodiments.

[0347] This disclosure also provides a DNA for preparing mRNA according to any of the above embodiments.

[0348] This disclosure also provides a DNA that can be transcribed into mRNA according to any of the above embodiments.

[0349] This disclosure also provides a DNA that can be transcribed and post-transcribed (e.g., capped) into mRNA according to any of the above embodiments.

[0350] This disclosure also provides a DNA that encodes an anti-CD8 antibody or an antigen-binding fragment thereof of any of the above embodiments, a multispecific antibody of any of the above embodiments, or a fusion protein of any of the above embodiments.

[0351] In some embodiments, the DNA comprises at least one of a 5'-UTR, a 3'-UTR, and a polynucleotide encoding a poly(A) tail, and a polynucleotide encoding an anti-CD8 antibody or an antigen-binding fragment thereof for any of the embodiments described above.

[0352] In some embodiments, the DNA described above comprises a 5'-UTR, a 3'-UTR, and a polynucleotide encoding a poly(A) tail, as well as a polynucleotide encoding an anti-CD8 antibody or an antigen-binding fragment thereof for any of the embodiments described above.

[0353] In addition, this disclosure also provides a gene engineering vector comprising DNA or nucleic acid of any of the above embodiments, or the gene engineering vector comprising a polynucleotide capable of being transcribed into mRNA of any of the above embodiments, or the gene engineering vector capable of being transcribed or transcribed and post-transcribed into mRNA of any of the above embodiments.

[0354] In some embodiments, the genetic engineering vector is an expression vector. In some embodiments, the genetic engineering vector is a plasmid, a granule, a virus (e.g., adenovirus, adeno-associated virus), a bacteriophage, or another vector conventionally used in genetic engineering. In one optional specific example, the genetic engineering vector is a plasmid. In one optional specific example, the genetic engineering vector is adeno-associated virus (AAV). In some embodiments, the genetic engineering vector further comprises at least one or more of the following: origin of replication (ORI), a marker gene or a fragment thereof, a reporter gene or a fragment thereof, and a restriction site allowing the insertion of a DNA element. In one optional specific example, the restriction site allowing the insertion of a DNA element is a multiple cloning site (MCS).

[0355] In some embodiments, the above-described genetic engineering vector comprises a promoter, a 5'-UTR, a polynucleotide encoding an anti-CD8 antibody or an antigen-binding fragment thereof of any of the above embodiments, a 3'-UTR, and a polynucleotide encoding a poly(A) tail, wherein the polynucleotide encoding the poly(A) tail, the promoter, the 5'-UTR, the polynucleotide encoding the anti-CD8 antibody or an antigen-binding fragment thereof, and the 3'-UTR are operatively linked to each other.

[0356] In other implementations, the aforementioned genetic engineering vector is a cloning vector.

[0357] This disclosure also provides a method for preparing DNA or nucleic acid according to any of the above embodiments, the method comprising the step of introducing (e.g., in plasmid form) a gene engineering vector according to any of the above embodiments into a host cell (e.g., Escherichia coli) and then culturing the host cell containing the gene engineering vector.

[0358] In addition, this disclosure also provides another method for preparing the above-described DNA, which includes the step of preparing the DNA using a chemical synthesis method based on the nucleotide sequence of the DNA according to any of the above embodiments. It is understood that the specific method of chemical synthesis can be a method known in the art, such as the solid-phase phosphorus amide method.

[0359] It is understood that the method for preparing DNA or nucleic acid in any of the above embodiments is not limited to the above, and may also be other methods.

[0360] In addition, this disclosure also provides a host cell comprising nucleic acid of any of the above embodiments, DNA of any of the above embodiments, or genetic engineering vector of any of the above embodiments.

[0361] In some implementations, the host cell is a separated cell.

[0362] In some implementations, the host cell is used to store and / or amplify the DNA of any of the above implementations.

[0363] In some implementations, the host cell is a bacterial cell. Bacterial host cells include *Escherichia coli* (E. coli) cells, which are well-known to those skilled in the art.

[0364] The host cells of this disclosure can be prepared by transforming competent host cells with a genetically engineered vector according to any of the above embodiments. Competent host cells are cells capable of taking up free extracellular genetic material (e.g., DNA plasmids) in a sequence-independent manner. Many bacterial cells known to those skilled in the art are naturally capable of taking up exogenous DNA from the environment and can therefore serve as bacterial host cells according to this disclosure. Furthermore, those skilled in the art know that competent bacterial host cells can be obtained from naturally non-competent bacterial cells using methods such as electroporation or chemicals (e.g., treatment with calcium ions accompanied by high-temperature exposure). After take-up, the exogenous DNA preferably neither degrades nor integrates into the genome of the bacterial host cell.

[0365] In addition, this disclosure also provides an anti-CD8 antibody or its antigen-binding fragment obtained by translating mRNA from any of the above embodiments.

[0366] In addition, this disclosure also provides a method for preparing an anti-CD8 antibody or its antigen-binding fragment, the method comprising: transcribing a polynucleotide (e.g., DNA or a genetic engineering vector) encoding an anti-CD8 antibody or its antigen-binding fragment into RNA; and translating the transcribed RNA into an anti-CD8 antibody or its antigen-binding fragment.

[0367] In addition, this disclosure also provides another method for preparing an anti-CD8 antibody or its antigen-binding fragment, the method comprising: translating an mRNA containing a polynucleotide encoding an anti-CD8 antibody or its antigen-binding fragment of any of the above embodiments into an anti-CD8 antibody or its antigen-binding fragment.

[0368] In some embodiments, the preparation of the anti-CD8 antibody or its antigen-binding fragment according to any of the above embodiments is carried out entirely or partially in vitro.

[0369] VI. Pharmaceutical Compositions and Lipid Nanoparticles

[0370] This disclosure also provides a pharmaceutical composition comprising an anti-CD8 antibody or its antigen-binding fragment according to any of the above embodiments, a multispecific antibody according to any of the above embodiments, a fusion protein according to any of the above embodiments, a nucleic acid according to any of the above embodiments, DNA according to any of the above embodiments, a genetic engineering vector according to any of the above embodiments, a host cell according to any of the above embodiments, a conjugate according to any of the above embodiments, or mRNA according to any of the above embodiments.

[0371] In some embodiments, the above-described pharmaceutical compositions further comprise a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means approved for use in animals and / or humans by a regulatory authority (e.g., the China Food and Drug Administration, the U.S. Food and Drug Administration (FDA)) or a recognized pharmacopoeia (e.g., the Chinese Pharmacopoeia, the European Pharmacopoeia). The term "pharmaceuticalally acceptable carrier" refers to a substance that can be administered with the anti-CD8 antibody or its antigen-binding fragment thereof, mRNA, fusion protein, multispecific antibody, nucleic acid, conjugate, DNA, genetically engineered vector, or host cell, including but not limited to delivery carriers, diluents, sweeteners, flavoring agents, wetting agents, adjuvants, flow aids, preservatives, dyes / coloring agents, surfactants, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers.

[0372] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, and an anti-CD8 antibody or antigen-binding fragment thereof of any of the above embodiments, a multispecific antibody of any of the above embodiments, a fusion protein of any of the above embodiments, a nucleic acid of any of the above embodiments, DNA of any of the above embodiments, a genetically engineered vector of any of the above embodiments, a host cell of any of the above embodiments, a conjugate of any of the above embodiments, or mRNA of any of the above embodiments.

[0373] In some implementations, the pharmaceutically acceptable carrier is a delivery carrier.

[0374] In some embodiments, the pharmaceutically acceptable vector is a delivery vector in which the mRNA, nucleic acid, DNA, or genetically engineered vector of any of the above embodiments is formulated (e.g., encapsulated).

[0375] In some embodiments, the delivery carrier is selected from a combination or one of the following: lipid nanoparticles (LNPs), liposomes, cationic proteins, vesicles, microparticles, polymers, and micelles. In some embodiments, the delivery carrier is selected from one of the following: LNPs, liposomes, cationic proteins, vesicles, microparticles, polymers, and micelles.

[0376] In some embodiments, the delivery carrier is a lipid nanoparticle. In some embodiments, the pharmaceutical composition comprises an mRNA of any of the above embodiments, the mRNA being formulated within the lipid nanoparticle.

[0377] In some embodiments, the pharmaceutical composition comprises multiple mRNAs of any of the above embodiments, with the multiple mRNAs co-formulated within lipid nanoparticles (i.e., a single lipid nanoparticle contains (e.g., encapsulates) multiple mRNAs).

[0378] In some embodiments, the pharmaceutical composition comprises multiple mRNAs of any of the above embodiments, with the multiple mRNAs individually formulated within lipid nanoparticles (i.e., a single lipid nanoparticle contains (e.g., encapsulates) one mRNA).

[0379] In some implementations, the above-mentioned pharmaceutical composition is a vaccine.

[0380] In some implementation schemes, the vaccine is a nucleic acid vaccine.

[0381] In some implementations, the vaccine is an mRNA vaccine.

[0382] This disclosure also provides lipid nanoparticles (LNPs) coupled with a targeting domain, the targeting domain comprising an anti-CD8 antibody or its antigen-binding fragment according to any of the above embodiments, a multispecific antibody according to any of the above embodiments, a fusion protein according to any of the above embodiments, or a conjugate according to any of the above embodiments.

[0383] In some embodiments, the lipid nanoparticles contain one or more of the following substances: ionizable lipids, auxiliary lipids, structural lipids, and polymer-lipids.

[0384] In some implementations, the lipid nanoparticles contain ionizable lipids.

[0385] In some implementations, lipid nanoparticles contain ionizable lipids, auxiliary lipids, structural lipids, and polymer-lipids.

[0386] In some embodiments, the polymer-lipid comprises both unfunctionalized and functionalized polymer-lipids. In other embodiments, the polymer-lipid is unfunctionalized.

[0387] In some embodiments, the lipid nanoparticles contain ionizable lipids, auxiliary lipids, structural lipids, and polymer-lipids, including unfunctionalized polymer-lipids and functionalized polymer-lipids.

[0388] In some embodiments, the lipid nanoparticles comprise ionizable lipids, accessory lipids, structural lipids, and polymer-lipids. The polymer-lipids include unfunctionalized and functionalized polymer-lipids. The ionizable lipids comprise 25 mol% to 75 mol% of the total lipids present in the lipid nanoparticles, the accessory lipids comprise 0 mol% to 45 mol% of the total lipids present in the lipid nanoparticles, the structural lipids comprise 0 mol% to 60 mol% of the total lipids present in the lipid nanoparticles, and the polymer-lipids comprise 0.5 mol% to 5 mol% of the total lipids present in the lipid nanoparticles.

[0389] In some embodiments, ionizable lipids account for 30 mol% to 65 mol%, 30 mol% to 60 mol%, 35 mol% to 60 mol%, 40 mol% to 60 mol%, 45 mol% to 55 mol% or 50 mol% to 55 mol% of the total lipids present in the lipid nanoparticles.

[0390] In some embodiments, the auxiliary lipids account for 1 mol% to 40 mol%, 5 mol% to 40 mol%, 5 mol% to 35 mol%, 5 mol% to 30 mol%, 5 mol% to 25 mol%, or 5 mol% to 15 mol% of the total lipids present in the lipid nanoparticles.

[0391] In some embodiments, the structural lipids account for 1 mol% to 60 mol%, 1 mol% to 55 mol%, 5 mol% to 55 mol%, 10 mol% to 50 mol%, 15 mol% to 50 mol%, 15 mol% to 45 mol%, 20 mol% to 45 mol%, or 25 mol% to 40 mol% of the total lipids present in the lipid nanoparticles.

[0392] In some embodiments, the polymer-lipid accounts for 0.5 mol% to 4.5 mol%, 1 mol% to 4.5 mol%, 1 mol% to 4 mol%, 1.5 mol% to 4 mol%, 1.5 mol% to 3.5 mol%, or 1.5 mol% to 3 mol% of the total lipids present in the lipid nanoparticles.

[0393] In some embodiments, ionizable lipids account for 45 mol% to 55 mol% of the total lipids present in the lipid nanoparticles, phospholipids account for 5 mol% to 25 mol% of the total lipids present in the lipid nanoparticles, structural lipids account for 25 mol% to 45 mol% of the total lipids present in the lipid nanoparticles, and polymer-lipids account for 1 mol% to 4.5 mol% of the total lipids present in the lipid nanoparticles.

[0394] In some implementations, the total lipids present in the lipid nanoparticles consist of ionizable lipids, accessory lipids, structural lipids, and polymer-lipids.

[0395] The term "ionizable lipid" refers to a lipid that becomes positively charged when the pH drops below the pKa of its ionizable groups, but gradually becomes neutral at higher pH values. Below the pKa, the positively charged lipid can bind to negatively charged nucleic acids. In some embodiments, ionizable lipids include zwitterionic lipids.

[0396] In some implementations, the ionizable lipid is compound 2-3, its salt, or an isomer thereof:

[0397] In some embodiments, the cofactor lipid includes phospholipids. Phospholipids are typically semi-synthetic, but may also be of natural origin or chemically modified. In one optional specific example, the cofactor lipid is a phospholipid. In some embodiments, the phospholipid includes one or more of the following: DSPC (distearylphosphatidylcholine), DOPE (dioleoylphosphatidylethanolamine), DOPC (dioleoyllecithin), DOPS (dioleoylphosphatidylserine), DSPG (1,2-octacosanoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol)), DPPG (dispalmitoylphosphatidylglycerol), DPPC (dispalmitoylphosphatidylcholine), DGTS (1,2-dispalmitoyl-sn-glycerol-3-O-4'-(N,N,N-trimethyl)homoserine), and lysophospholipids. In some embodiments, the cofactor lipid is selected from one or more of the following: DSPC, DOPE, DOPC, and DOPS. In some embodiments, the cofactor lipid is DSPC and / or DOPE.

[0398] In some embodiments, the structural lipid comprises sterols. In one optional specific example, the structural lipid is a sterol. In some embodiments, the sterol comprises one or more of the following: 20α-hydroxycholesterol, cholesterol, cholesterol esters, sterol hormones, sterol vitamins, bile acids, ergosterol, β-sitosterol, and oxidized cholesterol derivatives. In some embodiments, the structural lipid comprises at least one of cholesterol, cholesterol esters, sterol hormones, sterol vitamins, and bile acids. In some embodiments, the structural lipid is cholesterol. In one optional specific example, the structural lipid is high-purity cholesterol, particularly injectable high-purity cholesterol, such as CHO-HP (produced by AVT). In other embodiments, the structural lipid is 20α-hydroxycholesterol.

[0399] Polymer-lipid conjugates refer to conjugates comprising a polymer and a lipid coupled to that polymer.

[0400] In some embodiments, the lipids of the unfunctionalized polymer-lipid include one or more of the following: 1,2-dimyristoyl-sn-glycerol (DMG), distearoyl-phosphatidyl-ethanolamine (DSPE), diacylglycerol (DAG), dialkyloxypropyl (DAA), phospholipids, ceramide (Cer), 1,2-distearoyl-rac-glycerol (DSG), and 1,2-dipalmitoyl-rac-glycero (DPG).

[0401] In some embodiments, the unfunctionalized polymer-lipid polymer includes one or two of the following: hydrophilic polymers and zwitterionic polymers.

[0402] In some embodiments, the unfunctionalized polymer-lipid polymer is a hydrophilic polymer. In other embodiments, the unfunctionalized polymer-lipid polymer is a zwitterionic polymer.

[0403] In some embodiments, the hydrophilic polymer includes one or more of the following: polyethylene glycol (PEG), poly(oxazolines) (POX), poly(glycerols) (PGs), poly(hydroxypropyl methacrylate) (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), polyvinylpyrrolidone (PVP), poly(N,N-dimethyl acrylamide) (PDMA), poly(N-acryloyl morpholine) (PAcM), polyamino acids, glycosaminoglycans (GAGs), heparin, and hyaluronic acid. The following are listed: HA (acid), polysialic acid (PSA), elastin-like polypeptides (ELPs), serum albumin, and CD47.

[0404] Correspondingly, the unfunctionalized polymer-lipids include one or more of the following: polyethylene glycol-lipids (PEG-lipids), polyoxazoline-lipids, polyglycerol-lipids, polyhydroxypropyl methacrylate-lipids, poly-2-hydroxyethyl methacrylate-lipids, poly-N-(2-hydroxypropyl)methacrylamide-lipids, polyvinylpyrrolidone-lipids, poly-N,N-dimethylacrylamide-lipids, poly-N-acryloylmorpholine-lipids, glycosaminoglycan-lipids, heparin-lipids, hyaluronic acid-lipids, polysialic acid-lipids, elastin-like lipids, serum albumin-lipids, and CD47-lipids. It should be noted that "PEG-lipids" are conjugates of polyethylene glycol and lipids, "polyoxazoline-lipids" refer to conjugates formed by coupling polyoxazoline and lipids, and "polyglycerol-lipids" refer to conjugates formed by coupling polyglycerol and lipids; the same applies to other polymer-lipids. In an optional specific example, the hydrophilic polymer includes polyethylene glycol.

[0405] In some embodiments, the unfunctionalized polymer-lipid includes PEG-lipid. In one optional specific example, the unfunctionalized polymer-lipid is a PEG-lipid. In some embodiments, the PEG-lipid includes one or more of the following: myristoyl glycerol-PEG (DMG-PEG), distearate phosphatidylethanolamine-PEG (DSPE-PEG), diacylglycerol-PEG (DAG-PEG), dialkyloxypropyl-PEG (DAA-PEG), phospholipid-PEG, ceramide-PEG (Cer-PEG), 1,2-distearate-rac-glycerol-PEG (DSG-PEG), and 1,2-dispalmitoyl-rac-glycerol-PEG (DPG-PEG). The PEG-lipid is preferably DMG-PEG, DSG-PEG, or DPG-PEG. DMG-PEG is a polyethylene glycol derivative of 1,2-dimyristoyl glycerol. In some embodiments, the average molecular weight of the PEG in the PEG-lipid is about 2000 to 5000. In one optional specific example, the average molecular weight of PEG in the PEG-lipid is about 2000.

[0406] In some embodiments, the zwitterionic polymer includes one or more of the following: poly(carboxybetaine) (pCB), poly(sulfobetaine) (pSB), phosphobetaine-based polymers, and phosphorylcholine polymers. In some embodiments, the zwitterionic polymer includes one or more of the following: poly(carboxybetaine acrylamide, pCBAA), poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(methacryloyloxyethyl phosphorylcholine), poly(vinyl-pyridinio propanesulfonate), poly(carboxybetaine) based on vinylimidazole, poly(sulfobetaine) based on vinylimidazole, and poly(sulfobetaine) based on vinylpyridine.

[0407] Correspondingly, the unfunctionalized polymer-lipids include one or more of the following: polyhydroxybetaine-lipids, polysulfobetaine-lipids, phosphate betaine-based polymer-lipids, and phosphate choline polymer-lipids. In some embodiments, the polymer-lipids include one or more of the following: poly(carboxybetaine acrylamide)-lipids, poly(carboxybetaine methacrylate)-lipids, poly(sulfobetaine methacrylate)-lipids, poly(methacryloyloxyethyl phosphorylcholine)-lipids, poly(vinylpyridinylpropanesulfonate)-lipids, polyvinylimidazolyl betaine-lipids, polyvinylimidazolyl sulfobetaine-lipids, and polyvinylpyridinyl sulfobetaine-lipids.

[0408] In addition, in some embodiments, the unfunctionalized polymer-lipid can also be any one or more PEG-lipids in Hoang Thi, Thai Thanh et al., Polymers, 12(2), 298(2020), the PEG-lipids in the above literature are incorporated herein by reference.

[0409] In some implementations, functionalized polymer-lipids are used to couple lipid nanoparticles with target domains.

[0410] In some embodiments, the functionalized polymer-lipid is a modified version of an unfunctionalized polymer-lipid. In some embodiments, the modification is maleimide modification.

[0411] In some implementations, the functionalized polymer-lipid is a maleimide-modified PEG-lipid.

[0412] In some embodiments, the maleimide-modified PEG-lipids include one or two of the following: DSPE-PEG-Mal (DSPE-PEG-Maleimide) and DMG-PEG-Mal (DMG-PEG-Maleimide).

[0413] In some implementations, LNP contains ionizable lipids, phospholipids, cholesterol, PEG-lipids, and maleimide-modified PEG-lipids.

[0414] In some implementation schemes, lipid nanoparticles are encapsulated with active molecules. VII. Detection Reagents

[0415] As described above, the anti-CD8 antibody or its antigen-binding fragment, multispecific antibody, fusion protein, lipid nanoparticle, or conjugate of any of the above embodiments can bind to CD8. Therefore, it can be used for the qualitative or quantitative detection, localization, or imaging of CD8.

[0416] This disclosure also provides a detection reagent comprising an anti-CD8 antibody or its antigen-binding fragment according to any of the above embodiments, a multispecific antibody according to any of the above embodiments, a fusion protein according to any of the above embodiments, a nucleic acid according to any of the above embodiments, DNA according to any of the above embodiments, a genetic engineering vector according to any of the above embodiments, a host cell according to any of the above embodiments, a lipid nanoparticle according to any of the above embodiments, a conjugate according to any of the above embodiments, or mRNA according to any of the above embodiments.

[0417] In some embodiments, the detection reagent comprises a detectable marker, and an anti-CD8 antibody or its antigen-binding fragment of any of the above embodiments, a multispecific antibody of any of the above embodiments, a fusion protein of any of the above embodiments, a nucleic acid of any of the above embodiments, DNA of any of the above embodiments, a genetically engineered vector of any of the above embodiments, a host cell of any of the above embodiments, a lipid nanoparticle of any of the above embodiments, a conjugate of any of the above embodiments, or mRNA of any of the above embodiments.

[0418] In some embodiments, the detection reagent comprises an anti-CD8 antibody or its antigen-binding fragment from any of the above embodiments conjugated with a detectable marker, a multispecific antibody from any of the above embodiments, a fusion protein from any of the above embodiments, or a conjugate from any of the above embodiments.

[0419] In some embodiments, the detection reagent comprises lipid nanoparticles encapsulated with detectable markers in any of the above embodiments.

[0420] VIII. Application

[0421] In addition, this disclosure also provides the use of an anti-CD8 antibody or antigen-binding fragment thereof of any of the above embodiments, a multispecific antibody of any of the above embodiments, a fusion protein of any of the above embodiments, a nucleic acid of any of the above embodiments, DNA of any of the above embodiments, a genetic engineering vector of any of the above embodiments, a host cell of any of the above embodiments, a conjugate of any of the above embodiments, mRNA of any of the above embodiments, lipid nanoparticles of any of the above embodiments, or a pharmaceutical composition of any of the above embodiments in the preparation of a drug targeting CD8+ cells.

[0422] This disclosure also provides the application of any of the above-described embodiments of an anti-CD8 antibody or its antigen-binding fragment, any of the above-described embodiments of a multispecific antibody, any of the above-described embodiments of a fusion protein, any of the above-described embodiments of a nucleic acid, any of the above-described embodiments of DNA, any of the above-described embodiments of a genetic engineering vector, any of the above-described embodiments of a host cell, any of the above-described embodiments of a conjugate, any of the above-described embodiments of mRNA, or any of the above-described embodiments of lipid nanoparticles in the preparation of a detection reagent.

[0423] Furthermore, this disclosure also provides a method for treating or preventing a disease, the method comprising administering to a subject an anti-CD8 antibody or its antigen-binding fragment thereof from any of the above embodiments, a multispecific antibody from any of the above embodiments, a fusion protein from any of the above embodiments, a nucleic acid from any of the above embodiments, DNA from any of the above embodiments, a genetically engineered vector from any of the above embodiments, a host cell from any of the above embodiments, a conjugate from any of the above embodiments, mRNA from any of the above embodiments, lipid nanoparticles from any of the above embodiments, or a pharmaceutical composition from any of the above embodiments. It is understood that the method includes administering an active molecule for treating or preventing a disease, the active molecule being capable of or already conjugated to an anti-CD8 antibody or its antigen-binding fragment thereof, or being encapsulated in lipid nanoparticles conjugated with an anti-CD8 antibody or its antigen-binding fragment thereof, thereby enabling the active molecule for treating or preventing the disease to target CD8. + Cells, to achieve therapeutic goals.

[0424] In some embodiments, the method includes administering to a subject an anti-CD8 antibody or antigen-binding fragment thereof conjugated with or containing an active molecule, or a multispecific antibody of any of the above embodiments, a fusion protein of any of the above embodiments, a conjugate of any of the above embodiments, a lipid nanoparticle of any of the above embodiments, or a pharmaceutical composition of any of the above embodiments.

[0425] In some implementations, the disease is cancer.

[0426] In some implementations, CD8 + The cell is CD8 + T cells, such as tumor-invasive CD8 cells. + T cells.

[0427] In some implementations, the subject is a human. In some implementations, the subject is a non-human mammal, such as a rat, mouse, guinea pig, hamster, rabbit, dog, cat, cow, horse, goat, sheep, donkey, pig, monkey, ape, or other non-human primate. In some implementations, the non-human primate is a rhesus monkey or a cynomolgus monkey.

[0428] It is understood that the frequency and dosage of administration of the aforementioned anti-CD8 antibodies or their antigen-binding fragments, mRNA, fusion proteins, multispecific antibodies, conjugates, DNA, nucleic acids, genetically engineered vectors, lipid nanoparticles, or pharmaceutical compositions should take into account factors including the specific disease to be treated, the specific mammal to be treated, the individual patient's clinical condition, the cause of the disease, the delivery site of the agent, the method of administration, the timing of administration, and other factors known to the medical practitioner. The effective amount administered depends on the amount of anti-CD8 antibodies or their antigen-binding fragments present in or translated into the formulation, the type of disease or treatment, and other factors discussed above, and can be used at any dose and via any route determined empirically / clinically.

[0429] This disclosure also provides a method for targeting CD8+ cells with an active molecule, the method comprising administering to a desired subject an anti-CD8 antibody or antigen-binding fragment thereof from any of the above embodiments, a multispecific antibody from any of the above embodiments, a fusion protein from any of the above embodiments, a nucleic acid from any of the above embodiments, DNA from any of the above embodiments, a genetically engineered vector from any of the above embodiments, a host cell from any of the above embodiments, a conjugate from any of the above embodiments, mRNA from any of the above embodiments, lipid nanoparticles from any of the above embodiments, or a pharmaceutical composition from any of the above embodiments. It is understood that the active molecule can be or has been conjugated to an anti-CD8 antibody or antigen-binding fragment thereof, or is encapsulated in lipid nanoparticles conjugated with an anti-CD8 antibody or antigen-binding fragment thereof, thereby enabling it to target CD8+ cells. + cell.

[0430] In some embodiments, the method includes administering to a subject in need an anti-CD8 antibody or antigen-binding fragment thereof conjugated with or containing an active molecule, any of the above embodiments, a multispecific antibody of any of the above embodiments, a fusion protein of any of the above embodiments, a conjugate of any of the above embodiments, a lipid nanoparticle of any of the above embodiments, or a pharmaceutical composition of any of the above embodiments.

[0431] In some implementations, CD8 + The cell is CD8 + T cells, such as tumor-invasive CD8 cells. + T cells.

[0432] In some implementations, the method is performed in vivo or in vitro.

[0433] In some implementations, the method is performed in vivo.

[0434] In some implementation schemes, the required subjects are research subjects.

[0435] In some implementation schemes, the subjects are as described above.

[0436] In addition, a method for detecting the presence or absence of CD8 is provided, the method comprising: applying a detection reagent according to any of the above embodiments; and detecting the presence or absence of an anti-CD8 antibody or its antigen-binding fragment to determine the presence or absence of CD8.

[0437] In some implementations, the method includes detecting the presence or absence of CD8 in vitro or in vitro samples.

[0438] In some implementations, the method includes adding the detection reagent from any of the above implementations to an in vitro sample.

[0439] In some implementations, the method is based on Western blot, immunohistochemical analysis, or ELISA.

[0440] In some implementations, the method includes detecting the presence or absence of CD8 in vivo.

[0441] In some embodiments, the method includes administering the detection reagent of any of the above embodiments to a subject; and detecting the presence or absence of an anti-CD8 antibody or its antigen-binding fragment conjugated with a detectable marker in the body.

[0442] In some implementation schemes, the subjects are as described above.

[0443] In some implementations, detecting an anti-CD8 antibody or its antigen-binding fragment includes detecting a detectable marker conjugated to the anti-CD8 antibody or its antigen-binding fragment.

[0444] This disclosure also provides a method for detecting, locating, and / or imaging CD8+ cells, the method comprising administering a detection reagent of any of the above embodiments to a subject in need; and detecting the binding of an anti-CD8 antibody or its antigen-binding fragment to CD8.

[0445] In some embodiments, the method includes detecting the presence or absence of CD8+ cells in an ex vivo sample. In some embodiments, the method includes adding the detection reagent from any of the above embodiments to the ex vivo sample.

[0446] In some implementations, the method is based on Western blot, immunohistochemical analysis, or ELISA.

[0447] In some implementations, detecting the binding of the anti-CD8 antibody to CD8 includes detecting a detectable marker conjugated to the anti-CD8 antibody.

[0448] In some implementations, the method includes detecting, locating, or imaging the presence of CD8+ cells in vivo.

[0449] In some implementations, the method includes applying the detection reagent of any of the above implementations to the subject.

[0450] In some implementations, the method is administered orally, topically or locally, intravenously, or intraperitoneally.

[0451] In some implementations, the method includes applying a detection reagent to a subject and retrieving a sample from the subject for detection (i.e., detecting the binding of an anti-CD8 antibody or its antigen-binding fragment to CD8).

[0452] In some implementations, detecting the presence of CD8 in vivo includes detecting CD8 (e.g., CD8+) + Cells are located to organs or tissues.

[0453] In some implementations, the method includes targeting CD8 in the subject. + Cell imaging.

[0454] In some implementation schemes, CD8 in subjects + Cell imaging involves performing positron emission tomography (PET) scans on the subject.

[0455] In some implementation schemes, CD8 in subjects + Cellular imaging involves performing positron emission tomography / computed tomography (PET / CT) scans on the subject.

[0456] In some implementations, the method includes measuring CD8 in the subject's organs or tissues. + The number of cells.

[0457] In some implementations, the subject has cancer, and detecting the presence of CD8 in vivo involves... + Cells localize to the tumor.

[0458] In some implementations, CD8 + The cell is CD8 + T cells, such as tumor-invasive CD8 cells. + T cells.

[0459] In some implementations, the method includes measuring CD8 in tumors of subjects with cancer. + The number of T cells. In some embodiments, the method includes measuring CD8+ in tumors of subjects with cancer at multiple consecutive time points. + The number of T cells. Example

[0460] To make the objectives and technical solutions of this disclosure clearer, the following detailed description is provided in conjunction with specific embodiments. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the reagents and instruments used in the embodiments are conventionally selected in the art. Experimental methods not specifying specific conditions in the embodiments are implemented according to conventional conditions, such as those described in literature, books, or methods recommended by the manufacturer.

[0461] In the following examples, human CD8α&β protein was purchased from Peptasys (catalog number CDA-H52W5), cynomolgus monkey CD8α&β protein was purchased from Sinocare (catalog number CT077-C2508H), and mouse CD8α&β protein was purchased from Sinocare (catalog number CT076-M2508H).

[0462] Example 1: Preparation of anti-CD8 antibody

[0463] (1) Construction of protein expression vector: The anti-CD8 antibody OKT8 was humanized through CDR substitution and reverse mutation. The humanized anti-CD8 antibody is shown in Table 1. The anti-CD8 antibody in Table 1 consists of a heavy chain and a light chain. The heavy chain is composed of heavy chain signal peptide-heavy chain variable region-heavy chain constant region, and the light chain is composed of light chain signal peptide-light chain variable region-light chain constant region. For example, the heavy chain of antibody H1L1 is composed of heavy chain signal peptide-VH1-heavy chain constant region, and the light chain is composed of light chain signal peptide-VL1-light chain constant region.

[0464] Based on Tables 1, 2, and 3, and the amino acid sequences of the heavy chain signal peptide, heavy chain variable region, heavy chain constant region, light chain signal peptide, light chain variable region, and light chain constant region (underlined regions are complementarity-determining regions), the corresponding heavy chain and light chain gene sequences were synthesized. Then, the heavy chain and light chain gene sequences were constructed into the eukaryotic expression vector pcDNA3.1 via homologous recombination. After transformation into DH5α competent cells, the cells were plated and cultured. Single clones were selected for sequencing. Correctly sequenced single clones were cultured overnight at 37°C for plasmid extraction. Plasmids were then extracted using an endotoxin-free plasmid extraction kit (Qiagen).

[0465] (2) Protein expression and purification: The expression plasmid extracted in (1) was co-transfected into human embryonic kidney cell line 293 (Expi293F, Thermo Fisher Scientific) at a mass ratio of light chain to heavy chain of 1:1; after 5 days of culture at 37°C with 5% CO2, the antibody was purified from the cell culture supernatant using a protein A affinity chromatography column.

[0466] Table 1

[0467] Table 2

[0468] Table 3

[0469] Heavy chain signal peptide: MMVLSLLYLLTALPGILS (SEQ ID NO:30)

[0470] Light chain signal peptide: MDSQAQVLMLLLLWVSGTCG (SEQ ID NO:31)

[0471] VH:

[0472] VH1:

[0473] VH2:

[0474] VH3:

[0475] VH4:

[0476] VH5:

[0477] VL:

[0478] VL1:

[0479] VL2:

[0480] VL3:

[0481] VL4:

[0482] Heavy chain constant region (S228P F234A L235A):

[0483] Light chain constant region:

[0484] Example 2: Determination of antigen-binding activity of anti-CD8 antibody based on BLI technology

[0485] The binding activity of the anti-CD8 antibody from Example 1 to human CD8α&β protein, cynomolgus monkey CD8α&β protein, and mouse CD8α&β protein was determined using OctetRED96 (Sartorius). Specific steps included:

[0486] (1) Dilute the antibody to be tested to 10 μg / mL with PBST buffer (1×PBS with 0.02% Tween 20 added, pH 7.4). Dilute human CD8α&β protein, cynomolgus monkey CD8α&β protein and mouse CD8α&β protein serially with PBST: the initial concentration of human CD8α&β protein was 100 nM, and it was serially diluted 2-fold; the initial concentration of cynomolgus monkey CD8α&β protein and mouse CD8α&β protein was 500 nM, and it was serially diluted 2-fold.

[0487] (2) Add the diluted sample solution to a 96-well black ELISA plate. Add PBST buffer to the baseline, dissociation, and neutralization wells. Add 10 mM glycine-hydrochloric acid (pH 1.7) to the regeneration wells. The biosensor used is Protein A (Sartorius, catalog number 18-5010).

[0488] (3) Test the antibody and antigen using OctetRED96 under the conditions specified in Table 4. After obtaining the data, use the OctetRED96 analysis software to fit the binding and dissociation curves according to the 1:1 binding model to obtain the antibody-antigen binding rate constant (Kon), dissociation rate constant (Koff), and dissociation constant (KD).

[0489] The results are shown in Tables 5, 6, and 7. Table 5 shows the binding activity of the anti-CD8 antibody with human CD8α & β protein, Table 6 shows the binding activity of the anti-CD8 antibody with cynomolgus monkey CD8α & β protein, and Table 7 shows the binding activity of the anti-CD8 antibody with mouse CD8α & β protein. In this paper, the parameters used to represent the antibody-antigen binding activity are: KD (dissociation constant), Kon (binding rate constant), and Koff (dissociation rate constant).

[0490] Table 4

[0491] Table 5 ND means not detected, and the same applies below.

[0492] Table 6

[0493] Table 7

[0494] Example 3: FACS detection of antigen-binding activity of anti-CD8 antibody

[0495] The antigen-binding activity of the anti-CD8 antibody prepared in Example 1 was detected using FACS, specifically including:

[0496] 1. Cell preparation:

[0497] Human CD3 + T cells: Human CD3 cells were obtained by resuscitating frozen human PBMCs (Maisun Biotechnology, Donor: P122080805C) and isolating them using the Majosort human CD3 T cell isolation kit (Biolegend, catalog number 480131). + T cells.

[0498] 2. FACS testing:

[0499] (1) Cell blocking: according to 2×10 5 Take the cells required for the experiment from each well, resuspend the cells in DPBS containing 2% FBS, and seed 100 μL / well into a 96-well U plate. Incubate at room temperature for 15 min.

[0500] (2) Dilution of the antibody to be tested: Starting with a concentration of 300 nM, the antibody was serially diluted 3 times to 12 titers, with the 12th titer being 0 nM;

[0501] (3) Antibody incubation: Centrifuge the cells from (1) at 500g for 5 min, discard the supernatant, add the diluted antibody from step (2) at 100 μL / well, and incubate at room temperature for 1 h.

[0502] (4) Antibody washing: Centrifuge the cells from (3) at 500g for 5min, discard the supernatant, add DPBS to wash, 200μL / well, centrifuge at 500g for 5min, discard the supernatant, and repeat the washing twice;

[0503] (5) Secondary antibody incubation: Add the detection secondary antibody (Jackson, catalog number 109-036-098) to the cells in (4) and incubate at room temperature for 40 min;

[0504] (6) Antibody washing: Centrifuge the cells from (5) at 500g for 5min, discard the supernatant, add DPBS to wash, 200μL / well, centrifuge at 500g for 5min, discard the supernatant, and repeat the washing twice;

[0505] (7) Detection on the instrument: The cells from (6) were resuspended in 100 μL of DPBS and the fluorescence intensity was detected on the instrument (Agilent NovoSampler Pro).

[0506] The results are shown in Figure 1. As can be seen from Figure 1, the binding affinity of antibodies H2L4 and H4L4 to hCD3+ T cells is comparable to that of OKT8.

[0507] Example 4: FACS detection of the binding ability of anti-CD8 antibody to CD8+ T cells

[0508] 1. Detection of the binding ability of anti-CD8 antibody to human CD8+ T cells based on FACS method

[0509] Human PBMCs (Maisun Biotechnology, Donor: P123120707C) were revived from cryopreservation, and human CD8+ T cells (hCD8+ T cells) were obtained by sorting using a human CD8+ T cell sorting kit (Biolegend). The hCD8+ T cells were then processed at a concentration of 1×10⁻⁶ cells / mL. 5 Cells were seeded at a density of 100 cells per well in a 96-well plate, and serially diluted test antibodies were added. After incubation at room temperature in the dark for 30 minutes, the cells were washed, and 100 μL of a 1:1000 dilution of Goat F(ab')2 anti-human IgG Fc (APC) secondary antibody was added, followed by incubation for 30 minutes. The cells were washed, and the binding of the antibody to hCD8+ T cells was detected by flow cytometry. The results are shown in Figure 2A.

[0510] 2. Detection of antibody binding ability to monkey CD8+ T cells using FACS method

[0511] Cynocyprinid PBMCs (Yunqiao Purui Biotechnology, Donor: 190833C) were revived from cryopreservation, and cynocyprinid CD8+ T cells (cynocyprinid CD8+ T cells) were obtained by sorting using a non-human primate CD8+ T cell sorting kit (Stemcell). The cyno CD8+ T cells were then sorted at a concentration of 1×102 5 Cells were seeded at a density of 100 cells per well in a 96-well plate, and serially diluted antibodies were added. After incubation at room temperature in the dark for 30 minutes, the cells were washed, and 100 μL of a 1:1000 dilution of Goat F(ab')2 anti-human IgG Fc (APC) secondary antibody was added, followed by incubation for 30 minutes. The cells were washed, and the binding of the antibody to cyno CD8+ T cells was detected by flow cytometry. The results are shown in Figure 2B.

[0512] As shown in Figures 2A and 2B, the binding affinity of antibodies H2L4 and H4L4 to hCD8+ T cells is comparable to that of OKT8.

[0513] Example 5: Evaluation of the in vitro delivery capability of lipid nanoparticles (tLNP) conjugated with anti-CD8 antibody

[0514] In this embodiment, an anti-CD8 antibody was chemically coupled to the surface of lipid nanoparticles (LNPs) to obtain anti-CD8 antibody-coupled lipid nanoparticles (tLNPs), and the delivery capacity of tLNPs was tested in an activated T cell system.

[0515] 1. Preparation of tLNP

[0516] First, the anti-CD8 antibody buffer was replaced with PBS (containing 10 mM EDTA), SATA reagent was added, and the reaction was carried out at room temperature in the dark for 30 minutes. After ultrafiltration and centrifugation, the buffer was replaced with PBS (containing 10 mM EDTA). Then, hydroxylamine hydrochloride was added, and the reaction was carried out at room temperature in the dark for 30 minutes. After ultrafiltration and centrifugation, the buffer was replaced with PBS (containing 10 mM EDTA) to obtain the thiolized antibody. The lipid fraction was prepared in anhydrous ethanol to form an alcohol phase solution according to the following ratio: compound 2-3:DSPC:cholesterol:DMG-PEG2000:DMG-PEG2000-maleimide = 50:10:38.5:1.4:0.1 (molar ratio). The eGFP-encoding mRNA was prepared in acetate buffer to form an aqueous phase solution. The alcohol and aqueous phase solutions were then mixed using a microfluidic chip to obtain the LNP. Then, the thiolized antibody was mixed with LNP and incubated overnight at 4°C in the dark. Finally, the unconjugated antibody was removed by dialysis and the buffer was replaced with Tris buffer containing sucrose to obtain tLNP.

[0517] Following the steps described above, lipid nanoparticles conjugated with the antibody hIgG4 isotype were prepared, wherein the hIgG4 isotype was purchased from abinvivo (catalog number B21124101).

[0518] 2. Evaluation of tLNP delivery in activated T cells

[0519] Revive Human CD3 + T cells, after centrifugation and washing, were used in the prepared T cell expansion culture medium (OpTmizer). TM CTS TM T-Cell Expansion SFM basal medium, 2.6% OpTmizer TM CTS TM Resuspend cells in T-Cell Expansion Supplement, 100 U / mL IL-2 and 1× glutamine, 10% FBS, at a dose of 1.0 × 10⁻⁶ cells / mL. 6 Cells were incubated with 2.5 μL of CD3 / CD28 magnetic beads at a ratio of 2.5 μL each for 3 days at 37°C and 5% CO2. Afterwards, the magnetic beads were removed, cells were counted, washed once with D-PBS, and then conditioned on T-cell expansion medium to adjust the cell density to 1.0 × 10⁶ cells / year. 6 cells / mL. Inoculate at a rate of 1 × 10⁻⁶ cells / mL. 5Cells were transfected with tLNP at a ratio of 0.1 μg lipid per cell. After incubation at 37°C with 5% CO2 for 1 hour, the cells were centrifuged, the supernatant was removed, and the cells were washed with PBS before being cultured with T-cell expansion medium for 24 hours. After culture, the cells were collected by centrifugation, and the supernatant was discarded. Each well was then pre-treated with Human TruStain FCX. TM Fc receptor blocking was performed using a solution diluted 1:50 with PBS and incubated at room temperature for 10 minutes; the cells were washed with PBS, followed by the addition of a multicolor antibody mixture (Brilliant Violet 510). TM Anti-human CD3, PerCP / Cyanine 5.5 anti-human CD4, and BV650 anti-human CD8 antibodies were prepared with PBS at a 1:200 ratio. After incubation on ice in the dark for 30 minutes, the mixture was washed with PBS and then APC-Cy7-labeled Zombie NIR was added. TM Cells were stained using a Fixable Viability Kit (1:1000 PBS dilution) to differentiate between live and dead cells. Cells were incubated on ice in the dark for 10 minutes, followed by washing with PBS. Cells were then resuspended in 200 μL PBS and analyzed using flow cytometry. The total cell population was first screened using a gating strategy, followed by exclusion of dead cells using the viability stain. Further analysis of CD3+ in live cells was then performed. + T cells and their CD4 + and CD8 + The proportion of subpopulations and eGFP expression.

[0520] The tLNP delivery results are shown in Figures 3A and 3B. As can be seen from Figures 3A and 3B, lipid nanoparticles conjugated with anti-CD8 antibodies could not be delivered to CD4. + T cells, but can be specifically delivered to CD8 + T cells.

[0521] Sequences (excluding the sequences specifically listed above)

[0522] SEQ ID NO:8

[0523] SEQ ID NO:9

[0524] SEQ ID NO:10

[0525] SEQ ID NO:11

[0526] SEQ ID NO:12

[0527] SEQ ID NO:13

[0528] SEQ ID NO:14

[0529] SEQ ID NO:15

[0530] SEQ ID NO:16

[0531] SEQ ID NO:17

[0532] SEQ ID NO:18

[0533] SEQ ID NO:19

[0534] SEQ ID NO:20

[0535] SEQ ID NO:21

[0536] SEQ ID NO:22

[0537] SEQ ID NO:23

[0538] SEQ ID NO:24

[0539] SEQ ID NO:25

[0540] SEQ ID NO:26

[0541] SEQ ID NO:29

Claims

1. An anti-CD8 antibody or its antigen-binding fragment thereof, said anti-CD8 antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of the heavy chain variable region shown in SEQ ID NO:1, said heavy chain variable region further comprising a heavy chain framework region, said light chain variable region comprising LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO:5, said light chain variable region further comprising a light chain framework region, wherein: (1) The heavy chain frame region includes: HFR1 in the heavy chain variable region shown in SEQ ID NO:4; HFR2 in the heavy chain variable region shown in SEQ ID NO:4; or HFR3 in the heavy chain variable region shown in SEQ ID NO:1 or 4; Or, (2) the light chain framework region includes: LFR1 in the variable region of the light chain shown in SEQ ID NO:7; or LFR2 in the variable region of the light chain shown in SEQ ID NO:

7.

2. The anti-CD8 antibody or its antigen-binding fragment according to claim 1, wherein the heavy chain framework region comprises: HFR3 in the heavy chain variable region shown in SEQ ID NO:1; HFR3 in the heavy chain variable region shown in SEQ ID NO:4; HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:4; or HFR1, HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:

4.

3. The anti-CD8 antibody or its antigen-binding fragment according to claim 1 or 2, wherein the light chain framework region comprises: LFR1 in the variable region of the light chain shown in SEQ ID NO:7; LFR2 in the variable region of the light chain shown in SEQ ID NO:7; or LFR1 and LFR2 in the variable region of the light chain shown in SEQ ID NO:

7.

4. The anti-CD8 antibody or its antigen-binding fragment according to any one of claims 1 to 3, wherein the heavy chain framework region and the light chain framework region are a group of the following: (1) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:1, and the light chain frame region includes LFR1 in the light chain variable region shown in SEQ ID NO:5; (2) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:1, and the light chain frame region includes LFR2 in the light chain variable region shown in SEQ ID NO:

6. (3) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:1, and the light chain frame region includes LFR1 and LFR2 in the light chain variable region shown in SEQ ID NO:7; (4) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:1, and the light chain frame region includes LFR1, LFR2, LFR3 and LFR4 in the light chain variable region shown in SEQ ID NO:28; (5) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:2, and the light chain frame region includes LFR1 in the light chain variable region shown in SEQ ID NO:5; (6) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:2, and the light chain frame region includes LFR2 in the light chain variable region shown in SEQ ID NO:6; (7) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:2, and the light chain frame region includes LFR1 and LFR2 in the light chain variable region shown in SEQ ID NO:7; (8) The heavy chain frame region includes HFR3 in the heavy chain variable region shown in SEQ ID NO:2, and the light chain frame region includes LFR1, LFR2, LFR3 and LFR4 in the light chain variable region shown in SEQ ID NO:28; (9) The heavy chain frame region includes HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:3, and the light chain frame region includes LFR1 in the light chain variable region shown in SEQ ID NO:5; (10) The heavy chain frame region includes HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:3, and the light chain frame region includes LFR2 in the light chain variable region shown in SEQ ID NO:6; (11) The heavy chain frame region includes HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:3, and the light chain frame region includes LFR1 and LFR2 in the light chain variable region shown in SEQ ID NO:7; (12) The heavy chain frame region includes HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:3, and the light chain frame region includes LFR1, LFR2, LFR3 and LFR4 in the light chain variable region shown in SEQ ID NO:28; (13) The heavy chain frame region includes HFR1, HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:4, and the light chain frame region includes LFR1 in the light chain variable region shown in SEQ ID NO:5; (14) The heavy chain frame region includes HFR1, HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:4, and the light chain frame region includes LFR2 in the light chain variable region shown in SEQ ID NO:6; (15) The heavy chain frame region includes HFR1, HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:4, and the light chain frame region includes LFR1 and LFR2 in the light chain variable region shown in SEQ ID NO:

7. (16) The heavy chain frame region includes HFR1, HFR2 and HFR3 in the heavy chain variable region shown in SEQ ID NO:4, and the light chain frame region includes LFR1, LFR2, LFR3 and LFR4 in the light chain variable region shown in SEQ ID NO:

28. (17) The heavy chain frame region includes HFR1, HFR2, HFR3 and HFR4 in the heavy chain variable region shown in SEQ ID NO:27, and the light chain frame region includes LFR1 in the light chain variable region shown in SEQ ID NO:5; (18) The heavy chain frame region includes HFR1, HFR2, HFR3 and HFR4 in the heavy chain variable region shown in SEQ ID NO:27, and the light chain frame region includes LFR2 in the light chain variable region shown in SEQ ID NO:6; and (19) The heavy chain frame region includes HFR1, HFR2, HFR3 and HFR4 in the heavy chain variable region shown in SEQ ID NO:27, and the light chain frame region includes LFR1 and LFR2 in the light chain variable region shown in SEQ ID NO:

7.

5. The anti-CD8 antibody or its antigen-binding fragment according to any one of claims 1 to 4, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively; and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, respectively.

6. The anti-CD8 antibody or its antigen-binding fragment according to any one of claims 1 to 5, wherein the heavy chain framework region comprises: HFR1 as shown in SEQ ID NO:15; HFR2 as shown in SEQ ID NO:17; HFR3 as shown in SEQ ID NO:18 or 19; Alternatively, the light chain framework region may include: LFR1 as shown in SEQ ID NO:21; or LFR2 as shown in SEQ ID NO:

24.

7. The anti-CD8 antibody or its antigen-binding fragment according to any one of claims 1 to 6, wherein the heavy chain framework region comprises: HFR3 as shown in SEQ ID NO:18; HFR3 as shown in SEQ ID NO:19; HFR2 shown in SEQ ID NO:17, HFR3 shown in SEQ ID NO:19; or HFR1 shown in SEQ ID NO:15, HFR2 shown in SEQ ID NO:17, and HFR3 shown in SEQ ID NO:

19.

8. The anti-CD8 antibody or its antigen-binding fragment according to any one of claims 1 to 7, wherein the light chain framework region comprises: LFR1 as shown in SEQ ID NO:21; LFR2 as shown in SEQ ID NO:24; or LFR1 shown in SEQ ID NO:21 and LFR2 shown in SEQ ID NO:

24.

9. The anti-CD8 antibody or its antigen-binding fragment according to any one of claims 1 to 8, wherein the heavy chain framework region and the light chain framework region are a group of the following: (1) The heavy chain frame region includes HFR3 as shown in SEQ ID NO:18, and the light chain frame region includes LFR1 as shown in SEQ ID NO:21; (2) The heavy chain frame region includes HFR3 as shown in SEQ ID NO:18, and the light chain frame region includes LFR2 as shown in SEQ ID NO:24; (3) The heavy chain frame region includes HFR3 shown in SEQ ID NO:18, and the light chain frame region includes LFR1 shown in SEQ ID NO:21 and LFR2 shown in SEQ ID NO:24; (4) The heavy chain frame region includes HFR3 shown in SEQ ID NO:18, and the light chain frame region includes LFR1 shown in SEQ ID NO:22, LFR2 shown in SEQ ID NO:23, LFR3 shown in SEQ ID NO:25 and LFR4 shown in SEQ ID NO:26; (5) The heavy chain frame region includes HFR3 as shown in SEQ ID NO:19, and the light chain frame region includes LFR1 as shown in SEQ ID NO:21; (6) The heavy chain frame region includes HFR3 as shown in SEQ ID NO:19, and the light chain frame region includes LFR2 as shown in SEQ ID NO:24; (7) The heavy chain frame region includes HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:21 and LFR2 shown in SEQ ID NO:24; (8) The heavy chain frame region includes HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:22, LFR2 shown in SEQ ID NO:23, LFR3 shown in SEQ ID NO:25 and LFR4 shown in SEQ ID NO:26; (9) The heavy chain frame region includes HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:21; (10) The heavy chain frame region includes HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR2 shown in SEQ ID NO:24; (11) The heavy chain frame region includes HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:21 and LFR2 shown in SEQ ID NO:24; (12) The heavy chain frame region includes HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:22, LFR2 shown in SEQ ID NO:23, LFR3 shown in SEQ ID NO:25 and LFR4 shown in SEQ ID NO:26; (13) The heavy chain frame region includes HFR1 shown in SEQ ID NO:15, HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:21; (14) The heavy chain frame region includes HFR1 shown in SEQ ID NO:15, HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR2 shown in SEQ ID NO:24; (15) The heavy chain frame region includes HFR1 shown in SEQ ID NO:15, HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:21 and LFR2 shown in SEQ ID NO:

24. (16) The heavy chain frame region includes HFR1 shown in SEQ ID NO:15, HFR2 shown in SEQ ID NO:17 and HFR3 shown in SEQ ID NO:19, and the light chain frame region includes LFR1 shown in SEQ ID NO:22, LFR2 shown in SEQ ID NO:23 and LFR3 shown in SEQ ID NO:25 and LFR4 shown in SEQ ID NO:26; (17) The heavy chain frame region includes HFR1 shown in SEQ ID NO:14, HFR2 shown in SEQ ID NO:16, HFR3 shown in SEQ ID NO:29 and HFR4 shown in SEQ ID NO:20, and the light chain frame region includes LFR1 shown in SEQ ID NO:21; (18) The heavy chain frame region comprises HFR1 shown in SEQ ID NO:14, HFR2 shown in SEQ ID NO:16, HFR3 shown in SEQ ID NO:29, and HFR4 shown in SEQ ID NO:20; and the light chain frame region comprises LFR2 shown in SEQ ID NO:24; and (19) The heavy chain frame region includes HFR1 shown in SEQ ID NO:14, HFR2 shown in SEQ ID NO:16, HFR3 shown in SEQ ID NO:29 and HFR4 shown in SEQ ID NO:20, and the light chain frame region includes LFR1 shown in SEQ ID NO:21 and LFR2 shown in SEQ ID NO:

24.

10. The anti-CD8 antibody or its antigen-binding fragment according to any one of claims 1 to 9, wherein the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 1 to 4 and 27; and / or The light chain variable region contains an amino acid sequence as shown in any of SEQ ID NO:5-7 and 28.

11. The anti-CD8 antibody or its antigen-binding fragment according to any one of claims 1 to 10, wherein the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 6 or 7.

12. The anti-CD8 antibody or its antigen-binding fragment according to any one of claims 1 to 11, The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:1, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:5; The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:1, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:6; The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:1, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:7; The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:1, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:

28. The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:2, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:5; The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:2, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:6; The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:2, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7; The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:2, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:28; The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:3, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:5; The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:3, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:6; The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:3, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:7; The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:3, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:

28. The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:4, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:5; The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:4, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:6; The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:4, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:7; The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:4, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:

28. The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:27, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:5; The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:6; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:27, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:

7.

13. The anti-CD8 antibody or its antigen-binding fragment according to any one of claims 1 to 12, wherein the anti-CD8 antibody or its antigen-binding fragment is scFv, Fab, scFab, F(ab'), F(ab')2, scFv-Fc or microantibody.

14. The anti-CD8 antibody or its antigen-binding fragment according to any one of claims 1 to 13, wherein the anti-CD8 antibody or its antigen-binding fragment further comprises human Ig Fc; Preferably, the human Ig is human IgG1, human IgG2, human IgG3, or human IgG4.

15. The anti-CD8 antibody or its antigen-binding fragment according to claim 14, wherein the human Ig Fc is human IgG4 Fc; Preferably, the human IgG4 Fc has S228P, F234A and L235A mutations.

16. A multispecific antibody comprising the anti-CD8 antibody or its antigen-binding fragment as described in any one of claims 1 to 15.

17. A fusion protein comprising the anti-CD8 antibody or its antigen-binding fragment as described in any one of claims 1 to 15 and an active molecule.

18. A nucleic acid encoding the anti-CD8 antibody or its antigen-binding fragment as described in any one of claims 1 to 15, the multispecific antibody as described in claim 16, or the fusion protein as described in claim 17.

19. The nucleic acid according to claim 18, wherein the nucleic acid is mRNA.

20. A DNA that encodes the anti-CD8 antibody or its antigen-binding fragment as described in any one of claims 1 to 15, the multispecific antibody as described in claim 16, or the fusion protein as described in claim 17.

21. A gene engineering vector comprising the DNA of claim 20.

22. A host cell comprising the nucleic acid of claim 18 or 19, the DNA of claim 20, or the genetic engineering vector of claim 21.

23. A conjugate comprising the anti-CD8 antibody or its antigen-binding fragment as described in any one of claims 1 to 15 and an active molecule conjugated thereto, or the multispecific antibody as described in claim 16 and an active molecule conjugated thereto.

24. A lipid nanoparticle, said lipid nanoparticle being coupled with a targeting domain, said targeting domain comprising an anti-CD8 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 15 or a multispecific antibody as described in claim 16.

25. The lipid nanoparticles of claim 24, wherein the lipid nanoparticles comprise ionizable lipids; Preferably, the lipid nanoparticles further contain auxiliary lipids, structural lipids, and polymer-lipids; Preferably, the polymer-lipid includes functionalized polymer-lipids and unfunctionalized polymer-lipids; Preferably, the auxiliary lipid is a phospholipid, the structural lipid is cholesterol, the unfunctionalized polymer-lipid is a PEG-lipid, and the functionalized polymer-lipid is a maleimide-modified PEG-lipid. Preferably, the lipid nanoparticles are encapsulated with active molecules.

26. A pharmaceutical composition comprising the anti-CD8 antibody or its antigen-binding fragment as described in any one of claims 1 to 15, the multispecific antibody as described in claim 16, the fusion protein as described in claim 17, the nucleic acid as described in claim 18 or 19, the DNA as described in claim 20, the genetic engineering vector as described in claim 21, the host cell as described in claim 22, the conjugate as described in claim 23, or the lipid nanoparticles as described in claim 24 or 25.

27. The anti-CD8 antibody or its antigen-binding fragment according to any one of claims 1 to 15, the multispecific antibody according to claim 16, the fusion protein according to claim 17, the nucleic acid according to claim 18 or 19, the DNA according to claim 20, the genetic engineering vector according to claim 21, the host cell according to claim 22, the conjugate according to claim 23, the lipid nanoparticles according to claim 24 or 25, or the pharmaceutical composition according to claim 26, in the preparation of a drug targeting CD8. + Applications of cell-based drugs.

28. A detection reagent comprising the anti-CD8 antibody or its antigen-binding fragment as described in any one of claims 1 to 15, the multispecific antibody as described in claim 16, the fusion protein as described in claim 17, the conjugate as described in claim 23, or the lipid nanoparticles as described in claim 24 or 25.

29. The use of the anti-CD8 antibody or its antigen-binding fragment according to any one of claims 1 to 15, the multispecific antibody according to claim 16, the fusion protein according to claim 17, the nucleic acid according to claim 18 or 19, the DNA according to claim 20, the genetic engineering vector according to claim 21, the host cell according to claim 22, the conjugate according to claim 23, or the lipid nanoparticles according to claim 24 or 25 in the preparation of detection reagents.

30. A method for delivering an active molecule to CD8-positive cells, the method comprising administering to a desired object the fusion protein of claim 17, a nucleic acid encoding the fusion protein of claim 17, the conjugate of claim 23, or the lipid nanoparticles of claim 24 or 25.

31. A method for treating or preventing a disease, the method comprising administering to a subject an anti-CD8 antibody or antigen-binding fragment thereof comprising or conjugated with an active molecule as described in any one of claims 1 to 15, a multispecific antibody as described in claim 16, a fusion protein as described in claim 17, a nucleic acid as described in claim 18 or 19, DNA as described in claim 20, a genetically engineered vector as described in claim 21, a conjugate as described in claim 23, or lipid nanoparticles as described in claim 24 or 25 encapsulating an active molecule.

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